Methods of Experim en tal Physics
VOLUME 20
BIOPHYSICS
METHODS OF EXPE R I M ENTAL PHYS ICS:
C. Marton, EditorinChief
Volume 20
Bio phys ics
Edited by
GERALD EHRENSTEIN and HAROLD LECAR
Laboratory of Biophysics National Institutes of Health Bethesda, Maryland
1982
@
Toronto
ACADEMIC PRESS
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Library of Congress Cataloging i n Publication Data Main entry under t i t l e : Biophysics. (Methods o f experimental physics : v. 20) Includes bibliographical references and index. 1. Biophysics. I . Lecar, H. (Harold) 11. Ehrenstein, G. (Gerald) I I I . Series. PH505.8472 574.19'1 026642 ISBN 0124759629 AACRZ
PRINTED IN T I E UNITED STATES OF AMERICA
82 8.1 8 4 8s
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CONTENTS
CONTRIBUTORS .............................................. PUBLISHER’S FOREWORD ..................................... FOREWORD .................................................
xvii
xix xxi xxiii
.................................................... PREFACE
LISTOF VOLUMESI N TREATISE .............................. xxvii
1. Nuclear Magnetic Resonance by JOSEPH R . SCHUH AND SUNNEY I . CHAN
1 . 1 . Introduction
........................................
1 2 2 4 7 12 13 14 14 16 24 25 25 30 32 39 41
1.2. Phenomenon of Magnetic Resonance .................. 1.2.1. Quantum Description .......................... 1.2.2. Classical Description........................... 1.2.3. Detection of Magnetic Resonance ...............
1.3. Spin Relaxation ..................................... 1.3.1. SpinLattice Relaxation ........................ 1.3.2. SpinSpin Relaxation .......................... 1.3.3. Correlation Time .............................. 1.3.4. Relaxation Mechanisms ........................ 1.3.5. Effects of Anisotropic and Restricted Motions ......................................
1.4. Experimental Methods ............................... 1.4.1. Pulse Techniques .............................. 1.4.2. MagicAngle Spinning.......................... 1.4.3. Double Resonance ............................. 1.4.4. TwoDimensional FTNMR ..................... 1.5. Selected Studies on Biological Systems ................
V
vi
CONTENTS
1.5.1. Proteins: Probing the Active Site of an Enzyme ...................................... 1.5.2. Nucleic Acids: The Dynamic Structure of tRNA ........................................ 1.5.3. Membranes: The Restricted Motions of Lipid Chains ....................................... 1.5.4. Intact Cells: Metabolism Studied in Vivo . . . . . . . . .
41
44
47 49
2 . Nitroxide Spin Labels by GILLIAN M . K . HUMPHRIES A N D HARDEN M . MCCONNELL
2.1. Introduction
........................................
53 54 54 59 62 63
65
2.2. SpinLabel Theory: A Descriptive Treatment ........... 2.2.1. Fundamentals Including the Resonance Condition ..................................... 2.2.2. Effect of Orientation ........................... 2.2.3. Effect of Motion .............................. 2.2.4. Effect of the Electrostatic Environment . . . . . . . . . . 2.2.5. Effect of Distribution or Concentration of Spin Labels: SpinExchange Broadening . . . . . . . . . 2.2.6. Effect of Distribution or Concentration of Spin Labels: Dipole Dipole Interactions .......... 2.3. SpinLabel Theory: A Mathematical Treatment . . . . . . . . . 2.3.1. The Resonance Condition ..................... 2.3.2. Absorption Probabilities and Paramagnetic Relaxation ................................... 2.3.3. The Bloch Equations ......................... 2.3.4. Nuclear Hyperfine Structure . . . . . . . . . . . . . . . . . . . 2.3.5. The Spectroscopic Splitting Factor g . . . . . . . . . . . 2.3.6. The Spin Hamiltonian ......................... 2.3.7. Effect of Orientation: SingleCrystal Spectra ...................................... 2.3.8. Isotropic Distributions of Strongly Immobilized Labels: Powder Spectra . . . . . . . . . . . 2.3.9. Effects of Isotropic Motion on Spectra. . . . . . . . . . 2.3.10. Line Shapes with Fast Anisotropic Motion and Partial OrientationFrequency Amplitude Order Parameters . . . . . . . . . . . . . . . . . . . 2.3.11. Effect of ElectronElectron SpinSpin Interaction: Dipolar Interactions in
65 65 65 68 12 75 83 84
85
90 93 98
C0N T E N TS
vii
Biradicals with Fixed Distances between 102 Spins. ....................................... 2.3.12. Effect of ElectronElectron SpinSpin Interactions: SpinSpin Interaction Determined by Translational Motion and Resulting from, Colliding Spins . . . . . . . . . . . . . . . . . 104 2.3.13. Enhancement of Nuclear Relaxation by Spin Labels.. ................................ 109 2.4. Use of Spin Labels as Antigenic Determinants Capable of Reporting Their Physical State. . . . . . . . . . . . . . 2.4.1. Background. .................................. 2.4.2. SpinLabel Lipid Haptens ...................... 2.4.3. Determination of the Rate of InsideOutside Transitions of Phospholipids in Vesicle Membranes ................................... 2.4.4. Determination of Lateral Diffusion of SpinLabel Lipid Haptens in Membranes ...... 2.4.5. Determination of the Surface Area of Lipid Membranes ................................... 2.4.6. Antibodies Specific for SpinLabel Determinants ................................. 2.4.7. Physical Factors Affecting the Binding of SpinLabel Specific Antibodies to HaptenBearing Bilayers ....................... 2.4.8. Efferent Immune Responses Controlled by Antibodies Bound to AntigenBearing Membranes ...................................
110
I10 1 11 112 112
1 13
I14 116 121
3. Raman Spectroscopy by MARGARET R. BUNOW 3.1. Introduction ........................................ 3.2. Origin of the Raman Spectrum ........................ 3.3. Analysis of the Raman Spectrum. ..................... 3.4. Resonance Raman Scattering ......................... 3.4.1. ATerm Scattering. ............................ 3.4.2. BTerm Scattering ............................. 3.4.3. The Polarizability Tensor. ......................
123 124 125 128 129 130 130
Vlll
...
CONTENTS
3.5. Instrumentation ..................................... 3.5.1, Sample Handling .............................. 3.5.2. Optimization and Standardization of Signal . . . . . . . 3.5.3. Management of Fluorescence . . . . . . . . . . . . . . . . . . . 3.6. Strategy of Raman Spectroscopic Applications in Biology ............................................ 3.7. Conformational Studies ............................. 3.7.1. Conformation of Lipids in Biological Membranes .................................. 3.7.2. Conformation of Nucleic Acids . . . . . . . . . . . . . . . . 3.7.3. Raman Spectral Analysis of Polysaccharides .............................. 3.7.4. Raman Vibrational Analysis of Protein Conformation ................................ 3.7.5. Vibrational Markers of Protein SideGroups ................................. 3.7.6. Resonance Raman Studies of Porphyrins and Hemoproteins ............................ 3.7.7. Resonance Raman Parameters for Nonheme Metalloproteins .............................. 3.7.8. Extrinsic Chromophores for Resonance Raman Studies of Proteins., ................... 3.7.9. ResonanceEnhanced Vibrational Behavior of Biological Polyenes ........................ 3.8. Kinetic Studies .....................................
131 132 132 133 134 137 137 143 145 146 149
150
154 155 155 156 159 161 161
3 .9 . Nonlinear Phenomena ...............................
3.10. The Raman Microscope .............................
3.11. Conclusions and Prognostications ....................
4. Picosecond Laser Spectroscopy
by TAKAYOSHI KOBAYASHI 4.1. Introduction ........................................
4.2. Nanosecond and Picosecond Spectroscopy ............. 4.2.1. Comparison of Nanosecond and Picosecond Spectroscopy ................................. 4.2.2. Picosecond Light Pulses ........................
163 166 166 170
CONTENTS
ix
4.2.3. Various Methods for Picosecond Absorption and Emission Spectroscopy ..................... 4.3. Applications to Photosynthesis and Vision . . . . . . . . . . . . . 4.3.1. Photosynthesis ................................ 4.3.2. Vision ........................................ Appendix. Nonlinear Optical Phenomena. Optical Elements. and Detectors Related to Techniques of Picosecond Spectroscopy............................. A .1 . Nonlinear Optical Phenomena.................... A.2. Optical Elements and Detectors ..................
5 . Fluorescence Methods for Studying Membrane Dynamics by JOSEPHSCHLESSINGER AND ELLIOT L . ELSON
172 181 181 185
190 190 194
5.1. Introduction
........................................
197 199 201 203 204 205 208 208 209 212
5.2. Molecular Rotation in Membranes ..................... 5.2.1. SteadyState Fluorescence Polarization . . . . . . . . . . 5.2.2. Nanosecond TimeDependent Fluorescence Polarization ................................... 5.2.3. Decay of Transient Dichroism . . . . . . . . . . . . . . . . . . 5.2.4. Special Features and Limitations . . . . . . . . . . . . . . . .
5.3. Macroscopic Membrane Motions ...................... 5.3.1. Basic Concepts ................................ 5.3.2. Fluorescence Photobleaching Recovery . . . . . . . . . . 5.3.3. Fluorescence Correlation Spectroscopy . . . . . . . . . .
5.4. Applications ........................................ 216 5.4.1. Translational and Rotational Diffusion of Molecules in Membranes and Membrane Viscosity ..................................... 216 5.4.2. Possible Significance of Mobility and Immobility of Membrane Proteins . . . . . . . . . . . . . . . 222
5.5. Recent Developments ................................ 5.5.1. Technical Developments ........................ 5.5.2. Origin of Constraints on Membrane Protein Mobility ....................................... 5.5.3. Receptor Mobility ..............................
224 224 225 226
X
CONTENTS
6. Structure Determination of Biological Macromolecules Using XRay Diffraction Analysis by EATON E . LATTMAN A N D L . MARIOAMZEL 6.1. Introduction
.......................................
229 230 237 237 240 244 244 246 247 252 256 259 260 263 268 275 275 276 287 288 293 296
6.2. Diffraction by a General Object ....................... 6.3. Crystallography ..................................... 6.3.1, Diffraction by Crystals ......................... 6.3.2. The Patterson Function ........................ 6.4. Protein Crystallography .............................. 6.4.1. Introduction .................................. 6.4.2. Crystallization ................................ 6.4.3. Collection of XRay Diffraction Data . . . . . . . . . . . . 6.4.4. Phase Determination Using Isomorphous Replacement .................................. 6.4.5. Interpretation of Electron Density Maps . . . . . . . . . 6.4.6. Difference Fourier Syntheses . . . . . . . . . . . . . . . . . . . 6.4.7. Molecular Replacement ........................ 6.4.8. Refinement of Protein Structures . . . . . . . . . . . . . . . . 6.4.9. A Case History: Rubredoxin .................... 6.5. Fibers .............................................. 6.5.1. Definition and Description ...................... 6.5.2. Fiber Diffraction Theory ....................... 6.5.3. Solution of Simple Fiber Structures . . . . . . . . . . . . . 6.5.4. Tobacco Mosaic Virus Structure ................ 6.5.5. Structure of Microtubules ...................... 6.5.6. Conclusion ...................................
7 . Laser Light Scattering by RALPH NOSSAL
7.1. Introduction
........................................
299
7.2. Theory ............................................. 300 7.2.1. Classical Light Scattering ...................... 304 7.2.2. Intensity Fluctuation Spectroscopy . . . . . . . . . . . . . . 306
. 7. .....10......3.........1. .... 8. Characteristics of Measured Autocorrelation Functions ... Motility Measurements .. Large Particles .... 8........8....7. 333 8.. .... ........... 8..... ... 8.. . ........ Relationship between Photon Autocorrelation and Special Analysis ..............3..... ......... 7................ .. . .... 8. 8....... 326 330 331 7... X Rays ..8..... 337 337 338 338 339 340 341 341 342 343 344 345 346 347 349 8. ... .2............ Gels and Solutions of Fibrillous Proteins . .......... Flight Path .3......2.... Collimation .. ..... .3.3...................... ....2......... The Experimental Problem ............. The Scattering Signal .1. ... ...... Determination of Electrophoretic Mobilities . ....... ..2......... ..........4......... ....... 7.....6........2... .... SmallAngle Scattering Techniques for the Study of Biological Macromolecules and Macromolecular Aggregates by PETER B .. MOORE 8.. 324 7. Diffusion Coefficients ...7. . SrnallAngle Apparatus: General Features .....CONTENTS xi 310 310 312 313 316 319 7. The Sources of Current Interest .......... 7.. Blood Flow . ...... ... ..........................1..........1................ ............. 8..........2.. .... ......... ..... 7....2.... 8...... ... ....1............ 8.. .......4...... ..3..2......... Equipment ..2. .. 8.. ........ Monochromatization of X Rays ..... 7.............1..... Instrumentation and General Techniques .... Isolation of the Macromolecular Signal ..1..9............. 8..2..... Applications in Cell Biology ........ ................ 8...... ........ The SmallAngle Compromise . . ......... ..4.. Purpose ...... ..5... Historical Comments ... Introduction ... 8......6. ...........5.......1..2.. . ........ .........3. 7... 7.......... . .. Earlier Reviews ........5......2.......1.... The Nature of the Technique ..
........ . .. 8. .... ..... 8... . . Neutrons ... ..3... 8.... Data Analysis .... ..... .3...... .2. . ... . .... . ..18.. . .... 8.. 8.. ... .9. 8...14. . ......8.... .. ....... . . . ... Absolute Scale and Contrast Variation ...2...3....2. ... .. ... ... .. Quantities Related to the Radius of Gyration .2.. 349 350 351 352 353 355 356 357 358 358 360 362 362 364 365 367 368 369 371 372 372 374 374 315 376 378 380 381 383 385 8... .. .......... 8... ... ..2.. .. . .3............3. 8..3. .. . ... ............. Molecular Weight and Partial Specific Volume .3. Contrast Variation in Practice: Extended Scattering .... .. ..12. ........ ....... ....3.. ..3....... ...3..... Apparatus Designs . ............12. ..... ...3..... 8.... . ..... .. .. ........16. 8..13. . 8.. .....3. 8.. CONTENTS Detection .. ...... .....3.... ...17... .. . ....... .... . .... .. Beam Monitors ...... ..... ...9.......xii 8..... ... . Molecular Size and Shape: Radius of Gyration .4.... ............18... ... Available Instruments .. ......1.. ...... ....... ... ... .. Spherical Objects ......... The Dependence of Radius of Gyration on Contrast .....1 1......... ................. Characteristic Function Parameters .... 8..... ...14. ... 8.. 8. . Pulsed Reactors ... 8... .. .. ..... ...3.... ....... 8..... .... ..3.... Absolute Intensity Measurements .2.1 1 . ..3. ... Length Distributions ... . Flight Path and Detection .. . ... . The Hydrogen Exchange Problem ..... 8....6.. . .... .. 8............ ..16. .. ... 8.......... ....... ... Solution Scattering Studies on Molecules of Known Structure .7........ . Data Correction .5..... ... Contrast ... ....... Dependence of the Extended Scattering Profile on Contrast ..2. ......13. 8... Molecular Shape . .. .. .. ... ............... ... .........2... ..... ..3............ Macromolecular Aggregates: Distance Finding . ...... ...... .. ..2. ... ... Macromolecular Aggregates: Synthesis Methods ......... 8.........3.. ... ...10......19. . 8.. ......... ...... General Design: Collimation . Contrast Variation in Practice: Radius of Gyration . Monochromatization of Neutrons ..... .17... Neutrons versus X Rays . .2. ........... ............... . ... .. . . . 8..2.. . ......... . ....... 8.. 8..3...... ... . 8.. . .2....15.3.10.. 8.. . ...... .... . .15. ..... ...... ...... ...... ....
.. .........4.... Complications ............ 9. ............. The “Weak Phase Object” Model .... GLAESER 391 395 395 397 398 401 401 403 404 406 9... 416 418 419 9........ Contrast Transfer Function Theory ............ .. 9.2.. ThreeDimensional Reconstruction ..4.........3........ 9. A Simplified Picture: The Mass Thickness Approximation ................. 8................... 430 .............1......3..........2........C0N T E N T S xiii 388 388 389 8............ 9. Radiation Physics and Radiation Chemistry ... 9. 413 9.. Electron Microscopy by ROBERT M..... 9....... Image Formation in the Electron Microscope ... .. ..5..6. The Hollow Cone Problem . ...... .........3.....4.... ... .1......... ........3...... .........5...... .........2. . Summary . Concluding Remarks .3..3.. 430 9..4...6... ........... 430 9........2.......... Origin of Phase Contrast in Electron Microscopy ............... 9... Empirical Studies of the Radiation Damage Effect under Electron Microscope Conditions ... .... Radiation Damage ....1...4. Fourier (Crystallographic) Methods .... ...2.4....2... 422 427 427 9........ Complications.... ........2............. Solution of the ShotNoise Problem by Image Superposition .1............. 9...... .. Critique .2..........3..... Specimen Hydration within the Vacuum of the Instrument ..... 409 9..... 9...1... .... The Need for Maintaining the Hydrated State with Complex Biological Structures ..... Difficulty of the Problem ....................... ShotNoise Limitation for HighResolution Electron Microscope Work .. 9 ........... DirectSpace Methods ... . Image Restoration ......... The Envelope Function: Partial Coherence .4.... ... 409 9.. ..5....3........ .2. .......1.......... ..........3............................... 8. .2......6.......... ...... Electron Microscopy as a Tool for Structure Determination..........4.1.5.. ................ 9...............................5. 9....... 9.. 9..... 9.4...
1..... 10..1..... ... ........ . .. ... 10..... .... Three Microelectrodes . ...3. ...... 481 10.. .. .. ....6.5... Cable Theory of an Axon with Axial Wire in Voltage Clamp . 10..6.3..2.... ... . ..6......... Recent Innovations in Experimental Methods ..xiv CONTENTS 9...... Technical Solutions to the Hydration Ploblem .... .. JULIO VERGARA.3. .... ....... .....1.7.. 473 10.. .3.. SeriesResistance Compensation .... Two Microelectrodes ... 10.1.... .. 10..4.. . ......3. . ....... .. .... Electronics for the Voltage Clamp System ..........2.2.....2.. . AxialWire Voltage Clamp .. .. DarkField Methods .3. 9.... General Principles of Voltage Clamp . HighVoltage Electron Microscopy ... . ....... ......6...7...... 10....... .......... Internal Access ... .... . .. External Patch Isolation ....... . Voltage Clamp with Gap Isolation Techniques . .... 10. ..... .. .... .. 431 433 433 438 439 441 444 10.. .... ...... .... .......5 .... Measurement of Membrane Current .. 9.. Aberration Correction ... ..... ... 9... .. Voltage Clamping of Excitable Membranes BEZANILLA. 10..... ....... 473 10. . 10.. .1. ..4.4..... Node of Ranvier ... ...... ... Image Formation with Inelastically Scattered Electrons . . ..... ........ by FRANCISCO AND ROBERT E ...7.... .. 10...... . ........3.. 10. .7... . ..... 10. ....... .. . .5....... Voltage Clamp of an Isolated Patch Using External Pipettes .3.......3. Pulse Generation and Data Acquisition ........... ..... TAYLOR 10...... .......3........ ...4.3. ... 445 447 451 452 455 459 461 463 470 10. .... .... VaselineGap Techniques in Single Muscle Fibers . .. ... ..... .. .....4....2. ......5.....6.... ....... . ... . .. . 482 482 484 486 487 497 500 501 . .......... 10. . ...7..5...... .. . .... 9.... .. 10.. .. ....... ..... .... 9..6... 10.1.. SucroseGap Methods ....6. Giant Axon Preparation .. ...... ... ..............2. .. SingleSideband Images and Holography .. 9. Voltage Clamp with Microelectrodes ..... ...........4.. Errors Introduced by the Finite Length of the Gap ...7. .. .. Introduction .. . .. .. . .. .... .. ..
. ......5.... 1 1. CarrierMediated Ion Transport ......... 1 1.................. 11... 11... ..... .......... Lipid Model Membranes by G ............. 1 I ......... ................... Lipid Monolayers ....... .......... Ion Transport Mechanisms ...2..... C ............. .. . ....... 10.... ..A.4..9................. 10. 565 .....8.... .. .. SolventDepleted Bilayers ............... Techniques for the Measurement of Ion Transport .4...........7. Circuit Equations of VaselineGap Voltage Clamp .............. 10......2....2........... 536 1 1.. Multichannel Bilayers ............ ............. SolventFilled Bilayers ........ Spherical Lipid Bilayers ....... ...... Planar Lipid Bilayers ..... 11..... Concluding Remarks ......... Potential Distribution for a Fiber in a Gap Voltage Clamp ... ... 513 515 516 519 520 521 523 524 525 528 11.... 535 11.3........ ....CONTENTS xv 504 504 504 10........ ........ ......2.....................10....... ......... Relationship between Channel Structure and Function . 11... ...... 539 11.............A. ..7.... 1 1. 541 545 SUBJECT INDEX ..........1........... . Lipid/Solvent Systems ................. SZABO A N D R ... ....6....... ......A....4..............1 .3 .9.......... 11.. Direct Transport of Hydrophobic Ions 11..4.......... .....9.4......... ...... AUTHOR INDEX......... . ... ... 529 530 ....................... Single Channels in Bilayers .............. WALDBILLIG 508 11 1 ... .....4..... 11.......... .... . Molecular Channels .1.............. Pure Lipid Bilayers ...... Biological Membranes .............. ..... 11....... .. . ..... Appendix .. ...
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National Institutes of Health. San Jose. HARDEN M. Zthaca. MCCONNELL. Baltimore. Departments of Chemistry and Molecular Biophysics and Biochemistry. GLAESER. Baltimore. CaliforSUNNEY nia Znstitute of Technology. L. Los Angeles.* Department of Chemistry. Johns Hopkins University School of Medicine.National Institutes of Health. Yale University. t Present address: Institute for Medical Research. University of California. Stanford University. Stanford. Cornell University. A. LATTMAN. Louis. MARIO AMZEL. A. California 94720 (391) GILLIAN M. Connecticut 06511 (337) RALPH NOSSAL. Noyes Laboratory of Chemical Physics. California 94305 (53) PETERB. Missouri 631 10. Johns Hopkins University School of Medicine. California 95128. MOORE. Department of Physics. BunkyoKu. University of California. New York 14853 (197) Department of Biophysics and Medical Physics. xvii . ELSON . California 91125 ( I ) ELLIOTL. Maryland 20205 (299) * Present address: Department of Biological Chemistry. Maryland 21205 (229) Stauffer Laboratory for Physical Chemistry. Maryland 20205 (123) I. CHAN. Pasadena. Maryland 21205 (229) Department of Physiology. FRANCISCO BEZANILLA. HUMPHRIES. Bethesda. Washington University School of Medicine. California 90024 (445) MARGARET R. Stanford University. BUNOW. ROBERT M. K. New Haven. Department of Biophysics. University of Tokyo. Department of Biophysics. St. School of Medicine. Bethesda. California 94305 ( 5 3 ) TAKAYOSHI KOBAYASHI. Berkeley.~ Stauffer Laboratory for Physical Chemistry. Stanford.CONTRl BUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin. Tokyo 113 Japan (163) EATON E .
Israel (197) JOSEPH R. Department of Physiology.xviii CONTRIBUTORS JOSEPH SCHLESSINGER. Bethesda. National Institutes of Health. University of Texas Medical Branch. WALDBILLIG. Noyes Laboratory of Chemical Physics. National Institutes of Neurological and Communicative Disorders and Stroke. Laboratory of Biophysics. Maryland 20205 (445) JULIO VERGARA. School of Medicine. University of Texas Medical Branch. Department of Chemical Immunology. Galveston. Cal$ornia 91125 ( I ) G . SZABO. SCHUH. University of Calgornia. Department of Physiology and Biophysics. Department of Physiology and Biophysics. California 90024 (445) R. C. Texas 77550 (513) . Rehovot. The Weizmann Institute for Science. TAYLOR. Pudadena. A . Galveston. Texas 77550 (51 3 ) ROBERTE. Los Angeles. A . California Institute of Technology.
xix . Future volumes will be edited by Dr. National Bureau of Standards. Robert Celotta of the Electron Physics Group. Both Claire and her late husband. Washington. Claire Marton. Radiation Physics Division. Colorado. Their passing is both a personal and professional loss to us. University of Colorado. and by Dr.C.. were associated with Academic Press almost from its founding. Boulder. Judah Levine of the Joint Institute for Laboratory Astrophysics. We are fortunate to have such qualified and experienced people join us. Dr. D.PUBLISHER’S FOREWORD It is with sadness that we inform readers of Methods of Experimental Physics of the passing of Dr. Celotta and Dr. Levine had already started to work with Claire. Bill.
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MARTON xxi . in retrospect. This volume is a proud addition to the treatise. Given the subject matter. Certainly. C.FOREWORD It would seem that for more than any of the other volumes of this treatise the choice of topics to be included in this volume was the most difficult to make. this may not appear surprising. more volumes on this subject are called for. The high quality of this book is testimony to the effort and good judgment of the editors and the fine cooperation of the authors.
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and electrical measurements on cell membranes. xxiii . Almost every type of spectroscopy has been used with some success to study biological macromolecules. For systems without unpaired electron spins. We have emphasized methods which can be used to study molecular motion and conformational changes of macromolecules. Electron spin resonance (ESR) is similar to nuclear magnetic resonance in concept. Thus ESR spectroscopy is restricted to molecules with unpaired electron spins. Successful spin labeling requires that the label be incorporated into the desired part of the molecule under study and also that it not interfere with the normal functioning of that molecule. When these conditions are met. Part 1 includes an example of the N M R . A major theme of Part 1 is the use of nuclear magnetic resonance to measure relaxation times. Parts 10 and I 1 discuss methods for studying membranes. In nuclear magnetic resonance. other nuclei with magnetic moments are often studied. and any sampling of methods is somewhat arbitrary. and the first two parts are about these methods. the absorption frequency depends on the magnetic moment of a nucleus. Unpaired spins are present in biological molecules containing transition metals and in free radicals created as intermediates in biochemical reactions or by radiation damage. Parts 59 are concerned with methods used to visualize the structures and motions of large molecules and cells. either directly or by means of model systems. This approach. Parts 14 discuss several types of spectroscopy. With the improved sensitivity now available. Magnetic resonance spectroscopies have found considerable use in studies of biological systems. Since protons have large magnetic moments and are prevalent in biological material. it is sometimes possible to add covalently a group that contains an unpaired spin. is the subject of detailed discussion in Part 2. structure determination by the scattering of particles or radiation. early work centered on the proton. and are primarily concerned with the determination of molecular information.as well as other new and more sensiuse of natural abundance *T tive methods.PREFACE This volume describes three types of measurements that have been of intense interest to physicists working in biologyspectroscopy of macromolecules. called spin labeling. but utilizes the interaction of a magnetic field with an electron spin rather than a nuclear spin.
As previously mentioned. such as molecular motions. and the high sensitivity inherent in fluorescence measurements. Fluorescence spectroscopy has been very successful because of the prevalence of chromophoric groups in biological molecules. fluorescence can be a very valuable tool in the measurement of absorption spectra. Whether the objects are macromolecules. such as the possibility of recording from small samples. the possibility of incorporating fluorescent markers. the scattering and diffraction methods all share a common conceptual basisthe distribution of scattered radiation or particles gives the Fourier transform of the density of the object under study. the fluorescence methods are somewhat more flexible in that they can be used to determine motions over relatively long distances.xxiv PREFACE the spectrum of the spin label can be used to determine a number of important parameters. Picosecond laser spectroscopy. which is discussed in Part 4. are based not on the particular frequency of absorption. where previous methods have been inadequate to resolve the rapid transitions which occur immediately after the absorption of a photon. and lipid membranes. which has been the prime source of . Raman spectroscopy has been utilized effectively in studies of heme groups. Part 6 deals with xray diffraction. and sensitivity to homonuclear bonds. and circular dichroism (CD). is a relatively new method that has considerable promise for several types of biological problems. include fluorescence. The fluorescence methods presented in Part 5 . Other types of spectroscopies which have been used extensively to study macromolecules. and U V spectroscopies have been successfully used for biological studies. These methods allow the determination of molecular motion within a cell membrane. the lack of interference from liquid water. We have chosen to include a paper on Raman spectroscopy (Part 3) because it combines several important advantages. Part 4 discusses the new technology needed to produce and record pulses of light in the picosecond range and provides examples of applications to photosynthesis and vision. Parts 69 describe different approaches to the problem of obtaining images of small objects. A number of IR. and rates of reactions. cell organelles. visible. but are not described in this volume. nucleic acids. are especially valuable spectral indicators of conformation. optical rotatory dispersion (ORD). however. or whole cells. but on the bleaching of fluorescent molecules and the interdiffusion of bleached and unbleached molecules. ORD and CD signals. Thus these parts are representative of the current emphasis on Fourier optics. which are sensitive to helical content of macromolecules. Although similar information can be obtained from spin labeling (Part 2). distances between groups.
For those macromolecules that can be crystallized. viruses. In particular. Much of our pictorial knowledge of the relations between structure and function for proteins and for DNA comes from the xray crystallographic analyses of approximately 100 prototypical macromolecules. the scattering is confined to “small” angles near the forward direction. The last two parts describe methods for studying membrane transport. Different types of motion produce different Dopplershift characteristics. The laser. These shortwavelength methods provide information complementary to that obtained by crystallography. Consequently. Two examples are cytoskeletal architecture and membraneprotein complexes such as bacterial rhodopsin.PREFACE xxv our knowledge of the detailed structure of biological macromolecules. highresolution microscopy. Molecular structures of particular interest are the ionselective channels responsible for excitability and ion “pumps . Part 10 describes voltage clamping of excitable cell membranes. scattering by macromolecules is characteristic of objects much larger than the wavelength of the incident radiation. and these can be distinguished by means of autocorrelation calculations for the different motions. and ribosomes) that cannot be grown as crystals. since the wavelength of radiation is so small (of the order of l &. The concepts involved in smallangle xray and neutron scattering. has had a strong impact on all of optical technology. Highenergy . they can be used to analyze the shapes and interactions of macromolecules in solution. it complements the method of neutron diffraction (Part 8) to provide a good description of the structure of ribosomes. xray diffraction provides a wealth of information. and Part 11 describes methods for making lipid model membranes and for determining their properties. Part 9 emphasizes those aspects of electron microscopy that have grown out of physical considerations of the process of image formation. However. and electron diffractiontechniques that provide new and striking images of key supramolecular structuresare considered here. are similar to those involved in light scattering. For example. because of its ability to generate coherent light. xray diffraction can give a complete description of the spatial molecular structure to a level of resolution better than 3 A.” the . discussed in Part 8. Even for structures (such as fibrous macromolecules. scanning electron microscopy. Present membranetransport research focuses on characterizing those molecular structures in the membrane that are responsible for the various transport processes. Part 7 surveys the many applications to cell biology of laser light scattering and emphasizes a particularly promising areathe interpretation of light scattering from motile bacteria.
We hope that the sample of methods presented in this volume shows some of the technological ingenuity that biophysical problems have generated. in which various membrane proteins are embedded. As discussed in Part 10. GERALD EHRENSTEIN HAROLD LECAR . Without this control. and patience. This approach has contributed greatly to the currently accepted paradigm for membrane structurea lipid bilayer matrix resembling a twodimensional fluid. and photosynthetic reaction centers. significant progress has been made in reconstituting functional entities from biological membranes into lipid bilayers or lipid vesicles. such as nerve axons. In fact. lipid bilayer studies provided the first measurements showing that membrane ionic current passes through discrete channels. We should also like to thank the staff of Academic Press for their support. Part 11 discusses methods for using synthetic lipid bilayers as analogs of cell membranes. the many studies of synthetic ionophores implanted in bilayers have yielded major insights into the transport mechanisms of membrane channels and carriers. support. because it enables the experimenter to control the key variablethe transmembrane potentialand thus determine the time dependence and potential dependences of membrane conductance. The concept of voltage clamping has also been useful in several recent important advances in the study of membrane ionic channelsthe measurement of currents from individual channels. the use of noise analysis to determine the properties of the channels as they switch on and of€at random. In addition. postsynaptic acetylcholineactivated channels. have taken considerable interest in furthering interactions between physicists and biophysicists. making it virtually impossible to disentangle them.xxvi PREFACE membranebound enzymes that use metabolic energy to maintain concentration gradients of ions. Among the systems that have been reconstituted are NaK ATPase pumps. We should like to thank the editors of this series for their encouragement. For example. Finally. the electrical behavior is extremely nonlinear. and the measurement of gating current (the displacement current caused by molecular conformational changes during channel opening or closing). In electrically excitable cells. Marton. Claire Marton and Dr. both recently deceased. More recently. membrane potential and membrane conductance would vary in an extremely complex manner. the voltage clamp has proven to be the major tool for studying such highly nonlinear systems. L. the nonlinearity is crucial to excitability. Dr. we thank the authors for their effort and for their willingness to accommodate their individual papers to the needs of this volume.
0. Marton Volume 1. Atomic and Electron PhysicsAtomic two parts) Edited by Benjamin Bederson and Wade L. L. Lovberg Volume 10. Johnson Volume 7. Hornyak Volume 9. Yuan and ChienShiung Wu Volume 6. Second Edition (in two parts) Edited by E. Robinson xxvii Interactions (in . Plasma Physics (in two parts) Edited by Hans R. F. C . Schultz Volume 5. Atomic and Electron PhysicsPart A: Atomic Sources and Detectors. Griem and Ralph H. Hughes and Howard L. Marton and W. Classical Methods Edited by lmmanuel Estermann Volume 2. Bleuler and R.METHODS OF EXPERIMENTAL PHYSICS EditorinChief C. Solid State PhysicsPart A: Preparation. Mechanical and Thermal Properties. Physical Principles of FarInfrared Radiation By L. Nuclear Physics (in two parts) Edited by Luke C. Second Edition (in two parts) Edited by Dudley Williams Volume 4. Electronic Methods. Magnetic. Part B: Free Atoms Edited by Vernon W. Molecular Physics. and Optical Properties Edited by K. Part B: Electrical. Structure. Haxby Volume 3. Problems and Solutions for Students Edited by L. LarkHorovitz and Vivian A. Fite Volume 8.
Solid State Physics Edited by R. Richard Volume 18. PolymersPart A: Molecular Structure and Dynamics. Weissler and R.xxviii METHODS OF EXPERIMENTAL PHYSICS Volume 11. Tang Volume 16. Vacuum Physics and Technology Edited by G. L. W. Fava Volume 17. Edmonds Volume 20. Accelerators in Atomic Physics Edited by P. Ultrasonics Edited by Peter D. Biophysics Edited by Gerald Ehrenstein and Harold Lecar . L. Meeks Volume 13. Fluid Dynamics (in two parts) Edited by R. A. V. Part C: Physical Properties Edited by R. Spectroscopy (in two parts) Edited by Dudley Williams Volume 14. L. Carlson Volume 15. Emrich Volume 19. Carleton Part B: Radio Telescopes. AstrophysicsPart A: Optical and Infrared Edited by N. Quantum Electronics (in two parts) Edited by C. Part B: Crystal Structure and Morphology. Part C: Radio Observations Edited by M. J. Coleman Volume 12.
etc. we emphasize nuclear magnetic relaxation phenomena. Particular emphasis is placed on recently developed techniques and their usefulness in studies of biological macromolecules and systems.”’ this method has undergone continuous development toward the realization of its full potential. This review will be followed by a presentation of selected techniques that have found applications in biological NMR. when the first successful NMR experiments were reported. electroncoupled spinspin interaction. chemical shifts. particularly those techniques and features of NMR relaxation which permit *E ‘ F. NUCLEAR MAGNETIC RESONANCE By Joseph R. However. Purcell. Inc All rights d r c p r o d u c t i o n 111 . 127 (1946).1.idcmic Prc\*. Rrr. Schuh and Sunney I. In the next two chapters we briefly outline those aspects of magnetic resonance theory necessary to facilitate the discussions of these newer NMR techniques. and R. Phys. 69. on relatively simple chemical systems. PHYSICS.. VOI.37 (1946) 1 Ciipyright @ 1982 hy Ac. and M . out of necessity. Low spectrometer sensitivity and the increased spectral widths associated with larger molecules rendered it difficult to extend the method to macromolecules of biological interest.V. W. Phys. C. In the discussions to follow. including the concepts of magnetic shielding. H. 69. Hansen. Bloch. Introduction Nuclear magnetic resonance (NMR) spectroscopy has proven to be a most powerful tool for unraveling the fine details of molecular structure and interactions in solution. M . Torrey.. such as small organic and inorganic molecules. 20 ISBN 0124750629 . Initial studies were done. Instead. W . In this part we review some of the advances in NMR spectroscopy that have resulted in its successful application to problems in biology. We conclude Part 1 with a number of illustrative examples taken from the recent literature to highlight the achievements of the method to date. Rev. it is assumed that the reader is familiar with elementary magnetic resonance. as well as the manner in which these phenomena manifest themselves in liquidstate spectra and have proven useful for structural analysis.Packard. Chan 1.Pound. I . magnetic dipolar interactions. recently developed techniques and improved instrumentation have altered this state of affairs considerably. Since 1946.iny limn r c w v e d METHODS OF EXPERIMkNTAI.
Memory and G . “Nuclear Magnetic Resonance in Biochemistry. L.” Wilcy. especially those aspects which pertain to biological function. For a nucleus of spin I . Press (Clarendon). Rattle.I + 1) . New York.’ Dwek. A . Only half integral and whole integral values of 1 are allowed. Knowlcs. London and New York. E.2 1. New York. NUCLEAR MAGNETIC RESONANCE one to sample the details of motional processes in molecules.1) J . where h is Planck’s constant divided by 27t. ) I . “Magnctic Resonance of Biomolecules. 1.6 Wuthrich’.1. Press. Dwek. W. .I.* should be consulted. NorthHolland. Biomolecular structure has received considerable attention in biological NMR in the past. Quantum Description A large number of atomic nuclei possess an intrinsic spin angular momentum.I . 1961. Biophysics deals in part with the structure and dynamics of biological molecules and assemblies. 1976. 465 ( 1 974). as a number of recently developed techniques are bringing these measurements within the realm of possibility. P.2. London and New York.. Phenomenon of Magnetic Resonance 1. These quantized states correspond to the 21 + 1 projections of hI along an arbitrary axis in space. Amsterdam. ’ K. / m . The spin angular momentum of a nucleus imparts to it a magnetic moment p along an axis collinear with hI.” Oxford Univ. . Abragam. edited by J. S . Parker. “ N M R in Biological Research : Peptides and Proteins. W. . These two quantities are related by )r = $1. For a more comprehensive treatment of NMR in biology the books by James. (1. and Knowles et a1.1.” Academic Press. 1976. the angular momentum is denoted by J = hI and its magnitude is h . 197s. ’ K.2.2.’ Oxford Univ. F. We concentrate on the dynamical aspects here. Each of these projections will assume a value m I h with m. A . Jamcs. .” Elsevier. Wiithrich. T. Those readers interested in a more general and detailed treatment of magnetic resonance are referred to a previous chapter in this treatise3 as well as to the classical textbook by Abragam4 and the review series Advances in Magnetic Resonance. D. Physirs 3B. “Nuclear Magnetic Resonance (Nmr) in Biochcmistry.s Exp. D. Waugh. Marsh. “The Principles of Nuclear Magnetism. Meth0d. 1973. and H. the angular momentum vector of an isolated spin is characterized by 21 + 1 distinct spatial orientations. In quantum mechanical terms. = .
For an isolated spin. As a result of the interaction between the magnetic moment and the applied magnetic field.11 Spin Isotope (x) Relative sensitivityh 1.98 1.13 ' In a field of 84.00101 0. In the absence of electrostatic and magnetic interactions.4 93.833 0. . quantum mechanics allows only transitions between adjacent levels.6 kG.00470 0.0925 0. AE/h is in the radiofrequency (rf) range. so TARLE I. transitions will occur only at AE = y h H o . I78 0.49 338.80 48.64 100 0.26 26.2) Nuclear magnetic resonance is a spectroscopic method which permits one to monitor the transitions between the various spin states in a magnetic field.0291 0.28 16.01 59 0.037 10. In an NMR experiment one actually samples an assembly of spins. However. this degeneracy is totally lifted.52 36. when an external magnetic field Ho is applied.01 95.000508 0.00104 0.00268 0. Since contiguous spin states under this circumstance are separated by an equivalent amount of energy.00 90.0663 0.81 22. the projections of I will be quantized along Ho.08 0. (1.1.05 100 0.000 0.365 100 100 0. (1. For an equal number of nuclei at constant field.22 Natural abundance 99. PHENOMENON OF MAGNETIC RESONANCE 3 where y is the magnetogyric ratio.064 75.2.0156 99. Table I summarizes the NMR frequencies for a number of biologically important nuclei.23 24.2.74 55.ml.68 145.2.04 89.23 35. Properties of Some Biologically Relevant Nuclei NMR frequency" (MHz) 360. the 21 + 1 spin states are degenerate.00965 0.3) Typically. and each mr state will assume a different energy given by E = yhH.
2. Since the former affords a more graphic explanation. it should be noted that the classical approach is applicable only when discussing the net magnetic properties of a system.4 1. determincd by the Boltzmann distribution. (1. (1. . a limitation that has hampered the application of this technique to biological problems. ’ 1.2.2.n). Thus for an I = system (see Fig.kcal/mole of spins.I/ 2 j AE = YhHo Y I _ 0 I 1’0 (a) (b) FIG. . signal K (nt . la) we may write signal a (n.2.5) For a proton NMR experiment at room temperature and 360 MHz.eyfiHn’kT)/(l + eyfiHaikT). the measurement represents the net energy absorbed or emitted by a population of nuclei at the resonance condition. Classical Description Many aspects of NMR theory can be appropriately described by either classical mechanics or quantum mechanics. Inasmuch as the induced transitions between any two levels occur with equal probability. so that the energy change being measured is only about 10. . However. the signal one detects is proportional to the difference in the populations of nuclei occupying the different spin states. (a) Zeeman splitting of spin states for nuclei having I = : .. NUCLEAR MAGNETIC RESONANCE r n . the Boltzmann factor deviates from unity by only 3 parts in lo5. is along the z axis aligned with the field. Thus NMR is a spectroscopic method of low sensitivity. it will be used whenever possible. 1. (b) Classical depiction of precessing nuclear magnetic moments with I = +> in the presence of exterior field H. The nct rnagnctization M .4) When the driving rf field used to observe the transition is sufficiently small so as not to perturb the populations of the spin states from their values at thermal equilibrium. such as those relating to the properties of an individual nuclear spin. thus it will be necessary to turn to quantum mechanics when discussing other aspects of magnetic resonance.n?) = (1 .
In view of the relation1 and (p) = yAI. (Kp>/Wr.1. (1.1. (1.2.2.2.2. the sample will exhibit a net magnetization M.. The frequency of this motion is given by the Larmor equation 00 = yHo.6)does hold for a system of spins at thermal equilibrium with the external degrees of freedom.2.10) To demonstrate this result. = Y ( P ) x (Ho + dY>. Motion of a Spin in a Magnetic Field.8) where (p) denotes the classical magnetic moment. I = f. In order to illustrate the principles of NMR spectroscopy more fully.6) Although experimental situations may still be found where these components are nonvanishing.9) in a frame of reference The result is which rotates about the z axis at angular velocity a.2. The equation of motion is dJ/dt = (p) x Ho. Under these conditions. we rewrite Eq. each of the individual spins that make up M experiences a torque in the presence of H. Since the distribution of the azimuthal angles would then be uniform. where M = z i p i .2. The Concept of Magnetization. spins at thermal equilibrium is a vector of magnitude M. the effect of the torque on the magnetic moment ships J = A vector is d(P)ldt = Y ( P ) x H. while for those nuclei in the highenergy state p will oppose H. In the absence of other external forces an assembly of noninteracting spins exhibits no preference for the azimuthal angle. = t N y h tanh(yhHo/2kT) (1.2. PHENOMENON OF MAGNETIC RESONANCE 5 1..2. . we now consider a population of N identical nuclei having I = ).2. (1.11) . 1.. the nuclear magnetic moment vector p of each spin will assume one of two orientations with respect to the field. corresponding to the two quantum mechanically allowed energy states. (1. Classically. (1.7) aligned in the direction of the external magnetic field (see Fig.2. In the lowenergy state p will be aligned with the field. the result given by Eq. (1.. the population distributions among spin states are given by the Boltzmann factor.9) This equation describes the precession of (p) about an axis parallel to the imposed field.2. lb). (1. For a given distribution of spins between these two states. In the presence of the external magnetic field H.2. and the magnetization associated with the N .2.
2.13) where n = 1.rt . .1 5 ) dM. do not take into consideration relaxation processes which attempt to return the magnetization vector to its magnitude and direction at thermal equilibrium. In magnetic resonance one typically applies a linear H1 field perpendicular to Ho.14) When the effects of Ho and H1 on the components of M are explicitly spelled out. i. the equations become dMJdt = y(M. The Bloch Equations. (1. The component rotating in the same sense as the precessing spin will therefore appear stationary with respect to the magnetic moment when orf = wo.. while the counterclockwise component produces no net effect. 1. ( 1. In the rotating frame.2. including the Zeeman .H. These are the equations of motion for the macroscopic magnetization vector in the presence of a static magnetic field Ho and an oscillating rf field H I . oscillating sinusoidally with radio frequency orf x coo. HI cos u. sin urft + M .3./dt = y(M. H . . dM. . (1. This is the classical analogy to the quantummechanical transition that occurs between two nuclear spin states when I = $. .. Through interactions of the spins among themselves and with the surroundings. Since the energy of (p) depends on its orientation u i s .2. at resonance.6 1. NUCLEAR MAGNETIC RESONANCE where (d(p)/at).5.e. For such a system the torque equation is dM/dt = yM x H. axis at frequency w1 = yH.12) 1. sin urft).2..e.2..H.Ho).2.3. (1. Such a linearly polarized H1 field can be decomposed into two circularly polarized fields rotating in opposite directions.Ho + M./dt = y(M. These equations.2.. The above results can be generalized to an assembly of noninteracting or weakly interacting spins.M. when colt = nn.2.h i s the magnetic field. As a result of this precession the projection of (p) along the Zeeman axis will vary and it will become opposed to the external field every half cycle. the torque exerted on (p) by the H1 field will cause the magnetic moment to precess about the H. however. is the derivative computed in the rotating frame.4. Magnetic Resonance. there must be an exchange of energy between the rf field and the nuclear spin as it precesses about H I . i. From this equation it can be readily seen that (p) will be stationary in the rotating frame when Ho + o / y = 0 or o = yHo = wo. cos wrft).
t) y ( M . Bloch noted that magnetic resonance could be observed in two ways. Bloch. H . is the time constant for the return of the M . In this manner. both the precessional frequencies of the spins as well as their lifetimes in the spin states can be affected.M.. consequently. dM. thus. After the pulse is turned off.. H . and M y components does not.. the relaxation of the M . R w .16)  . powerful pulse of rf energy at the resonance frequency.t) ( M .is the time constant for the complete decay of the net magnetization in the x y plane.M.Ho)  M. These component equations of motion for the magnetization vector have proven to be extremely useful in the development of advanced experimental techniques. we shall have reason to return to them in subsequent sections. Both the FID and the frequency spectrum contain the same F. cos O ..2. denoted T. denoted T2. .1.' In the first method. . (1. a frequency spectrum is obtained when the magnetic field H. It is important to note that these two types of relaxation processes are intrinsically different. Associated with each decay is a characteristic time constant../dt dMJdt dM. H . all the nuclei of interest are excited simultaneously by a short. 1. component to its equilibrium value M./dt = = = y ( M y H o + M .Mo)/T.y ( M . PHENOMENON OF MAGNETIC RESONANCE 7 field.. . for example. between the spins and the surroundings. ( M y ) components of the magnetization. 70. cos o. and M . The spinlattice or longitudinal relaxation time. The spinspin or transverse relaxation time. component involves an energy exchange. and explicit solutions are given in Part 2 of this treatise. sin w. Detection of Magnetic Resonance In the introduction to his classic paper on nuclear induction. This procedure is called the continuous wave (CW) method because of the requirement for an uninterrupted rf field.2. is slowly varied through the resonance condition while a radio frequency field H I is imposed. Taking these relaxation processes into consideration one obtains the wellknown Bloch equations. sin q f t + M y H ./T2. called the freeinduction decay (FID).3. f t . while the decay of the M . In the second method./TZ. the electronic cloud. vibrational/rotational/translationaldegrees of freedom. The Bloch equations are also useful for describing the effects of molecular motions on electron spin resonance signals. etc. the spins can readjust their relative phases and redistribute themselves among the Zeeman levels of the system.2. Two types of relaxation processes may be distinguished corresponding to the decays of the M . Phys. . magnetic resonance is observed as the decay of the induced signal. 460 (1946).
A.. tips toward the xy axis under the influence of the continuous H1 field.e. Ernst and W. cancel each each other [cf. o/y. it is more convenient. 1. this is done by measuring either the energy exchange lo R. as the resonance condition is approached. necessary to acquire a frequency spectrum by the CW method. one can obtain the NMR frequency spectrum of a sample from its FID. the signaltonoise ratio in the transformed spectrum is increased by a factor of N/"? It was previously noted that the low sensitivity of NMR spectroscopy is one of the major limitations of this method when it is applied to biological systems. The spectrum is then obtained by varying either the Ho field strength (field sweep) or the rf frequency (frequency sweep). while the signal due to the background noise will fluctuate randomly. 1. 107.and H. However. Lvwe and R. the signal response from a population of resonating nuclei is consistent for each FID. Subsequent averaging of the accumulated data produces a marked improvement in the signaltonoise ratio. the introduction of techniques utilizing FTNMR to increase sensitivity has had a particularly significant impact on biological studies.E. RBZI. On the other hand. to obtain a frequency spectrum experimentally it was necessary that a continuous H1 field be imposed. for the same data acquisition time. As a result. Prior to the development of FTNMR.2. the FID (the time domain function) is obtained in a fraction of the time (10. using a mathematical operation. This gives FTNMR a distinct advantage over the CW method in that. RCLI. Thus by averaging the data from N freeinduction decays. Anderson. Norberg. the spectrum). Sci. the net magnetization will follow HeR." Thus. 46 (1957). the fictitious field.210. However." This method is based on the finding that the Fourier transform of an FID is the frequency response spectrum acquired from a C W experiment. Under these conditions.17) Initially the system is far from resonance.1. This component is the resonance signal that is detected experimentally.2.3. 93 (1966).2. Insirurn. Phys. ContinuousWave NMR. (1. the effective field in the rotating frame is ( 1. Ernst and Anderson introduced Fouriertransform (FT) NMR spectroscopy in 1966. R. since this information is more explicit when expressed in the frequency domain (i. Eq.10)] and He. Since this method is the basis for many of the techniques that will be discussed subsequently. This enhancement is due to the fact that under appropriate experimental conditions. If the sweep rate is slow enough.. thus giving M a measurable component in the xy plane.3. . the experiment can be repeated many more times. Typically. In order to take advantage of the more efficient FID signal. its theory is included in this section. so that HeRz Ho.8 1. 37. J . NUCLEAR MAGNETIC RESONANCE information characterizing the system under study.
the strongest signal will be observed when the duration of H. In addition.1. (1. and T ' % Tf. Similar pulse methods have also been designed .21) Immediately following a 90" pulse. In order to obtain the intrinsic T2 values a number of multiplepulse techniques have been developed which eliminate the contribution from magnetic field inhomogeneity. However.2. % 1/4A. the rf field H . i. and magnetic nuclei of the same kind exhibit different resonance frequencies depending upon the chemical environment.2. = n/2yH1. and the restriction in Eq. The H. is t . t.. where the asterisk indicates that this T2 value includes contributions from field inhomogeneities and may be dominated by them.2. This dispersion of the N M R frequencies according to chemical environment has been widely exploited for structural analysis and determination. Hefi s HI for the period in which H I is turned on. The length of this 90" pulse. In order to accomplish this.3. Hi % H.* is quite short and we have both T.. is long enough to rotate M by 90" (n/2).18) where A is the frequency range spanned by the nuclei under examination. In general. field for a pulse experiment is usually several orders of magnitude larger than it is in the C W method. must have a magnitude such that yH.18) requires that t . Since the dephasing occurs in the xy plane. (see Fig. As is well known. the individual magnetic moments will begin to dephase freely.is dictated by the range of nuclear frequencies to be observed.Instead M precesses about H. because of field inhomogeneity effects and other instrinsic relaxation processes.. T. H. Pulse NMR. the pulse experiment is always done very close to resonance. In general. (1. since the duration of the rf pulse is usually on the order of microseconds. its duration is of the order of Tf.2. 2). A is determined by the range of chemical shifts for a given type of nucleus. The pulse method differs from CWNMR in that all the nuclei of interest are excited and observed simultaneously rather than individually.' 1.20) In practice. PHENOMENON OF MAGNETIC RESONANCE 9 between the spin system and the rf coil' or the voltage induced in a separate receiver coil placed perpendicular to both Ho and H.2. (1. % 2 4 (1. (1. This results in the decay of the induced voltage in the receiver coil (the FID).2.19) Accordingly.2.2.e. + a/y. a nuclear moment is magnetically screened by the electrons in a molecule. Inasmuch as the detector only measures the components of M in the xy plane. the magnetization vector cannot follow He#.
(b) Net magnetization at resonance with rf field H. The latter is a measure of the frequency components that comprise the response function f(t). which allow one to accurately determine the longitudinal relaxation time.1. following a 90" pulse. TI. is to tip Mtoward theyaxis. NUCLEAR MAGNETIC RESONANCE 2' X' / (a 1 (b) FIG. FourierTransform NMR.2. These interactions cause the transition frequencies to be different for different resonant nuclei. (a) Net magnetization Maligned along the Zeeman axis in a rotating frame of reference. Classically.givingthemagneticvectoracomponent alongboth thez and the y axes. Mathematically.3.10 1. this variation in the transition frequencies corresponds to differences in the Larmor frequencies of the precessing magnetic moments under the influence of H . arises from variations in local electrostatic and magnetic interactions. one can describe a time domain function as the Fourier transform of a frequency domain function F(o).2.2. rotating at the Larmor frequency. It is these various frequency components that interfere constructively and destructively to give the decay function in the time domain. the FID. In keeping with the properties of Fourier integrals these two functions are merely Fourier transforms of each other .4. of course. that the Zeeman interaction dominates). (provided.22) and .We discuss these techniques in more detail in Section 1.3. The decay of an NMR signal in the x y plane. thus (1. The effect o f H . 1.1.
Inward spiraling is caused by transverse relaxation of the moments in the x y plane.24) Taking the Fourier transform of this.3. . we obtain the response function (1. (1./[l + T.equal to 2/T2. we consider a simple spin system which exhibits an NMR transition at frequency o = o. these two functions contain the same information about the nuclear spin system.(w .. To illustrate this.2. We shall assume that the resonance line is Lorentzian with a full width at half height Ao. so that F(w) = AT.1. (b) FID of the system depicted in (a). The recorded signal is induced in a phasesensitive receiver coil positioned t o sample the net magnetization along y. PHENOMENON OF MAGNETIC RESONANCE 11 Naturally. . The numbered points on the FID correspond to the points in (a) marking positions of the net moment in the x y plane.2. The trace is circular because the resonant nuclei precess at a frequency different from that of the rotating frame. (a) Path followed by the net magnetization in the xy plane after a 90" pulse.25) X I Time ) (b) FIG.2.wO)'].
of course.3..In this frame of reference. Spin Relaxation Following an rf pulse one typically creates a nonequilibrium magnetization along the Zeeman axis and in the xy plane. From James. is merely a sinusoidal function with frequency w o . and ( c )multiline spectra.12 1. In practice. but since its magnitude is concurrently decreasing exponentially because of the T2 relaxation processes. following a 90" pulse. where the FIDs for three different samples are shown. Alternatively. Each of these will contribute to the induced signal.wo1. (b) twoline.' This result indicates that the FID signal. 3b). The return of the magnetization to its equilibrium value along the Zeeman axis is described by the spinlattice or TI relaxation time. In Fig. the precesssing magnetization will describe a spiraling arc in the xy plane. one is usually sampling more than one nuclear species exhibiting various transition frequencies.4. and the FID will appear to be quite complex.except that the amplitude decays with a time constant of T. NUCLEAR MAGNETIC RESONANCE (C 1 FIG. Note that from the periodicity of this decay and a knowledge of o we can calculate the frequency of the NMR transition w o . This is illustrated in Fig. 1. one can plot either the inphase or outofphase component of the induced voltage across a receiver tuned to frequency o as a function of time (see Fig. 4. the magnetization will be precessing with frequency I o . Freeinduction decay signals (lefthand side) and their Fourier transforms (righthand side) for (a) singleline. The decay of each of these two components is described by a distinct characteristic time. The resulting interference or beat pattern one observes here measures the decay of the transverse component of the precessing magnetization as it is being detected at the receiver. 3a we plot the magnetization of our simple spin system as it appears in a frame of reference rotating at w. Since this component of the . where o # wo. along with their respective Fourier transforms. This important result can be graphically illustrated in two ways.
except that only those Fourier components which match the frequencies of the quantum mechanically allowed transitions will be effective. SPIN RELAXATION 13 magnetization is a measure of the distribution of spins among the energydependent Zeeman states there must necessarily be an energy exchange between the spin system and other degrees of freedom. where we have a spin manifold of weakly interacting nuclei of I = 5. For molecules in solution the fluctuating nature of these fields is caused by the rotational and translational motion of the molecules as they undergo thermally induced Brownian motions.3. This characteristic time constant is the wellknown correlation time zc. These fields act on the nucleus in a manner analogous to the applied H I field.3. The fluctuating local magnetic field acting at the site of the resonant nucleus can be decomposed into its Fourier components and written 1 H ( t ) = __ & s_ “ “S(o)eiw‘ dw7 (1. which we discuss in more detail in Section 1. Since energy exchange between spin and lattice systems can only occur in quantized units of the spacing hwo between the two spin levels. Such a condition obtains only when the stochastic modulation time of the fluctuating field is approximately (ao)l . In the simplest case.3.3.2) Therefore spinlattice relaxation will be most efficient when the value of the distribution function at the Larmor frequency is at a maximum. SpinLattice Relaxation The nonradiative decay mechanisms responsible for TI relaxation have their origin in the interactions between resonant nuclei and fluctuating local electric and magnetic fields arising from the atoms and molecules present in the sample. it follows that 1/Tl = Is(wo)12.3. a nonequilibrium distribution of the spin population can only return to equilibrium through transitions between the two Zeeman levels. The application of an H1 field in the xy plane can also perturb the phases of the wave functions for the nuclear spins and lead to a nonzero component of the magnetization in the xy plane.. . (1. Although in T2 relaxation there is no energy exchange between the total spin system and the surroundings. This nonequilibrium state decays to zero with a characteristic time T. Thus the efficiency of any relaxation mechanism depends upon the nature of the interaction and the frequency with which it fluctuates.3.1. which is distinct from the spinlattice relaxation time since only the phases are involved.1) where g(w) describes the distribution of frequencies in the field.1. 1. the phases of the spins can be modified via energy exchange among the individual spins themselves.
SpinSpin Relaxation For an assembly of noninteracting spins the equilibrium value of the net magnetization in the xy plane is exactly equal to zero. if the spinlattice relaxation rate for the system is sufficiently fast. The magnitude of this homogeneous contribution to T2 depends upon both the time scale and nature of these fluctuations. In many cases.3. only local field components collinear with H . 1. The value of z. NUCLEAR MAGNETIC RESONANCE 1. component will decay to zero according to this time constant..However. Dephasing of an otherwise homogeneous set of spin vectors can also occur due to random fluctuations in local electric and magnetic fields. . Correlation Time In the preceding discussion of spinlattice relaxation. the positional correspondence is lost and the motion appears to be random. In discussing the relaxation mechanisms responsible for T. this contribution to the dephasing will be included as part of the homogeneous T. Thus the magnitude . that the energy levels will be broadened by a width of the order of AV z 1/2Ti (1. caused by transient variations in the frequency of precession for individual spins. In this case the spin vectors precess about the field at the same average frequency but exhibit a statistical oscillation in their phase coherence. This means that for the interval zc. magnetic moments lose phase coherence by precessing at different frequencies.2. along the zaxis contribute to homogeneous T2processes. For the socalled the decay of M. The length of this period may be considered the “memory time” of a molecule. since there can be no phase coherence of the individual magnetic moments under these conditions. This is caused by static field inhomogeneity across the volume of the sample and by firstorder interactions such as those responsible for chemical shifts and spinspin splitting. The introduction of phase coherence by an appropriate rf pulse results in the creation of a nonzero xy magnetization in the rotating frame.. Of course. M x y .which will decay to zero in a characteristic time T2..3. then the nonzero M x ymust decay within the limit of TI. under this condition. .. motional changes in the position of the nucleus bear some relation to each other.. such as in liquids and gases. It follows. it is useful to distinguish two major contributions to this process.3) and the M. occurs as the individual inhomogeneous contribution to T.14 1. At times longer than z. can be approximated as the time required for the molecule containing the resonant nucleus to either rotate 1 radian (rotational correlation time) or diffuse a distance equivalent to its own dimensions (translational correlation time).3. analogous to the way local fields affect TI.3. we introduce the concept of a motional correlation time z.
say Em and E k .. (b) Proton spinlattice relaxation times for glycerine at different temperatures and two different frequencies. (). thus T~ becomes longer." ..1. This condition obtains = w o . A measure of the correlation between X 1values as they vary with time is the autocorrelation function G(z). described by the Hamiltonian operator Zl.3.4) where Gmk(z) is the autocorrelation function.3. but for t + z 9 t.(sec) (5. From Farrar and Becker. (. the viscosity of the solvent. ' = w.+ ' w. For a nuclear magnetic moment which can occupy two energy states. AS the temperature decreases. the viscosity 1 increases..) FIG.5. (TJ' 9 w.as shown graphically in Fig.6) which describes the probability that a fluctuating field will occur at a particular frequency. its value at time t.. (). will differ from that at Xl(t + T).5) While the function G(z) tells us something about the rate at which a randomly fluctuating field loses its positional correspondence. and the temperature of the sample. we now consider the energy of a local fluctuating field. will be a function of the size of the molecule. its Fourier transform gives the spectral density function (1. Since relxation will be most efficient when the frequencies of the fluctuating fields lie close to the Larmor frequency of the resonant nuclei.3. the probability W. described by Gmk(r) = (mIXl(t + z)I')('I*l(t)lm). SPIN RELAXATION 15 of z. (1.3. (a) The spectral density function J((. For very small values of z. for three different values of T ~ : ( T ~ ) .. In order to illustrate this more quantitatively. TI will be at a minimum when J ( o 0 )is maximal. If this field fluctuates in a random manner... X l ( t ) . of a transition induced by XI between these states can be shown to be related to G(z) by (1. the similarity will be lost. we expect S l ( t ) x Xl(t + z).) . 5a where J(w) is plotted for when (zJ T..
8 MHz because the maximum value for J(wo) increases as the resonant frequency decreases (note that the area under the spectral density function in . the spinlattice relaxation rate becomes frequency dependent. including wo. may yield a surface exhibiting multiple minima.)approaches the resonant frequencies. with a rigid molecule undergoing axially symmetric anisotropic motion (where zll << zL) two minima may occur. when z . M . E. is short. However. At lower temperatures. . At high temperatures.8MHz. The TI minimum is shorter at 4. Phys. For this reason. Thus far we have assumed that molecules undergo isotropic Brownian rotational and translational diffusion in the liquid state. 5b are greater at 29 than at 4. l 3 N.679 (1948). the transition probability will be equally low for a wide spectrum of frequencies. 7 J(o. Farrar and E. As zC increases so that (z. 73. but the transition probability decreases at higher frequencies. Tl is relatively long and frequency independent. Pound. ' 1. " Academic Press. However.) begins to decrease. reaching a maximum at wo. although TI may exhibit an additional dependence on the average orientation of the molecule relative to the magnetic field. Beckcr. V. C. Reo. corresponding to temporally distinct correlation times.' Thus when z. and R . For convenience we assume that all motions are sufficiently rapid to average the various interactions to first order. where the proton spinlattice relaxation time for glycerine is plotted at two different frequencies as a function of temperature. and Fig.4. multiple correlation times can contribute to the spinlattice relaxation. first at the higher frequencies then at the lower ones. where thermal motion is rapid. z. 1971. is well demonstrated by an experiment from the classic work of Bloembergen et ~ 1 . it should be noted that for large macromolecular complexes exhibiting long correlation times T . the increasing Tl values depicted on the right side of the curves in Fig. such as those found in membrane systems. is short and the spectral densities of the random fields at either 29 or 4. Relaxation Mechanisms In this section we discuss a number of mechanisms that have been found to contribute significantly to nuclear magnetic relaxation in biological systems. for motionally restricted molecules. "Pulse and Fourier Transform N M R .16 1.3. Bloembergen. For example. in the case where molecular reorientation or translational diffusion is anisotropic.8MHz are equally low. increases further. 'This ~ is shown in Fig. New York. accordingly. Purcell. a plot of TI versus z . NUCLEAR MAGNETIC RESONANCE three different correlation times. However. J(w) increases for low frequencies. 5a remains constant !). D. 5b. is long. Similarly. The dependence of TI on z.
1. F = a[(Z.3. + iZ. D = &Ix . 3 .1.)](l .3.3 cosz O).3 cos2 O). Magnetic DipoleDipole Interactions. (1. (1.g.)Iz](sin 8 cos 6 23. For a twospin system consisting of p1 = yJhI and pz = yshS separated by a distance r. it can be shown that the dipolar contribution to the longitudinal relaxation rate for nucleus I is while for transverse relaxation the rate is l4 C . .)(S.iI. Slichter.8) ZZSZ(l . If r makes an angle 0 with respect to the field and 4 is the azimuthal angle. P.3.$ [ ( I . Berlin and New York. + is. 1978. + i~.8b) (1.iZ.3.  (1.8~) + iZZ)sz + (s.)]sin’ 0 e”+.8f) iI.3.) + ( I . The predominant relaxation mechanism for nuclei having spin number I = $ (e.i~.” SpringerVerlag.)(S.3. For this Hamiltonian.)Sz + ( S . 31P.8e) (1.)]sin~ O eZi+. + iZ.iS.)(S. 13C. E = .)(s..3.8d) (1. . SPIN RELAXATION 17 the expressions used here to describe relaxation will not necessarily be valid.”F) is through magnetic dipoledipole interactions with other nuclear magnetic moments.8a) B = $C(Z. 1.3. ZD = (yrysh2/r3)(A +B where A = + C + D + E + F). . + i ~ . the energy of interaction between the spins is14 Z D= (pi * p z ) / r 3 . “Principles of Magnetic Resonance.3. (1. ‘H.iS. . ) ~ ~ ] ( sOi n cos 8 e’”).7) This dipolar Hamiltonian can be conveniently rewritten in a polar coordinate system involving the interspin vector r and the applied magnetic field.3.3(v1 * r)(c~z r)/r5. (1.4. C = $[(I.
/(l + w2t. the spectral density functions for Eqs.14) From this result we may gain some insight into those factors that influence the intramolecular dipoledipole relaxation rate. Komoroski.3. Chem. and 5") are the spectral density functions associated with the fluctuations in F'O' = (1  3 cos2 Q r . where r is fixed. D. that is. J")./(l 022..9) and (1. (1. Phys. (1. a situation which is not always satisfied for large biological macromolecules.11) For intramolecular dipolar relaxation.3. 4 1.13) where q is the viscosity of the medium in which the molecule is embedded and a is the radius of a hypothetical sphere used to approximate the molecule. of course. and R . J")(o) = +$r6[zc/(1 + J'O)(w) = $$6[z.12) It is generally assumed that molecular reorientation in the liquid state satisfies the StokesEinstein relationship. J'"(w) = &r6[z. In any case. In order to illustrate how these ideas may be exploited we have selected some results from the work of Allerhand and c o . This is.2)].18 1.I('). 55. t.6 . If we may assume that any rotational anisotropy in the molecular motions is small.2)]. This is the socalled extreme narrowing condition. (1. for example.3. = T~ = 4nr]a3/3kT. J .2)].h2S(S + 1)TcP.~ o r k e r s on ' ~ the Tls for the I3C nuclei of cholesteryl chloride dissolved in CCl.3. Frequently. (1. when the extreme narrowing condition obtains.3 .3. (1. whose magnitude determines the strength of the microscopic magnetic field acting at the resonant nucleus. when they are covalently bonded to each other. NUCLEAR MAGNETIC RESONANCE .10)can be shown to be + 02z. Allerhand. to be expected since y is proportional to the nuclear magnetic moment p. the dipoledipole interaction will be maximal when I and S are in closest proximity. all the l5 A . 189 (1971) . For example.3. as. woz. since the spectral density functions are proportional to r .. Tl = Tz and the intramolecular dipolar relaxation rates are given by @:)intra = (Ri)htra= $y:y. It can also be noted that the dipolar relaxation rate will be highly dependent upon the gyromagnetic ratios of the interacting nuclei. Doddrell.
)y14h2W + 1)N/dD.49. namely.5 sec. which shows that carbons with 0. 13C spinlattice relaxation times (sec) for cholesteryl chloride in CCI.”) . SPIN RELAXATION 19 carbons located in the rigid ring structure will have the same correlation time 7. Within the Stokes formulation D and d are related by D = kT/6naq. The situation is therefore somewhat more complex. and 2 protons have average Tl values of 3.0. d is the distance of closest approach. here the increased rotational freedom of the methyl rotor results in a faster effective z .0 sec for these carbons. (1. Accordingly.16) It should be evident that the intermolecular contribution to the relaxation rate can be suppressed by diluting the molecule in a magnetically inert solvent. and D is the translational diffusion coefficient..6 . in which case the r values will be similar as well. but if the extreme narrowing condition is applicable and the spins are of the same kind. we predict that Tl for the ring carbons should decrease with the number of protons bonded to them. An apparent anomaly is the TI value for the ringbonded methyl carbons.. This effect of z..15) where N is the density of spins per cubic centimeter.3.3. the Tl values are about 2. in additional to 6 and 4. which despite the three directly bonded protons is 1. it can be shown that @:)inter = (A. the number and the gyromagnetic ratio of the nuclei coupled magnetically to the carbon atoms and the respective internuclear distances. This expectation is clearly borne out in Fig.1. 2 7 FIG. on the relaxation rate is even more pronounced in the case of the methyl carbons attached to the terminal end of the aliphatic tail segment. CI 0 .26sec. (From Allerhand et a1. (1. the only significant dipolar contribution to the 13C Tl should come from directly bonded protons. Finally.3. for intermolecular dipoledipole interactions the interspin distance r is also time dependent. and 0. than t. respectively.4.6 dependence of the relaxation rate.1. 6. However.The Tls for these nuclei will then be a function of structural factors only. Because of the low natural abundance of 13C (about 1%) and the strong r . d = 2a.
and water (50:40: 15).18) where J is the wellknown scalar spinspin coupling constant.  (1./(l and R2 1 .3.3. there can also be indirect interactions between nuclei which are mediated via the bonding electrons. 1238 (1981). In solution. or if the coupling between I and S is modulated by rapid chemical exchange for one of the spins.1j J 2  + (0. Murari and W. 7. An example of how spinspin coupling manifests itself in the frequency spectrum is illustrated in Fig. SpinSpin Coupling.3.3. J. corresponding to the three possible orientations of the 14N spin in a magnetic field.3. It should be evident that X j can become time dependent when either one of the spins undergoes rapid nuclear relaxation.4. Baumann. (01 (1. 13Cis chemically bonded to 14N. when this condition does not obtain. spectral splitting vanishes and the timedependent scalar coupling affords another mechanism for relaxation. Soc. and the two nuclei are spin coupled with J / h of 3.20 1. 103.16 Here. Scalar relaxation of the first kind occurs when the chemical exchange rate of a coupled nucleus is greater than both J and the nuclear relaxation rate. The spin Hamiltonian that describes this interaction between two spins is given by Xj = I * J *S.0. For this mechanism only a single correlation function is involved.1.20) where re is the chemical exchange time constant. In addition to the direct dipolar coupling of nuclear magnetic moments. (1. only the trace of J can be measured and the spin Hamiltonian simplifies to X J = J(1 S). . . + (xe/{l + . Therefore J splittings will only be manifested in the NMR spectrum when J / h is greater than either the nuclear relaxation rate or the frequency of dissociation between I and S. This effect is called spinspin or scalar coupling and can lead to fine structure in the resonance signals. Since I = 1 for 14N. describing the probability that S and I are still coupled at t + T. mI = .ws)2z:>]s(s + 1) CT. where we show the ' j C spectrum of the choline methyl resonance from a solution of phosphatidylcholine dissolved in a mixed solvent of chloroform.~ s ) ~ t f ) > ] S+ ( SI). Assuming that I is always bound to some S spin.19) (1.J. If the spin coupling interaction fluctuates in time it can also act as a mechanism for relaxation.the effect of this coupling is to split the I3C resonance into three lines. Of course. Chrm.2. NUCLEAR MAGNETIC RESONANCE 1. one can show that the relaxation rates for I are RI1 _ _ :J2cT.7 Hz. Am.17) where J is the tensor describing the bilinear coupling of nuclei I and S through interactions with their electrons. l6 R. + 1. methanol. namely.
3.79 ppm).45 ppm). A second type of scalar relaxation can exist when the relaxation rate for one of the nuclei is much greater than both J and/or the exchange rate l / ~ ~ .3) is predominant for the second nucleus. (1.P (64.R. e. Protondecoupled 20MHz 13C NMR spectra of egg yolk phosphatidylcholine in (a) CDCl3/CD3OD/D2O(50: 50: 15).23) + l)[TS/{l + + (uI .3. The contribution to the spinlattice relaxation rate for I is simply R: = 25"'(w. SPIN RELAXATION 21 75 70 65 60 55 50 PPM FIG.16 Copyright @ 1981 American Chemical Society..65 ppm).(35 2 2 )S(S + J J ~ S (+ S l)eiosr e71Tf. only the timedependent components of the spinspin interaction having frequency wI will contribute to T i .4. = J(')(w1) ~J'"'(O). sn1 CH. sn3 C H 2 .3. For spinlattice relaxation. (63.1.0 . Insert is an expansion of the N(CH.21) where J(')(w)is the Fourier spectrum of the autocorrelation function G(~)(T) The result is R I1 .24) . quadrupolar (see Section 1.~s)'(Ts)~}].3. CH2N (66. This may occur when a different relaxation mechanism. Reprinted with permission from Murari and Baumann.).3.94 ppm). and (b) D 2 0 . (1.)3 peak. For the transverse relaxation rate.26ppm). and sn2 CH0R2 (70.7. POCH2 (59. Therefore R'.O.g. and in fact only those components of the S magnetization not aligned with the Zeeman field will be important. Under these conditions the coupling between the two spins is rapidly modulated by fluctuations in the S spin as it makes frequent transitions among its energy states.02 ppm)..3. (1.Peak assignments in (a) are given for: N(CH3)3 (54. it is necessary to include a second autocorrelation function involving the z components of S.22) (1. where stationary fields also can contribute.
Chem. (1. c ) of Q or V. Is + q(I. Commun. 1. Expanding X a in the principal axis system (a.3. 7 (1979).I6 which we cited earlier in this section (Fig. while in chloroform. even in the absence of an external magnetic field. The interaction of this asymmetric nuclear charge distribution with the electron cloud can partially lift the 21 1 spatial degeneracy of the nuclear spin.Zz) . 49.1)]V.1)][(3I: " J.1)][Kc(31: . Res.06 and 0. . M.3.22 1. E.9 .V.I . T. by Q = [eQ/21(21 . London. In accordance with this expectation. R.3. Wilson.3. the 14N T. Lipids 25.25) (1. the I4N splitting in the choline methyl resonance of the I3C spectrum are collapsed. (1. and water (50: 50: 15). D.Z2) + (KO . Matwiyoff.27) where Q is the quadrupole energy tensor and is related to the electric field gradient tensor at the nucleus. b. Phys. and T2 values for phosphatidylcholine liposomes in D 2 0 are 0. and N. the corresponding values are 0.3. Walker.3. ) ] . a medium where phospholipids do not aggregate.4. in turn. methanol. } Scalar relaxation of the second type can be illustrated from the phospholipid studies of Murari and Baumann. This coupling is usually referred to as the electric quadrupole interaction. V.15 and 0. Biochem. I573 (1972).3.26) Thus the expression for the transverse relaxation rate is Ri = [+J'S(S + l)][T:/{l + .0006 sec.)(Z: . Biophys. E. Quadrupolar interactions. Behr and J .' / ~ ? . Lehn.28) where e is the electron charge and Q the electric quadrupole moment of the nucleus.)] P. respectively.29) (1.. 1 .30) which may be simplified to X'.3. (wI (1.177 sec. 7). . The Hamiltonian operator for this problem is given by + &?a= 1 . (1. The spinlattice and spinspin relaxation rates for the I4N nucleus in phosphatidylcholine are controlled by the rotational rate of the CH2N which. Concomittant with the increased relaxation rates of the I4N in the liposomes.W ~ ) ' ( T $+ )~ T:]. Atomic nuclei having I 2 1 possess an electric quadrupole moment because the charge on the nucleus is distributed asymmetrically.I. = [e2qQ/4Z(2Z . M. depends upon the aggregation state of the phospholipids in solution. we get X Q= [eQ/41(21 . NUCLEAR MAGNETIC RESONANCE where J(O)(w)is the Fourier transform of the correlation function G(O)(~) = [&Ps(s + ~ ) ] e . A.
. g 1.4 x lO”/V m2. This screening effect arises from the electronic currents induced in the molecule by H. however.33 respectively.4..Q)27 .32) where u is the anisotropic chemical shielding tensor. 23Na. Chemical Shift Anisotropy. Since the electronic environment is usually asymmetric about the nucleus. If the rotational reorientation of the molecule is sufficiently fast.and 35Cl. in fact. This mechanism is. For example.40 Z’(21 .33) + + . the electric field gradient.(l  u). Generally. the magnitude of the shielding will depend on the orientation of the molecule relative to the external field. measures the deviation of the electron charge distribution from spherical symmetry at the nucleus. in units of e x cm2. Obviously. Similarly. the greater the nuclear charge and the degree to which this charge distribution deviates from spherical symmetry. Q for ’H.1) y) (e2.31) 1. cause mixing of the nuclear spin states. 14N. For the 35Clanion. exhibits the following angular dependence: oz = coo sin’ 8 cos2 4 obb sin2 8 sin’ 4 of. such that ooz. the Zeeman Hamiltonian is modified to XZ=p.016.4. and 79Br are 0. as in CH3Cl. the contribution of electric quadrupolar interaction to the relaxation rates can be shown to be R. SPIN RELAXATION 23 by taking advantage of Tr V = 0 and introducing V..0028.&b)/l/cc* The importance of the electric quadrupole interaction is determined by the magnitudes of Q and q. In the presence of an external magnetic field. For solid samples this orientational dependence gives rise to the wellknown “chemical shift anisotrop y. (1. and promote nuclear spin relaxation. the effective field seen by a nucleus will differ from H.=R 3 (21 + 3) (l + ’.3. (1. the most frequently studied quadrupolar nuclei are 14N.3.1. When a quadrupolar nucleus is placed in a magnetic field. In biological systems. and 0. In terms of the principal values of u. q = 0. the larger Q is. broaden their energy levels.” With magnetic shielding. reorientation of the electron cloud uisuuis the magnetic field via rotational tumbling of the molecule will “confuse” the nucleus. ~ * (1.‘H.3.3. there will be a competition between the Zeeman interaction to align the magnetic moment of the nucleus with the magnetic field and the electric quadrupole interaction to align the nuclear quadrupole with the electron cloud of the molecule.H.the effective magnetic shielding oz along H. by a small amount due to magnetic screening by the surrounding electron cloud. the most important spin relaxation pathway for quadrupolar nuclei.0. = eq and q = (KO . q.3. when the chlorine is covalently bound. the electric field gradient has a value of 38. cos’ 8.
M. B. Holm.35) &.uL. NUCLEAR MAGNETIC RESONANCE where 0 and 4 define the polar and azimuthal angles of Ho relative to the principal axes of u.3. J . The characterization of a molecule undergoing anisotropic motion requires at least two rotational correlation times. In solution. Although we do not treat anisotropic motion here. 104. Woessner. R w . the approximation of isotropic motion is often a gross oversimplification. McConnell and C.3. it is important to emphasize that. In less rigid systems.. "F. Gutowsky and D. 1. If we assume for sake of simplicity that u is axially symmetric. i. if we consider the motions of a rodshaped molecule in solution. it can be seen that rotation about the long axis will be much more rapid than molecular rotations perpendicular to this axis. but with quite different rates of reorientation about the various molecular axes. motions which take the molecule over all directions in space with equal probability. In studies of biological systems.5. Cliem. the relaxation rates for this mechanism are R1 = & y 2 H i ( A ~ ) 2 ~ . More likely. Phys. Anisotropic Motions. H. Observations of spectral width and T1dependence on H i having been reported for I3C. anisotropic shielding effects are likely to become more prevalent. in the interpretation of relaxation data. this modulation of the shielding field can contribute to spinlattice and transverse relaxation.3.5.e. and 31Pnuclei in a number of c o m p o ~ n d s . ' indicating ~*~~ that anisotropic shielding can indeed contribute to nuclear relaxation in some systems.1. The inclusion of such motional details can have a profound effect on the NMR relaxation times.JH~(AB)~T.34) (1. For example. E. and Rz = (1. Effects of Anisotropic and Restricted Motions 1. 843 (1956). treatments based on single correlation times are l9 2o H. we expect that molecules will undergo anisotropic motions. = a l land aaa= obb= al. a part of the molecule may also undergo segmental motion at rates quite distinct from those motions of the whole system. . and the condition of extreme narrowing holds. crZ is time dependent because of the rapid reorientation of the molecule. 1289 (1956) H. however.3. Phys. We have assumed in our discussion of relaxation mechanisms that the molecular motions that modulate the various interactions are rapid and isotropic. 25. S. With the advent of superconducting solenoid NMR spectrometers capable of NMR observations at magnetic fields greater than 10T. where Aa = a l l ..24 1... that is.
Such motional restrictions lead to firstorder dispersions in the NMR spectrum due to chemical shift anisotropy and incompletely averaged dipolar and quadrupolar interactions.4.1.4. will begin to relax back toward equilibrium along +z. where motional restrictions prevent the averaging of certain spin interactions. Following this pulse. The initial height of this signal is a measure of the magnetization along the z axis at time z after the first pulse. EXPERIMENTAL METHODS 25 not applicable for systems where the molecules are undergoing anisotropic motions. While these effects are not difficult to treat.4.1. 1. In studying complex biological systems one is often interested in molecules that are found in large.1. Since TI characterizes the time required for the M . TI can be determined by utilizing a twopulse sequence.3 an example to outline some features of the NMR of motionally restricted systems. such as those involving cell membranes. component to return to equilibrium.4. Restricted Motions. M . while others have proven especially useful in studies of biological systems. rotating M by 90" and thereby placing it in the xy plane. In such an environment these molecules may undergo motions that are not only anisotropic but also restricted in the angular spatial orientations that can be assumed. By repeating this sequence for different values of . Pulse Techniques 1. macromolecular assemblies and arrays and exhibit a high degree of order. and T. 8a). In addition some recent advances in solidstate NMR have been included. TI.5.2. however. T. 1..5. Some of these methods were chosen because they are of general utility. It was previously noted that one of the most useful ways to detect magnetic resonance is by measuring the freeinduction decay signal following a 90" pulse.. Experimental Methods In this section a number of experimental techniques are described. after time T a second pulse is applied.. .. If. Measurement of T. One such procedure. we present in Section 1. it cannot be directly measured from the FID. a signal will be induced in the receiver coil. which reflects only the decay of M X yHowever. These techniques hold great promise for studies of complex biological phenomena.1. 1. However.3. space limitations preclude the discussion of these details in the present chapter. often referred to as the 180"t90" method. calls for an initial pulse which rotates the magnetization vector M 180" from its position along the Zeeman axis (see Fig.
= .180" pulse sequence and is illustrated in Fig. Rrz. Integrating d M . If the height of the ith signal in the partially relaxed spectrum is A:.( M . (1.M&TI (1. Carr and E. the TI values of any particular species can be obtained by a varient of the scheme described above if their signals are spectrally resolved in the frequency spectrum. NUCLEAR MAGNETIC RESONANCE FIG. .nT. wherein T2 is related to the linewidth at half height by Av = l/(. / d t = . After a period t there will be a loss in the phase coherence of the magnetic moments.A:) versus t.'' t..z . (1. processes will '' H. T. inhomogeneous T. 94.M . (b) after a time T. Following a 90" pulse. then ln(AL  Af) = ln(2AL) . The intrinsic T2 is best determined by the CarrPurcell spinecho techniques.4.2) For a heterogeneous spin system containing a number of distinct populations of resonant nuclei. and that the linewidth contribution from magnetic field inhomogeneity is not significant. . one obtains a measure of the rate at which M . may be determined by plotting In(Ab. at t = 0 gives the following expression for the magnetization recovery: M .. The transverse relaxation time T2 is often measured directly from the linewidth of a resonance signal. M ." This method employs a 90".4. Phys.1) with the initial condition M . 8b. t. 630 (1954). Purcell. . rotating M . Measurement of spinlattice relaxation by the 180°~90" method: (a) H I is applied along x'. The method calls for Fouriertransforming the FID observed after the 90" pulse to obtain the socalled partially relaxed frequency spectrum at each time t following the magnetization inversion.8a. H I is applied long enough to rotate the net magnetization along y'. .).4. This procedure assumes that the line shape of the resonance is Lorentzian.26 1. to M . = M.Z I T . but if at this time a 180" pulse is applied. Y .3) and its spinlattice relaxation rate l/Ti. returns to equilibrium. a condition not normally attained in highresolution NMR.(1  2er'"'1). M is rotated onto the x y plane.
under appropriate . they are limited in that spinlattice relaxation is only effected by relatively rapid motions. measurements provide a convenient parameter to sample motion in biological systems. Although T. the efficiency of relaxation will depend upon the amplitude of local fields fluctuating at y H . occurring near 1081010 sec. (e) maximum magnetization along .relatively slow molecular motions can be sampled. in the laboratory frame. He. H... the sensitivity of the method does not extend beyond times slower than about sec.For systems having more than one spin species. and applying a second 180" pulse to refocus the spins and obtain nuclear induction at the receiver coil. there exists a third relaxation time TIP (referred to as T. However.. rather than yH. under the spinlocked condition yields TI.2 r i T z because of homogeneous T. EXPERIMENTAL METHODS 27 FIG. Tl.1.8b. When the spins are locked by H.1. inhomogeneity across the sample volume. Although T2 measurements do permit one to sample slower motions.4.4. It is evident that the amplitude of the induced signal decays exponentially with a time constant equal to half the homogeneous T2.12 bring the moments back in phase after another period 2. TlPis measured by switching the phase of H I following a 90" pulse so that it becomes aligned with M x .2.. (d) nuclei rephasing towards . The diffusion of molecules can introduce significant errors in the measurements of T2 because of H.y ' . processes. Spinecho method in the rotating frame: (a) 90" pulse H I along x' rotates M . (f) dephasing of spin echo. The decay of M. this is rarely done because of the difficulties in obtaining a properly phased spectrum.y ' half of the x'y' plane. However. the maximum signal induced in the receiver coil at this time will have decayed by e . However. (c) 180" pulse along x' rotates dephasing magnetization into the . the peak heights in the frequency spectrum may be measured after Fourier transformation of the FID at 22.. Measurement of Diffusion Coefficients.. at resonance and the spins become locked to H. in the rotating frame. very much like the alignment of M along H. However. in the rotating frame) which allows one to sample motions as slow as lo4 per second. . (b) magnetic field inhomogeneity causes nuclei to lose phase for a time T. 1. The cycle can be repeated by allowing the spins to dephase for another time 2. to the y' axis.. Thus by appropriate choice of HI. particularly from complex systems with coupled spins.y'.
3.1.4. J .22 1.. 9b. Pulse scqucncc and data acquisition pcriod for a pulsed gradient experiment. Decay of the proton signals from a dimcthyl sulfoxideH. The angular dependence of dipolar interactions is of no consequence for molecules undergoing rapid. is given by M ( 2 r .9a.4) The selfdiffusion D can be measured by comparing M(2r) determined in the presence and absence of G at various values of 2.28 1. For a sample in the presence of magnetic field gradient G . NUCLEAR MAGNETIC RESONANCE Field Gradient . random reorientation since the dipolar interactions are averaged to zero by such '' T.. James and G . MultiplePulse Methods. 11." 0 1 0 0 200 T 0 100 200 (rnsec) Fic. 9a).2 r / T 2 ) k 2 / 3 7 2 G 2 D r 3 (1. the spinecho amplitude at 2 2 . 58 (1973) . G . Mot.4.O ( 1 : 1) solution as a function of the echo development time T shown in (a).= M o e ( .A + ~ Data Acquisition FIG. R m m . M q n . Echo development occurs in thc prcscnce of a gradicnt which is then turned off to sample the echo decay envelope. McDonald. b . Spectra were obtained in the presence [(b) and (d)] and absence [(a)and (c)] ofa pulsed field gradient. L. A significant improvement in this method can be realized by utilizing a pulsed gradient imposed on the sample following each rf pulse (see Fig." conditions one can take advantage of this effect and use it to measure the molecular diffusion coefficient D (cm2/sec). 9b). Fourier transformation of the resulting FID allows one to calculate the diffusion coefficient from any spectral peak that can be resolved (see Fig.
Details about these further developments may be found in HaeberleqZ5 Mehring.z6 and Vaughan. S.28 For dipolar broadening in a twospin system consisting of I = S = 3.. In 1966 it was reported that dipolar effects in solids could be reduced by a modification of the CarrPurcell pulse s e q ~ e n c e . we can disregard the terms C .^^.22 . Phys. Waugh. and F in Eq. such as chemical shifts and spinspin couplings. EXPERIMENTAL METHODS 29 motions. Chem. .2. such as solids and membranes. Suppl. R E D . Unfortunately. J. However. Rewriting the simplified Hamiltonian. then P. Phys.90. Haeberlen.and sixteenpulse sequences. E. Inasmuch as this pulse sequence over the period 62 of the cycle aligns the magnetization along all three axes x. 29.22 . Ado. 26 M.4. (1. Phys.Unfortunately.8) since they serve only to induce relatively weak absorptions at frequencies O.900. L. this effect masks many of the weaker spin interactions. z for durations of 22 each. ~~*~~ there was a concomitant loss in the chemical shift and spin coupling effects. . in systems where this condition does not obtain. Ostroff and J. D. Huber. However. Since these parameters hold important information concerning molecular structure. Prog. S. we get (1. 133 (1966). 11 (1976). Huber. Reti. Lett.S.7) where the subscripts denote the axes along which the pulses are applied. which also contribute to the spectrum. it is of great interest to have a method which allows them to be observed. these sequences permit unmasking of the weaker interaction^. ** R.1. and U. 391 (1978). W. this led Waugh and coworkers to formulate a number of multiplepulse sequences that allow for the coherent averaging of dipolar effects in solids. (1.3. Annu.4. and 20. The purpose of the WHH fourpulse cycle is to averageout XD through an appropriate choice of pulses so that (31. y . D. 180(1968). Mehring. Reson. N M R : Basic Princ. 22. Lett. Ware. this averaging does not occur and the signal is broadened. 23 24 *’ ’’ . (1.4. Haeberlen.5) From this result.6) This averaging in spin space may be accomplished by imposing the rf sequence 90: .4.I ’ S) = 0. M. 1096 (1966).2 . Waugh. R w . . U. M q n . Phys. and Haeberlen (WHH). 20.90Yx .27 although better averaging is now possible by utilizing eight.E . we will briefly discuss the initial fourpulse sequence developed by Waugh.^^ To illustrate this. I (1976). it is evident that XD can be averaged to zero both in real and spin space. Vaughan. 16. Lrtr. Mansfield and D.
for the timedependent .. 10. to minimize dephasing effects t must be chosen to be much smaller than T 2 . The spin Hamiltonian for an ensemble of interacting nuclei in a solid undergoing rapid rotation about an axis can be conveniently written as 3P = 2 + X(t). In a typical multiplepulse experiment.& = +I * S and Eq. (1.z6 I.4.F. NUCLEAR MAGNETIC RESONANCE ~ 0 . which averages the lattice operators in real space. it was shown how dipolar broadening could be removed by coherent averaging of the spin operators in spin space.30 1. (1. . 1. Resolution of the ”F rnultiplet from polycrystalline C.4.6) is satisfied.4.FI2 by application of a WHH fourpulse sequence. GAUSS5 C652 200K 2000 1000 0 1000 2000 3000 400C dHz) FIG. 10 the striking effect of coherent averaging in spin space on the I9F spectrum of polycrystalline C. the magnetization is sampled once every cycle at a 6t interval. and the averaged spectrum is subsequently obtained by Fourier transformation of the accumulated decay signals.. .8) where 2 is the timeaveraged value and %(t) is the timedependent Hamiltonian having a mean value of zero.9) It can be seen from this equation that when fl is set at the “magic angle” of 54’44’ the value of ZD goes to zero. Clearly.the timeaveraged dipolar contribution to this system is (1.4.2. Similarly. We illustrate in Fig. MagicAngle Spinning In the preceding discussion. In this section we describe the technique of magicangle spinning. If the axis of sample rotation forms angle p with H.
pij is the angle between internuclear vector rij and the axis of sample rotation. Inc. Lu. resulting in the loss of information about these tensors. by courtesy of Marcel times of co.’ 207. 4 .10) the value will average out to zero when the frequency of sample rotation o. Waugh. 1 1 . Dekker.4.01 FIG. Rapid spinning also removes the chemical shift anisotropy. The major reason is probably that biological systems always display some motion. then it can be shown3’ that. R. dipolar term x [sin 2p sin 2pij COS(O. Prog.'^ At the present time. magicangle spinning experiments result in highresolution spectra such as those observed in liquids and solution^. in order to obtain further narrowing by sample spinning. S. eds. 1 (1971). magicangle spinning has not received the attention it deserves in NMR studies of biological systems.t+ 4ij)]. p. If the frequency of such motions is on the order of l/zc. New York. Accordingly.1 0. Opella and P.~ 4ij) + + sin’ fl sin’ pij cos 2(o. Effect of magicangle spinning rate v . In both expressions. (1.1 .). Thus. is greater than the linewidth A of the rigid lattice. and lo’ sec. 1979. J . Dekker. one can effectively remove dipolar (as well as quadrupolar) interactions from the spectrum of a solid. ~ p. Reson. 203. Andrew. EXPERIMENTAL METHODS 1/r 31 (kHZ) 1 0 0 0 . 1 I 1 0 1000 10. Magn. Reprinted from W a ~ g h . J. 2y 30 . Nucl.000 v I N 73 3 0. in “ N M R and Biochemistry” (S. the condition or > l/zc > A E. under appropriate conditions of sample spinning. 8. on linewidths from samples having correlation sec.
Doubleresonance techniques employ a second rf field which acts on one of the coupled nuclei while the other is being sampled. . such as a phospholipid multilayer. 25. change the spinstate distribution for one type of spin by 3' D.87 HOD L . FEBS Lett. and B. Double Resonance Numerous methods have been devised to take advantage of the coupling interactions (dipolar and scalar) that exist between nuclei. 261 (1972). D. Proton N M R spectrum of a sample containing 33 wt. This effect is illustrated in Fig. 11.32 1.O at 25" and rotating at 500 Hz about an axis perpendicular (a) or at the magic angle (b) to the external field H . I I I I J I I I 600 400 200 0 0 200 400 600 I I c/s c/s FIG. egg yolk lecithin in D. for systems displaying l/z. 3 1 The resolved peaks in (b) at 9 . l ~ and 6 . 6 ~ are assigned to the fatty acid terminal methyl group protons and the choline methyl group protons.3 HMD5 MDS (a 1 (b 1 6. Chapman. Of course. Doskocilova. respectively. Under appropriate conditions these methods can remove the spinspin splitting between scalar coupled nuclei (spin decoupling). where the theoretical linewidth for a dipolar broadenined sample is depicted as a function of sample spinning in the presence and absence of internal motions. Schneider. z A. HMDS is an cxtcrnal reference. NUCLEAR MAGNETIC RESONANCE must be met.3.12. a significant reduction in linewidths may be observed when samples are spun at the magic angle (see Fig. 12).4. 1. E. "/. Oldfield. .
EXPERIMENTAL METHODS 33 equalizing the spinstate populations of a coupled species (saturation transfer or nuclear Overhauser effects. Childers. This is because the dipolar interaction provides a mechanism for transferring magnetization from one spin species to the other.4. spin decoupling may be used to simplify complex spectra. for example by setting H . we now observe only a single peak at the center of the multiplet of the undecoupled spectrum.1. which experience only weak longrange coupling (I 15 Hz). however. In our discussion of spinspin coupling above we noted that the signal from one coupled nucleus will be split into a multiplet because of the different spatial orientations of the other nuclear magnetic moment. Allerhand. However the linewidths Av of these carbons are all slightly broadened due to the strong J coupling ( 2 120 Hz). Since the 32 A .4. facilitating spectral assignments. Oldfield.3. 1. and simultaneously sample the species of interest.2. If. $ nJ. Since the area under the spectral peaks is proportional to the number of nuclei. 1335 (1973). Spin Decoupling. Saturation Transfer. 13. The technique of double resonance can be taken to great advantage when two spins are dipole coupled. The multiplet is observed only because the rate at which transitions occur in the coupled nucleus causing the splitting is slow. and the spinspin splitting is collapsed. Unlike spin decoupling. Biochemistry 12. are easily resolved and unambiguously assigned. R. off resonance from the proton frequency. The usefulness of spin decoupling is apparent. An interesting example is presented in Fig. the magnetization transfer experiment employs a second field which is strong enough to equalize the spin populations (saturate) of the irradiated species.32 The peaks from the protonbonded carbons all appear as singlets due to proton decoupling.3. . In addition. and increase the signal intensity of a dilute spin species by transfer of polarization from an abundant species (cross polarization).1. Here the I3C spectrum for the aromatic region of tryptophan is complicated by interference between signals from protonbonded and nonprotonated carbon atoms. 13b. 1.4. Under these conditions the peaks arising from nonprotonated carbon atoms. Since Av oc J 2 / H i .. any protonbonded carbon resonance can be effectively broadened by reducing H. F. In this section we briefly describe these techniques and illustrate their usefulness. and E. What has happened is that the second rf field induces rapid transitions between the various Zeeman states of the coupled nucleus which cause the splitting to begin with. where a relatively weak rf field is used to excite one of the scalarcoupled nuclei. we impose a second rf field H. at the resonance frequency of the coupled nucleus such that y H . the collapse of spinspin splitting can result in considerable signal enhancement. The effect of this incomplete proton decoupling is shown in Fig.
J . brought about by the equalized populations.SO). Reprinted with permission from Allerhandet Copyright @ 1973 American Chemical Society. New York. Allerhand. respectively." Academic Press. is manifested by a change in the intensity of the NMR signal from the unsaturated spins when the latter are amp led. 14.and lowenergy spin orientations. "The Nuclear Overhauser Effect.10).( w z.13.34 1. Phys. H. 'I' I ' FIG. ) is described by34 d(J. The coupled Zeeman levels are shown in Fig. Noggle and R. This effect. Under these conditions. 56. (b) Offresonance protondecoupled spectrum of tryptophan. .'^ For the sake of discussion we shall consider a simple twospin system consisting of I = S = +. V. Doddrell.+ wz)((Iz) . which couple the spin states to the lattice degrees of freedom. the resulting increase in the relaxation rate of the saturated spins. called the nuclear Overhauser effect (NOE).wo)(<sz) . NUCLEAR MAGNETIC RESONANCE I I I I I I 55 I 60 65 70 75 00 05 COOH J 90 PPM FROM CS2 . Schirmer. as modulated by the dominant spin interactions.4. E. 3 4 D. Chem.11) 3 3 J. and A. Glushko. Zeeman states of the two nuclei are coupled by dipolar interactions. (a) Fully protondecoupled "C N M R spectrum of the aromatic resonances of tryptophan in aqueous solution. will cause a redistribution in the Zeeman populations of the nonirradiated species.)/dt = (wo + 2 w 1 .3683 (1972). 1971. Also shown are the transition probabilities K. (1. the equation of motion for the observed I spin magnetization ( I . where a and /?denote the high.
Thus for dipolar relaxation of small molecules undergoing rapid motions.14. Chem. the NOE is also influenced by the onset of nuclear spin relaxation mechanisms other than dipoledipole interactions.4. a and I . Phys. Overhauser effects have been used to sort out the relative relaxation contributions in several welldefined ~ysterns.12) The effect of saturation is therefore to change the steadystate value of ( I .. Energy level diagram for coupled spins I = S = i. where fr(S) = C(wz .1. ) and (&). (1. (1. and So are the equilibrium values for ( I . + W2)[(I. 52. J . ) = 0. or 2 representing the quantum of energy absorbed or emitted by I or S or both. W. % Wo and the fractional enhancement reduces to ys/2y.4. However. the zero quantum transition probability will also be important and the NuE modified accordingly. K . but also by the relative probabilities of the various relaxation pathways.4. Harris. and R. Eq. In fact. M. For nuclei exhibiting a significant scalar contribution to the relaxation rate. because of the dependence of f r ( S ) on the relaxation mechanism. Grant. Kuhlmann.1.4. D.~ 35 K. F. 1.Transition probabilities between fi (high and low) energy levels are denoted W. If we now impose a saturating rf field at the S spin frequency. with subscripts 0.13) For I = S = f the transition probabilities are where K = hysy.) .3439 (1970) . so that ( S .11) becomes d(Iz)/dt = (wo + 2w1. and J("(o) are the spectral density functions discussed in Section 1. EXPERIMENTAL METHODS 35 FIG. ) from its value at thermal equilibrium by the factor 1 + fr(S).4.wo)/(wo+ 2Wir + wz)l~s/~r. From these relationships. (1.3.Zo(1 + fr(S))]. it can be seen that the net NOE is determined not only by the relative signs and magnitudes of the nuclear gyromagnetic ratios.
2042 (1962). the dependence of the fractional enhancement on z. A. ~ . 3 . A. J . In this experiment the abundant spins are irradiated with an rf field of amplitude H i at the Larmor frequency of I .4.3. 94. For this reason.36 1. Dadok. Chmi. Breslow. . 128.w. but for long correlation times. Bioclzemistry 12.4695 (1973).4w42:). and E. where (w. Hartmann and E. Phys. .4.1. 94. A. A . Soc. A. J . A.G 1) results in f.3. Am.(S) = .4017 (1972) .4015 (1972). This result unambiguously places the protein tyrosine residue at the hormone binding site and further suggests that a specific interaction occurs with the peptide phenyl group. and J. these studies usually take advantage of the fact that spin interactions are distance dependent. For a system of two dipolarcoupled protons. A m . Balaram. The usefulness of this approach is illustrated by the work of Balaram et a1. L. Rreslow. (1. over W. Chern. and E. NUCLEAR MAGNETIC RESONANCE The interpretation of saturation transfer experiments on biological molecules is complicated by both the size and intrinsic complexity of the macromolecules involved.'*P. '"S. P.)'zf < 1. Cross Polarization of Dilute Spins. Based on preliminary studies showing a differential broadening of certain aromatic protons in position 2 or a tripeptide hormone analog.. these workers attempted to further investigate the binding interaction by measuring the proton NOE of the peptide while irradiating chemically shifted protons on the protein. Balaram. The physical basis for the negative Overhauser effect associated with slow motions is the predominance of W. BothnerBy.15) which for rapid motions ( w .5.4w42:)/(10 + 2 3 ~ ' ~+. Balaram. and 4 phenyl group protons in the presence of a saturating rf field at a frequency corresponding to the ortho ring protons on the lone tyrosine of neurophyrin 11. f.show a decrease for the resonance intensities of the 2 . is given by fi(S) = ( 5 + w'TZ. Soc. Hahn. any measurable NOE can then be taken to infer that two nuclei are in reasonably close proximity.. Under these conditions both spin magnetizations may P. The negative Overhauser effect observed in the above hormone/neurophyrin I1 study is due to the long correlation times of the protons associated with the protein. Rrv. 1. R ." . Their data. In 1962 Hartmann and Hahn3' proposed a doubleresonance method for observing the magnetic properties of an isotopically or chemically rare spin S coupled to an abundant spin species I .(S) = 0. BotherBy. These spins are then brought into contact with the rare S spin system by imposing a second field H T at the S frequency. BothnerBy.3638 who used proton NMR to characterize the hormone binding site on the protein neurophysin 11. obtained by monitoring the aromatic protons of the peptides SMeCysPheIleNH. 3h 3' .
4.. the interactions of the xy components of the precessing spin magnetizations are negligible. the z components. only results in a small decrease in the I magnetization.569 ( 1 973). which are generated as the spins precess about their respective H. G . A further decrease in I magnetization occurs if the S spins are now allowed to reach equilibrium with the lattice (for example by turning off Hf) and then are brought back into contact with the I spins. the observation of a dilute spin signal still presents difficulties for solid samples.3. . Although the advent of FTNMR and signal averaging techniques have alleviated many of the difficulties associated with investigations of rare spins. Repetition of this cycle ultimately results in a measurable decrease in the I magnetization. as described in Eq.4. there will be a net transfer of magnetic polarization from the abundant I spins to the rare S spin system. the use of lengthy signal averaging is impractical. Phys. In the laboratory frame.fields so that one can effectively match the Zeeman splittings of the two nuclei along their respective HI fields. # ysH. This exchange. Similarly. (1. J .17) By adjusting the amplitudes of the H. Gibby.1%).98 %) to enhance the signal from relatively rare spins. M . for studies of labile systems or transient effects. S. fields in the rotating frame. it is the rare S spins that are sampled directly. where dipolar interactions are not averaged out. since yIH. Early applications of the technique utilized isotopically abundant protons (99. of course. precessing about the imposed HI fields with frequencies Qr = Y~H: (1. thus allowing one to sample the rare S spins indirectly.1. will have the same frequency. (1. however. As the two spin systems reach internal equilibrium. EXPERIMENTAL METHODS 37 be considered as vectors in the xy planes of their respective rotating frames. In an effort to mitigate this problem a technique based on the aforementioned HartmannHahn experiment was introduced in 1973 by Pines. Waugh. However. in this case. and W a ~ g h .8a).4. and J. Chem. 4n A . such as 13C(1. ~ In ' this experiment. Gibby. Pines. as previously described.16) and sZs = ys H s . However. 59. This allows energy exchange to occur through the z components of the dipolar interaction. the abundant I spins are polarized and brought into contact with the S spin system.
Only in this way was it possible to define the chemical shift tensor in relation to the plane of the membrane... Powers.4. denoted t . FIG. During this period. this method is commonly known as protonenhanced nuclear induction spectroscopy. rN 1 I * time t I . Grifin. Pulse sequence for a cross polarization experiment in which the magnetization from the abundant spins (M.. in Fig..38 1.~' In this experiment the enhancement by cross polarization was necessary because of the small amount of sample (about 4 pmole PO4) that could be oriented between glass slides. 17. The FID of Msis sampled during the periods denoled by T ~ . This cycle is then repeated until the rotating frame magnetization of the I spin system becomes depleted. Biochmiistrj. The two systems are next brought into contact so that energy can be exchanged via dipolar cross relaxation. Finally. One version of the protonenhanced nuclear induction spectroscopy experiment is shown in Fig. as in the HartmannHahn experiment. 15. the accumulated FIDs are averaged and transformed to yield a highresolution spectrum of the rare S spins. by imposing Hf such that the conditions of Eq.1 I * T I  2 i " i '72' 3 i ~ i 1 N. 15.18) are satisfied. L. (1... Pcrshan. A recent application of this technique included measurement of the angular dependence of the * P chemical shift for oriented phospholipid bilayer~. the I magnetization is locked along Hi so that it decays in a time Ti. 15. G. " As a result. NUCLEAR MAGNETIC RESONANCE I 1 .2718 (1978) . This is done..) is transferred to the rare spins (M\).I S Spins . S. and P. 41 R. The rare spins are then sampled by tion along H turning H Y off and recording the FID while continuously irradiating the abundant I spins in order to decouple the remaining dipolar interaction.. Following a 90" pulse. the energy conserved from the longitudinal relaxation (in the rotating frame) of the I spins causes an increase in the S spin magnetizaY in the rotating frame.
Phys. 16. Hahn and D.1. Jresolved spectrum. 64. W. J .4226 (1976). This is the rationale for the development of multidimensional NMR spectroscopy. thus much important information is lost. P. Mugn.4. Ernst. Another approach is to disperse the signal along different dimensions in frequency space. .g. P.4.4' a new method which holds great promise for unraveling the complex spectra of biological macromolecules. P. K. Nagdyama. each direction displaying the effect of a single variable (e. L.4. Most of the techniques that have been developed to simplify complex spectra do so by suppressing some of these effects. J .2229 (1976). R. spinspin coupling). J. C % m . The experimental scheme for obtaining a 2D. Ernst. 1070 (1952). K. Chern.133 (1978). R. Wuthrich. EXPERIMENTAL METHODS 39 1.43 is simply the spinecho method repeated at different values of t l . Ernst. Pulse sequence for obtaining a 2D. shown in Fig. R . Bachman. E. and R. Maxwell. 88. Rev. Karhan. For the sake of illustration. chemical shift. the basis for it can be outlined. where t l is the echo development time. Although the mathematical development of this method is beyond the scope of this chapter (see Ref..45This is due to the fact that dephasing effects from 42 43 44 4s W. Phys. and R. E. In its simplest form. Rrson. 64. Phys. J . twodimensional (2D) NMR. spectra are obtained by Fourier transformation of a signal possessing more than one independent time variable. E. as we have noted. based on the spinspin coupling and chemical shift (a technique referred to as 2D. 31. 90" 180" I FIG. and R. If the coupling constants J are small relative to the chemical shifts between coupled nuclei. 42). Bartholdi. 16. Jresolved spectrum. we consider the case in which a complex spectrum is resolved by effectively spreading it in two dimensions. TwoDimensional FTNMR Many of the difficulties that arise in NMR spectroscopy occur because the method characterizes energy absorption as a dispersion along a single variable. either time or frequency. then the spinecho amplitude will be a function of J. a sample can actually be characterized by many useful variables which act on chemically identical nuclei to split and spread their signal in frequency space. Aue. Aue. Jresolved spectroscopy43344). However.
decoupled peaks along 6.4.v j k ) 1. The contribution arising from the resonance described in Eq. 0) cos(vjkt1 . (a) Standard proton NMR spectrum of bovine pancreatic trypsin inhibitor in the highfield region.4. The decaying echo signal is then sampled for time t . ~ ~ chemical shifts and magnetic field inhomogeneity are reversed following the 180" pulse. (b) 2D.19)can be shown to be IS(jk(W1.8 0.8 / / 0.Ojk)21112 vjk (1. (1. projected in trace ( c ) . displaying the unresolved multiplets arising from 19 of the 20 methyl groups present. .0 2 ) = lMjk(O. Repetition of this experiment using different values for the parameter t .40 1. Jresolved spectrum of the same sample with the individual multiplets displayed along the J axis and the chemically shifted. NUCLEAR MAGNETIC RESONANCE / 1.which is a running variable of constant length.jk C1ITT?k + (01 .2 1A 1. t z ) = Mjk(0.4.20) where 0jk = nj + v j k and = 2n 1 Jj.O / 0. .1 j 2 2 > + (O2 . The contribution each resonance line k in the multiplet of nucleus j makes to the matrix is given by Mjk(t1.17. results in a data matrix of the transverse magnetization components as a function of t l and t 2 . 0)[1/T.Wjkt2)er1'T2Jk12'TtJk. m{k.19) This data matrix can then be Fourier transformed in two dimensioils to give a corresponding matrix in frequency space.6 / 1. (1. Thus by changing the spinecho development time one can obtain a measure of the spinspin couplings in the absence of chemical shifts.4 / / 6( p p w FIG.
w 2 ) along the o2axis one obtains the chemical shift information. Nagayama.1. 1. Reson. Biochem. while the spectrum projected along o1shows the spin coupling multiplets. 218 ( 1 979). and K. Thus by displaying S ( o . In this case the onedimensional spectrum contains overlapping spin multiplets from nineteen chemically shifted methyl resonances.5. Lauterbur. J . 34. Cornmun. in two dimensions the data are well resolved. In these efforts much information has been derived from the investigation of enzyme reaction rates and substrate binding constants. that the few described here will stimulate researchers to seek out and develop other applications in order to capitalize on the vast wealth of knowledge that NMR techniques hold for biology. where the proton spectrum of bovine pancreatic trypsin inhibitor is shown.425 (1979). R. C . Res. Ernst. 1. R. It is hoped. We have also decided to forgo any discussion of NMR zeugmatography since NMR imaging studies are highly technical and require specialized equipment. 47 D. P. and mlk are the magnetic quantum numbers of the coupled nucleus 1. Biophys. many excellent and important examples must necessarily be omitted in this limited treatise. Hoult and P. However. ~ ~ This type of information can be used for the unambiguous assignment of resonances from similar groups located in different parts of the macromolecule. Readers wishing to learn more of this most interesting application of NMR to medical imaging are referred to a recent paper by Hoult and L a ~ t e r b u and r ~ ~the literature cited therein. For many years biological chemists have worked at unraveling the molecular mechanisms responsible for enzyme catalysis. Proteins: Probing the Active Site of an Enzyme Practically every chemical reaction occurring within a cell is catalyzed by an enzyme. . Mayn. Bachman.1. Selected Studies on Biological Systems In this section we discuss a limited number of published studies on biological applications of NMR in order to illustrate how the technique can be uniquely useful for investigating biological systems. 86.5. Wiithrich. J j . SELECTED STUDIES ON BIOLOGICAL SYSTEMS 41 ojis the Larmor frequency of j . Unfortunately. allowing investigators to directly observe the effects of spin decoupling on each r e ~ o n a n c e . More recent studies employing both xray crystallography and NMR have also allowed the definition 46 K.5. . The effectiveness of this technique in resolving complex spectra is illustrated in Fig. 17. I. is the spin coupling constant. however.
Roberts. Two schemes have been proposed for the involvement of this triad in the proteolytic cleavage (Fig. 49 . Chem.4~ Since 13C and "N are normally present in low isotopic abundance (1. the enrichment provides an unambiguous assignment of the reasonances arising from this residue. whereas the second approach can be used to reveal the orientation of the substrate when it is bound to the active site of the enzyme. Smallcombe. and J . 100. the chargerelay mechanism depicted in Scheme I is possible only if the pK. value.5. On the other hand. specific enrichment allows for greater sensitivity. 12. R. 18a).9) and then only if the histidyl residue has an unusually low pK. J . of the aspartyl residue is unusually high (6. the pK. In addition to their similar functions. and serine) in close proximity.1. In addition. W. with enzymes and substrates remaining in solution. The first technique allows one to characterize the protein residues at the active site. Richards. Based on the pH dependence of the enzymatic activity (maximum at 6. histidine. For discussions of these methods.8041 (1978). D. Am. Whitaker. Soc. for the histidyl group should be normal. W. D.365 %. because this enzyme has only one histidine residue. H. H. 1.9).42 1. ~ NMR '~~~ studies of this enzyme have recently been reported using histidine enriched in either 3C at the C2 ring position4' or "N for the imidazole nitr0gens. S. In the following section we describe two approaches for using NMR to study enzyme mechanisms. This approach has been used to investigate alytic protease isolated from cultures of M y x o b ~ c t e r . 6. Of course. 4732 (1973). containing the same triad of amino acid residues (aspartic acid. the NMR approach has an additional advantage in that it can be done under physiologicalconditions. respectively). NUCLEAR MAGNETIC RESONANCE of the activesite residues of enzymes and the orientation of the bound substrates. While both xray crystallography and NMR provide detailed information at the molecular level. Hunkapiller. The mechanism of action of Elytic protease is of interest because it is representative of a ubiquitous group of enzymes called serine proteases. In the elegant work of Bachovchin and these investigators exploited the sensitivity of the I5N chemical shift of the imidazole 15N 48 M.9. In some cases. a host of other NMR techniques have been successfullyapplied to the study of proteins. if Scheme I1 is operative.11 % and 0. Bachovchin and J. Two of the major problems associated with using NMR techniques in biology are the low natural abundance of many nuclei of interest and the difficulty of assigning resonances in a complex spectrum. Isotopic Labeling.1. both of these problems can be obviated by incorporating labeled precursor molecules into a biosynthetic system which produces the macromolecule to be studied.1. the reader should consult the biological NMR texts cited in Chapter 1. these proteases share a common type of active site. Biochemistry W.
(iii) the  50 51 52 F. Chem.1. Chem. J. Two interactions can contribute to the nuclear spin relaxation : (a) throughspace magnetic dipolar interaction between the nuclear spin and electron spin .5. Chem. 99.1. Phys. indicate that the histidyl residue does have a pK. Luz and S . resonances to their state of p r o t ~ n a t i o nto ~~ examine the pH titration behavior of this residue.8149 (1977).__ *1=Ir) 1 6 8 I 1 0 I PH ( 0 ) fb) FIG. Phys. SELECTED STUDIES ON BIOLOGICAL SYSTEMS 43 SCHEME I FI k t \ \ \ \ \ \ SCHEME Ii \ \ \ \ tetrohedral \ \ '\ mlermediale 140 4 I . and A. J . (a) Proposed mechanisms for the catalytic activity of the serine protease family of enzymes. thus disproving the popular chargerelay mechanism.49 Copyright @ 1978 American Chemical Society. SOC. Maurer. 1. E. I5Nenriched at N3. 18b. 7. shown in Fig. 40. as well as the extensive application of NMR relaxation t h e ~ r y ~ ' .2686 (1964). . The results of these 15N experiments. respectively.(ii) the binding stoichiometry or coordination number of the electronnuclear complex (4).for ~ ' paramagnetic systems. and (b) isotropic nuclear hyperfine coupling of the unpaired electron at the nucleus under consideration. Connick. Z. Because of the large magnetic moment associated with unpaired electrons (on the order of lo3 times larger than that for protons) the relaxation for any ligand nucleus with I = at the active site will be dominated by the paramagnetic interactions. (b) Effect o f p H on the I5Nchemical shifts of "Nenriched histidine nitrogens in alytic protease: 0 . Reprinted with permission from Bachovchin and Roberts. . Paramagnetic Probes. and H . J. The approach outlined in this section requires that an unpaired electron. be present at the active site where the substrate binds. Ruterjans. from either a paramagnetic ion or a spin label. Blomberg. The longitudinal relaxation rate is primarily determined by five parameters: (i) the ratio of the concentration of paramagnetic species to ligand ( f ) . Am. See text for details.5. J. T. 37. Meiboom. Swift and R.18.2. 307 (1962). W.enriched at N I and N3.
S. (iv) the correlation time modulating the dipolar interaction between the nuclear and electron spins (T. however. frequently TIM9 z M . 19a. The dipolar contribution to TIMprovides a means of determining r. The value of z. In this case the longitudinal relaxation rate has been shown to be (1. (1. The general structure of tRNA is illustrated in Fig. NUCLEAR MAGNETIC RESONANCE lifetime of binding at the active site (z.2. and in this manner the orientation of the substrate relative to the paramagnetic probe at the active site of the enzyme can be ascertained. one determines l/Tlp. z M must still be known. For an enzymebound paramagnetic center. only. one does not measure TIM directly. ’’ A. unlike proteins. is usually large enough so that ~ f z : 9 1 and the contribution from modulation of scalar nuclear hyperfine interaction in Eq. Gupta. the paramagnetic contribution to the longitudinal relaxation rate. provided that the dipolar mechanism dominates the relaxation. and (v) the distance between the paramagnetic center and the nucleus (r).5. A is the nuclear hyperfine coupling constant.). The nuclear spin relaxation is then dominated by magnetic dipolar interaction between the nuclear spin and the paramagnetic center. z. (1.. Mrthuds Enzyrnul.1) Here S is the electron spin.44 1.Note at two frequencies is a function of z.). assuming that t. . Nucleic Acids: The Dynamic Structure of tRNA Transfer RNAs (tRNAs). 1. 322 (1978). from the difference between the relaxation rate measured in the presence and absence of the paramagnetic probe and takes advantage of the following relationship between Tlpand TIM: 1lfTIp = 4/(TlM + TM). p is the Bohr magneton. is probably best determined by ~ ~ that the ratio of TIP measuring T IPat two or more f r e q ~ e n c i e s . all display a similar structure since they all perform an identical function in the process of translating messenger RNA. In a typical experiment. Instead. or the experiments can be done at temperatures where this condition is fulfilled.5.5.5. is the effective correlation time for the scalar interaction.2) Of course. This procedure may be repeated for several substrate nuclei. K.1) may be neglected. 44G. Mildvan and R. g is the electronic “g” factor. the distance between the nuclear spin and the paramagnetic center. can be obtained. and z.
. 52 (1978). 238. Copyright @ 1978 by Scientific American. Rich and S. 100. by courtesy of Marcel Dekker.D &*a A I) " LOOP 1 7 M ANTICODON STEM . H.) The numbered assignments for the cloverleaf (secondary) base pairs correspond from Reid et to the diagram (inset). Inc.I3 12 I1 10 9 PPM FIG. 511 . (a) Schematic representation of the threedimensional structure of tRNA. 19. Inc. Reprinted from A. . Kim. .(a) i LCIOP h i m (b) I SlEhl I _ > CND ACCEPTOR STEM so5. . ! 15 14 . (b) 360MHz proton NMR spectrum of yeast tRNAPhe (Reprinted p." C. A11 rights reserved. . I I I . . Sci. while the resonances from the tertiary base pairs are marked with asterisks. . .. " em \ 9RIABLE I . Am. .
1979. Dekker. The signal intensity is then measured from the frequency spectrum taken after the saturating field has been turned off for various time intervals z. Redfield. However. New York.11 to . 3599 (1977). D. the expotential recovery of the saturated signal can be followed.” (S. Hilton. E. Annu. Lu.55In this method. Biophys. In addition. the rate may be determined from deuterium exchange studies. Woodward and B. that the rate of exchange is greater than the intrinsic longitudinal relaxation rate of the resonance monitored. although features characteristic of the tertiary folding are often discerned54 (see Fig. E. Accordingly. a saturating rf field is imposed at a specific frequency corresponding to the resonance of the specific hydrogen bond to be sampled. p. These hydrogen bonds resonate at very low fields in the ‘H NMR spectrum (.15 ppm from DSS). 4. ’‘ ‘’ . K . the hydrogenbonded protons of a particular kind are spectrally dispersed according to base proximity relationships determined by the sequence. in “ N M R and Biochemistry. 8. Azhderisn. N u c l ~ i cAcids . Rcid. Bincng. The rate of this recovery measures the rate of replacement of the hydrogenbonded proton by the unsaturated water proton. since the nucleic acid bases consist of unsaturated ring molecules which can induce ringcurrent shifts in adjacent spins. eds. the ‘H NMR spectrum provides a fingerprint for the secondary structure of the tRNA. 9 1 . The cross rungs represent hydrogen bonds between complementary base pairs.46 1. Rev. Hurd. G. Initial studies have utilized a technique called saturation recovery to measure these hydrogenbond proton exchange rates. Rcs. J. These proton exchange rates can be an extremely sensitive probe of conformational fluctuations in tRNAs and offer some insights into the pathway of conformational changes that might be followed in tRNA binding interactions in solution or on the ribosome surface. NUCLEAR MAGNETIC RESONANCE It is an Lshaped molecule with an acceptor site for binding the amino acid to be transcribed to the polypeptide during protein synthesis and a recognition site (in the anticodon loop) for binding the complex to the appropriate sequence of messenger RNA. there have been attempts to measure the rates of proton exchange between tRNA hydrogen bonds at specific sites and the solvent. For hydrogenbonded protons which exchange very slowly compared with TI. and R. R. D. following the decrease in signal intensity when D 2 0 is substituted for H 2 0 in the medium. The assignment of the individual hydrogenbond resonances in the lowfield spectrum has received considerable attention in recent years. C.99 (1979). Generally these studies have confirmed the cloverleaf model for the secondary structure of tRNAs. Johnston and A. s 5 P.56 B. 19b). By varying z. provided. Some important insights into the tertiary folding of these molecules in solution have also emerged. Opella and P.). of course.
1) + ‘ 1sin2 e cos 241.4) For a CD bond in an alkyl chain. The results for the case of 2H ( I = 1) are shown in Fig. when the Zeeman interaction dominates over the quadrupolar interaction. A number of NMR techniques have been applied to probe the orientational order and the molecular mobility of the phospholipid molecules in bilayers. + [e2qQ/8Z(2Z . Membranes: The Restricted Motions of Lipid Chains The important role played by cell membranes in controlling biological functions is firmly established. the electric field gradient is essentially axially symmetric about the CD bond.2 x [(3 cos2 e . 20. L. particularly the effects which lipids might exert on the proteins and vice versa. )175. in order to provide benchmark information on this system. Nicholson. not only between the cytoplasm and the extracellular medium. . (1.3) where the angles 8 and r#. but also in compartmentalizing many activities within the cell (e. C . Singer and G .1)][3rn. e2qQ/his typically 170 kHz. The ’H NMR spectrum of a specifically deuterated ” S.1) + q sin2 0 cos 241. that is. These 2H NMR studies take advantage of the quadrupolar interaction of 2H and the manner in which these interactions are manifested in the NMR spectrum of a motionally restricted system. there has been considerable interest in the structure and motional state of phospholipids in bilayer membranes. Science (Washington. The current dogma of membrane structure is the fluid mosaic model. nuclear and lysosomal membranes) while providing a highly specialized surface to promote certain biological reactions.vo = k$(e2qQ/h)[(3 cos2 8 . J.3. and q is near zero. with proteins intercalated into a lipid bilayer matrix.. are given by E(.1.g.5.I) = yhHorn.5.5.” This model raises many questions about the role of lipids in cell structure. SELECTED STUDIES ON BIOLOGICAL SYSTEMS 47 1. The resultant NMR spectrum can be seen to be a doublet displaced symmetrically about the Zeeman frequency by v . They serve as highly specialized barriers. The energy levels of the combined Zeemanquadrupolar Hamiltonian.  Z ( Z + l)] (1. D . To answer some of these questions.5. Of these. 720 (1972).~ define the orientation of the major and minor axes of the electric field gradient tensor relative to the external field. the most informative has been deuterium NMR studies on phospholipids with perdeuterated or specifically deuterated methyl and methylene groups.
. E. Heidelberg. 21b. but it is significantly smaller than the value of 170 kHz expected for motionless acyl chains. NUCLEAR MAGNETIC RESONANCE ml (a) . The effect of motional averaging on the electric quadrupole interaction is usually expressed in terms of an order parameter. is defined in Eq.406 (1974). In a phospholipid bilayer. Res. 21a. SpringerVerlag. Seelig. Commun.+) Zeeman + Quadrupolor YO FIG. SOC. The ’ H NMR spectrum of a powder sample of dipalmitoyl lecithin specifically deuterated at carbon 5 in both acyl chains is shown in Fig.$) . (1. From “Membrane Spectroscopy” (E. 5 9 Note that one frequently deals with unoriented multilayers in membrane studies and only a powder spectrum is observed. Niederberger.+) \___= El= yhHo+$ e2qQx(B.ldideuterooctanol) intercalated into hydrated sodium octanoate and oriented at various angles with respect to the external magnetic field is shown in Fig. the chain motion takes a molecular axis symmetrically about a director perpendicular to the bilayer surface.5. 57. (b) The NMR spectrum expected for a single crystal of a molecule with an isolated I = 1 nucleus at an arbitrary orientation in the magnetic field. SCDshave been measured for methylene segments located at different ’* J. Grell.I).58A quadrupolar splitting is indeed observed. ed. of course. (a) Nuclear energy levels of an I = 1 nucleus in a strong magnetic field.l= yhHo + $e2qQX(B. p.5.I. The angular . The relative contributions to the splitting from the function ~ ( 04) nuclear Zeeman effect and the nuclear quadrupolar interactions are not drawn to scale. +I E~=  $&ox(e. 12. The origin of the reduced splitting is. (1.5) where 8’ is the angle between the applied magnetic field and the director and ScD is the order parameter for the CD bond relative to the director. Seelig and W.3).).. 59 J.48 1. and the ’H quadrupolar splitting may be rewritten as A V Q = $((e’qQ/h)sc~(3 COS2 8’ . Biophys. motional averaging. Chem. 96. Biochem. . Seelig and A .2069 (1974). longchain alcohol (1. .20. __c I_____ . J . Am. \ Q.
2069 (1974).8 MHz) of specifically deuterated fatty alcohol intercalated into hydrated sodium octanoate and oriented at various angles with respect to the magnetic field. Reu. Soc. From these data the motional state of the phospholipid bilayer membrane can be quantified and expressed in terms of the flexibility profile. 10. C. Chern. 1. 6 1 J. The angle 0' is between the magnetic field and the bilayer normal. Seelig. 353 (1977). Q. F. The interested reader is referred to a review by Seelig6' and to the Specialist Periodical Reports on NMR published by the Chemical Society. C. Copyright 1974 American Chemical Society. as depicted in Fig. Polnaszek.1. 22. .4. Biochemistry 15. (b) 'H NMR spectrum (13. Biophys. A. SELECTED STUDIES ON BIOLOGICAL SYSTEMS 49 . (From Seelig and Seelig. Am. Temperature 60°C. and I. Smith. F. 96. 10KHz .60 'H NMR is now widely used in biophysical studies of biological membranes. Stockton. J .5. FIG. Intact Cells: Metabolism Studied in Vivo Despite what may have appeared to be insurmountable difficulties involved in utilizing NMR as a tool for biological studies. P. Tullock. W. Reprinted with permission from Seelig and Niederberger.8 MHz) of a powder sample of dipalmitoyllecithin specifically deuterated at carbon atom 5 in both chains.59) positions along the hydrocarbon chains of a phospholipid bilayer. investigators have G. The sharp signal in the center of the spectra arises from a small fraction of molecules which form an isotropic phase when the oriented multiplayers are prepared.5. (a) 'H NMR spectra (13. 954 (1976).21. P. Hasan.
The great usefulness of phosphorus NMR in studying cell metabolism arises from the fortuitous combination of its nuclear magnetic properties and its presence in highenergy substrate molecules. In this section we briefly review two recently published studies in this area. We view the outlook for exciting breakthroughs and giant strides in this field with some degree of optimism.5. being overcome. has . NUCLEAR MAGNETIC RESONANCE 35 r 30 t 0 0 250 20 QVQ (kHz1 1 5 0 0 0 0 0 0 Carbon Number FIG. but also presents interpretational hurdles. 31PNMR. are gradually. and tissues and organs not of interest. 1. Both of these studies have used NMR to characterize the metabolism of living cells. Such work involves not only the immense technical challenge of maintaining the sample under conditions compatible with life. Quadrupolar splittings from specifically deuterated steatic acid in egg yolk lecithin dispersions plotted as a function of the hydrocarbon chain position.22."" persevered to the point where today living tissues and whole animals are finding their way into the spectroscopists' magnetic field.50 1. if only slowly. such as those involving magnetic field inhomogeneity caused by air bubbles.4. interference from resonant nuclei located in organelles. Data taken from Stockton et a1. many problems.1. 31Pis an abundant nucleus (natural abundance of loo%). However. even when the data are successfully gathered. but they illustrate the diverse range of problems the technique is capable of tackling.
G .5. stimulated for 1 sec every 60 sec and recorded at the time intervals indicated. inorganic phosphate displays a characteristic pHdependent "P chemical shift62which can be used to simultaneously determined pH changes within the cell medium during metabolic studies. Gadian. R. (London) 267. J. Dawson. To illustrate the utility of 31P NMR for monitoring metabolic changes. Wilkie. Wilkie.~ o r k e r s . D. we cite the recent studies of Dawson and c o . SELECTED STUDIES ON BIOLOGICAL SYSTEMS 51 FIG. In addition. with short correlation times and concomitantly long relaxation times. render 3 1 P NMR an ideal tool for the study of cell metabolism. 8. 862. These properties.1. D. From Dawson e t a / . J. Dawson. Richards. and D. Moon and J. H. together with the fact that metabolically important molecules bearing 31P are usually small. 248. 7276 (1973).703 (1977) h4 R . a spin of ). J . Chrm. 861 (1978). B i d . Physiol. In b2 '' M . and exhibits a large chemical shift range. (d) Decline in the isometric force development of the stimulated muscle during the time period of the experiment. J . and D. Nature (London) 274.23. (a)(c) "P NMR spectra from anaerobic frog gastrocnemius muscles. . M. p. R.These ~ ~ * investigators ~~ were interested in delineating the chemical changes occurring in frog muscle as it fatigues due to repeated contractions under anaerobic conditions. Gadian. G. 6 4Reprinted by permission from Nature 274. Copyright @ 1978 Macmillan Journals Limited.
Ugurbil. G . Acknowledgment This work was supported by USPHS NIH Grant GM22432 and American Cancer Society Grant PF1628. U. A . . A.^^ Taken together these results established an unambiguous correlation between muscle fatigue and the energy state of the cells. This approach may also prove fruitful in unraveling the origins of various metabolic diseases. K . Acad. using [l”C] glucose and [6’3C] glucose as substrates. stimulate. In addition. 6 5 J. and determine the strength of muscle contraction. while simultaneously using 31PNMR to record the level of various metabolities as well as the cellular pH.1 we noted the great advantages of enriching an isotopically rare nucleus at a welldefined position in a protein. 76. and R. This approach has recently been utilized by den Hollander et aL6’ to investigate anaerobic glycolysis in yeast cells. Although the NMR experiments permitted only the direct measurement of ATP. S. NUCLEAR MAGNETIC RESONANCE their pioneering experiment. 1. Because of the isotopic enrichment they were able to obtain wellresolved spectra at oneminute intervals. this method can be useful in following the fate of a labeled substrate molecule as it is metabolized within a cell.52 1. 23. and inorganic phosphate (Pi) levels from the observed peak areas in the 31P NMR spectrum and the cellular pH from the inorganic phosphate chemical shift. and lactic acid. Brown. Natl. Shulman. T. they suspended the muscles in a specially designed chamber which allowed them to perfuse. Some of their data are reproduced in Fig. R.6bisphosphate.2.5.5. In Section 1. based on their quantitative relationships to the directly measured metabolite^. the production and consumption of the metabolic intermediate fructose 1. it was also possible to infer indirectly the concentration of creatine. Thus by exploiting NMR as a noninvasive probe one can investigate reaction kinetics and the stereospecificity of enzymes in their natural environment. Sci. the use of 13Cglucoselabeled at the 1 or 6 carbon made it possible to follow the knetics of label scrambling caused by the enzymes aldolase and triose phosphate isomerase.4.1. Proc. 3CLabeled Substrates. den Hollander. phosphocreatine (PCr). The successful application of NMR in studies of this type shows great promise for future investigations into the direct effects of various metabolites and antimetabolites in v i m . This capability allowed them to directly measure the rates of consumption of glucose. free ADP. and the production of the fermentation end products ethanol and glycerol. Similarly.6096 (1979).
with a view to discovering selection rules which control immune responses to membranes.1. They aspire to the eclectic and are especially dependent upon the lines of communication between themselves and the specialists in areas of their interest. there is no room for esoteric science. Inc All rights o f reproduction in any form rrsrrved. Guided by this belief. This latter phenomenon should not be surprising because many such boundaries are not natural but exist as a convenience to scientists and librarians. McConnell 2. physicists. but has not been published previously.2 spinlabel theory is presented in a simplified form with the aid of copious diagrams rather than mathematical equations. it is vital that information be communicated in a manner which is palatable to biologists. Those who engage in highly interdisciplinary science are usually searching for unifying principles rather than specifics. at two levels. If biophysical chemistry is to be effective and intellectually stimulating. This method has been used many times by Humphries to present spinlabel theory to medical and biological scientists. Boundaries are no longer convenient if they are allowed to become barriers to creative science. Our thesis is that the physical state of antigenic determinants associated with membranes must have a marked effect on afferent and efferent immune responses to those membranes and that the use of determinants capable of providing direct information regarding their physical state. NITROXIDE SPIN LABELS By Gillian M. Introduction Systematic natural science has advanced by means of two interdependent processes. In Chapter 2.the former being concerned with how things differ from one another and the latter with how they are the same. which is a review of recent work in the McConnell laboratory concerned with using spin labels as antigenic determinants. but also for the increasing difficulty with which we construct boundaries between “different ” scientific disciplines. as do spin labels. specialization and generalization . ISBN 0124759629 .2. 20 Copyright 0 1982 by Academic Press. we have taken the unusual step of presenting a physical method having great realized and unrealized potential in biophysical applications.4. VOL. The recent course of science has been remarkable not only for its tremendous growth. is essential in 53 METHODS OF EXPERIMENTAL PHYSICS. K.2 will enable any scientist to follow Chapter 2. Humphries and Harden M. We hope that Chapter 2. and chemists alike.
J . either with respect to work from this laboratory or from others. ed. SpinLabel Theory: A Descriptive Treatment 2. 161 (1974). rate of motion.. 267 (1972).’’’ However. 1.” Wiley. Bolton. EPR enables us to reach conclusions regarding the distribution. New York. Methods Enrymol. Likhtenstein (P. 83. 1976.” New York. H3CQH3 H3C 0 2. Dwek. “Biological Applications of Electron Spin Resonance. NITROXIDE SPIN LABELS any study designed to test this proposition. Biomembranes 3. We also hope that Chapter 2. B. and refer the reader to other recent monographs or reviews which accomplish that purpose. M.4dimethyl2. in “Structure and Function of Biological Membranes”(L. R. Fundamentals Including the Resonance Condition The essential structure of the type of spin label discussed here is a nitroxide group adjacent to two tertiary carbon atoms.1. Shelnitz. p.(3. M. 214 (1971).Carboxypropyl)4. J. J.. Waggoner. G. Adz$. We do not endeavor to provide an encyclopedic review of the use of spin labels. 1973. 1971. Relaxation Processes 4. C. 32B.tridecyl3 oxazolidinyloxyl L.2. R. A. S. London and New York. R. H. Cliem. L. Dwek. Arc. F. our discussion will touch upon certain of the most common uses of spin labels. Reuben. Berliner.). New York. orientation.6.2. “Spin Labeling Methods in Molecular Biology. The choice of this topic allows us to illustrate the main features of electron paramagnetic resonance (EPR) spectra of nitroxide spin labels.Mol. A. ’ ’ . and 0.3. Rothfield. Berliner. Academic Press. Press. In brief.). ed. P. Grifith. Gaffney.2. Res. and dielectric environment of spinlabel antigenic determinants. trans. Swartz. Cohn and J . 1976. New York.1 oxyl I CH3 (TEMPO) 2. “Spin Labeling.) in Biochemistry. Seelig. H. eds. Methods Enzymol. Jost.2 will provide an easily read introduction to spinlabel theory for those who wish to pursue the subject in greater depth by reading the mathematical treatment written by McConnell and presented in Chapter 2. Theory and Applications. Borg. 49G. 4. 1. and D. 2. Examples are shown below. C.6Tetramethylpiperidine. “Nuclear Magnetic Resonance (N. R. Such a molecule is host to an unpaired electron: it is a stable free radical and therefore paramagnetic.54 2.” Academic Press.” Oxford Univ. J . 418 (1977). J . M. 1972. A. I (1972).
Similarly. The figure is drawn to indicate that the unpaired electron is solely associated with the nitrogen atom. it is also significantly associated. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 55 Such molecules are stable in the p H range 3.2. also indicated in the diagram. with the oxygen atom. occupying a 2p n orbital.OL . 1 and legend).?.to )( 0 I FIG. The conventional assignment of z and x axes is given. Diagrammatic representation of the nitroxide group in a typical spinlabel molecule. 0 OH I The products are no longer spin labels because they do not contain unpaired electrons. chemical reduction by appropriate agents such as ascorbate or glutathione destroys the paramagnetic properties of spin labels as shown below. This is an oversimplification. it is partly governed by the y 3 CN?. t h e y axis is normal to the page. so generating the charge separation indicated in the diagram. This leaves a single unpaired electron.2. This sharing has a concomitant effect on the extent of charge separation (also indicated by the structural representation). If the carbon atoms adjacent to the nitrogen atom are not tertiary. disproportionation takes place as shown below. but to a lesser extent. In simple terms one may consider that two of the five nitrogen valence electrons are required for the formation of the two CN bonds (only one of which is shown here) and two form a coordinate bond with the oxygen.10.1. in fact. It is helpful to look at a structural representation of the important end of TEMPO in order to understand why it is a free radical (see Fig. 0 OH Certain oxidizing agents and photoinduced addition reactions can also destroy the paramagnetism. which is predominantly associated with the nitrogen atom. .
4.” designated +$ and 9. One consequence is that the diflerence AE in energy levels between the ++and spin states increases with the strength of a magnetic field in which the molecules are placed (see Fig. on the energy level E of the permitted orbitals of unpaired electrons having spin states S of +f and +. The unpaired electrons exist in one of two possible “spin states.3. . (The population difference at equilibrium is controlled by the temperature and the difference between the energy levels ofthe two spin states. it demonstrates appreciable interaction with any magnetic field to which it is subjected. 2).56 2. by unpaired electrons not influenced by nuclear hyperfine splitting. (So long as the EPR spectrometer is adjusted correctly. If an electron is not paired to another of equal and opposite spin in the same orbital. (a) Effect of external magnetic field strength H . there will be a population difference.2. (b) Diagrammatic representation of resonance: absorption A of microwaves of frequency v as a function of field strength H .) In a manner analogous to the induction of a magnetic field by an electrical current passing through a coiled wire. As a result. This is covered in detail in Section 2. they can be thought of as little bar magnets. unpaired electrons in the ++state being slightly in the minority with respect to those in the state.2. it may absorb it and assume the state. the rate at which +fstate electrons revert to 4 electrons by “spinlattice + 4 4 ++ FIG. If an electron in the $state is supplied with a quantum of energy exactly equal to the energy level difference between the two spin states. with the microwave power set low enough. This is the condition of resonance and is the essential step for generation of EPR spectra of the type which we consider here. NITROXIDE SPIN LABELS dielectric properties of the environment and is an important factor leading to certain spectral changes which we discuss in Section 2. spinning electrons are each associated with their own magnetic moment. The energy levels of these two spin states are not identical in the presence of an applied magnetic field ( 4 being higher than 4). The energy of a quantum of electromagnetic radiation is given by its frequency v multiplied by Planck’s constant h. for a sample of spinlabel molecules at equilibrium.2.
2. 3. 9. are illustrated in Fig. a machine which is designed to detect the resonance condition.3.3. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 57 relaxation” is fast enough that the system does not “saturate.) The principal features of an EPR spectrometer. . Principal features of an EPR spectrometer. quanta ofenergy susceptible to absorption by the unpaired electrons of spin labels are associated with CIRCULATOR KLYSTRON DETECTOR L 1 ? I AMPLIFIERS AND P H A S E SENSITIVE DETECTION SYSTEM VARIABLE POWER SUPPLY FOR MAGNET J 1 MODULATION UNIT XY RECORDER d F I E L D SCAN U N I T r I I FIG.” This is covered in detail in Section 2.2. The axes are the same as in Fig.2. If the spacially fixed magnetic field is sufficientlystrong.
The reason why nitroxide spinlabel spectra are interesting is that the unpaired electrons are strongly influenced by the nitrogen nuclei. and the wavelength of electromagnetic radiation (supplied by a klystron) is kept constant at approximately 3 cm (i.” a carefully engineered structure suspended between the poles of the magnet with dimensions appropriate to the wavelength. which have three possible A + dA d 7 FIG 4 (a) Effect of external magnetic field Ha and spin state of nitrogen nuclei ( I = + I . 0. . or dielectric environment of the spin labels (see Fig. In summary. 2b). If the only relevant facts were those which have already been presented. in the “microwave” range). Typically. EPR spectrometers are usually designed to measure the absorption of microwaves (supplied at a fixed frequency) as a function of increasing (or decreasing) magnetic field strength.58 2. which would vary little in shape or position with changes in motion. The spinlabel sample is placed within the cavity and its absorption of a portion of the microwave radiation which would otherwise be allowed to leave the cavity is responsible for the signal detected by the spectrometer. NITROXIDE SPIN LABELS electromagnetic radiation of a convenient wavelength. a measurable net absorption of microwaves by the sample can occur when this allows electrons associated with spin labels to move from the lower permitted energy state to the higher. the strength of the spacially fixed magnetic field is varied at around 3000 G. we would expect a field versus absorption spectrum showing a single narrow absorption peak or “line”: a very boring bump.e.. orientation. for the spinlabel work. This is experimentally convenient because the radiation is supplied as standing waves in the “cavity. +) (b) Absorption A of microwaves of frequency Y ds a function of field strength Ha by unpaired electrons an example of nuclear hyperfine splitting (c) Firstderivative spectrum taken from (b) “6 .I ) on the energy level E of permitted orbitals of unpdired electrons (spin states S = ++. distribution.
However. corresponding to the + 1. This is convenient for the instrument's design and because firstderivative spectra provide information in a more measurable form than absorption spectra do. the CNO angle being a function of the particular nitroxide molecule considered.. but as the first derivative of the absorption with respect to field strength d A / d H .e. as 7 there is a small difference between the splittings when H . versus H . [In fact. Effect of Orientation Figure 1 illustrates the disposition of the 2p n orbital of the nitrogen atom. and the minimum ' . EPR spectra are usually not recorded as microwave absorbtion A versus magnetic field strength H .0.2. the maximum value for the hyperfine splitting between adjacent peaks is known as IT.and two tertiary C atoms are placed in a single plane in the diagram although this is not usually strictly correct. This shows that resonance conditions are obtained at three different positions of the field sweep. because the electron is partially shared by the oxygen atom. with which the unpaired electron is predominantly associated.. This phenomenon is an example of nuclear hyperfine splitting.2. absorption spectra are easier to understand theoretically. and the conventional manner of assigning Cartesian coordinates to the molecule.. As a simplification. the N.l). is parallel to x and when it is parallel to y.2. onethird of all the unpaired electrons in a spinlabel sample are associated with nitrogen nuclei in each of the three spin states. but usually it is sufficient to consider an average of the . 0. 2. and we therefore continue to use them as models in the following discussion. In actual practice. (as shown in Fig. This is a consequence of the fact that the extent of splitting is positively correlated with the influence of the nitrogen nucleus on the electron. . is governed by the orientation of the spinlabel group with respect to the direction of H.2. Because of the anisotropic disposition of the unpaired electron (i. If the orientation of such a crystal in the cavity of the EPR spectrometer is rearranged in many different ways.1 spin states ofthe nitrogen nuclei. 4. the observed hyperfine splitting (the distance between the absorption peaks) changes such that it is maximal when the external field H . this is maximal when H . .0. in the p orbital) its interaction with the large external applied magnetic field H . and therefore exhibit magnetic properties themselves. (as shown in Fig. Therefore. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 59 spin states (+ 1. The effect on the permitted energy levels of the electrons is illustrated by Fig. 4c). is parallel to the z axis of the nitroxide group and minimal when it is perpendicular to the z axis. 4b). Spin labels have been oriented in single host crystals so that all the spinlabel axes are aligned the same way with respect to the crystallographic axes. Because the populations of nitrogen nuclei in each spin state are approximately equal under normal experimental conditions. and z (the long axis of the 2p n orbital) are parallel.
. the hyperfine splitting values are intermediate. If we assume that the various positions indicated by the lines in Fig.. 5b are all occupied by spin labels but. 0 is the angle between the xy plane of the nitroxide groups (see Fig. ( 2 ) The curve associated with the outer lines has been removcd. we consider labels with similar positions as groups or “packets” k‘rc.] Figure 5a illustrates the true change in hyperfine splittings and line positions as a crystal is rotated about the spatially fixed external magnetic field axis H. i.5). for further simplification. with concomitant shifts of the outer lines. 1 ) and H . (a)Truechangein nuclear liyperfincsplittingsand linepositionsasacryslalcontaining oricnted nitroxide spin labels is rotated about a spatially fixed external magnetic field of variable strcngth If. the signal generated by such a sample would be a composite of contributions from the randomly oriented labels.3 but need not be of concern for the purpose of the present discussion.. Note that the center line is equidistant from the two outer lines at all values of 0. is parallel to any line drawn in the xy plane. Figure 5b is a simplified version which approximates to Fig.e.3. For orientations of spinlabeled crystals other than H . (1) The “ y fiictor. 5a and allows us to present a version of spinlabel theory without recourse to mathematics. when 0 = 90” or 90” the z axis of thc labels is parallel to the magnetic field. Iz . If we were to take the carefully prepared crystal that we have been discussing and crush it to a powder.” is shown to be constant (see Section 2. (b) Simplified version of(a). This is nut the true case because orbital magnetic contributions by the electrons cause variation of y with orientation and give rise t o the nonlinearity of the center line in (a). Two simplifications have been madc. . . It is also true that there is another shift in the position of the set of all three lines (as a group) when the crystal is rotated.” which is discussed in Chapter 2. NITROXIDE SPIN LABELS values obtained when H . 11 z or H . and when 0 = 0”it is perpendicular. This is due to a change in the “g value..60 2. 5.
(b) Superimposed absorption spectra of all packets of labels shown in (a). 5b but indicating anoncontinuous distribution of labels as an aid to interpretation. the EPR spectrometer records spectra not as field versus A . 6d) of the lowfield shoulder shown in the composite absorption spectrum (Fig. The firstderivative spectrum (Fig.). we arrive at the prediction illustrated by Fig. be used to determine both T. therefore. (Fig.  "0 FIG. 6c) has the same shape as the absorption spectrum of the packet of labels with z /jH.8. These field positions are those at which A for that particular packet (i) commences to increase and (ii) is at its maximum value (see Fig. as shown in Fig. 6b). (c) Composite absorption spectrum of all packets of labels.2.6. This fact is also derived mathematically in Section 2.2. both commence at the same lowfield position and achieve their maximum value at the same position. etc.e.and TL. 6d. the spectrum may.. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 61 which generate absorption spectra of fairly narrow width.. H . 6b). . that is to say. (a) As Fig. If we consider the lefthand (lowfield) side of the composite absorption spectrum (Fig. versus d AIdH. This shows that a powder spectrum is related in a very interesting way to spectra derived from single crystals rotated relative to H o . 6. obtained by adding the individual spectra shown in (b). The composite firstderivative spectrum may similarly be used to detect contributions from the packet of labels having their z axes perpendicular to H. (d) Firstderivative spectrum taken from (c). is responsible for the field positions at which C A commences to (i) increase increasingly rapidly and (ii) increase less rapidly. but as field versus the first derivative of A (i. Simplified version of the generation of a powder spectrum. However. See Fig.3. 6c) we see that the packet of labels that includes those with z 1) H . 4 for use of symbols..
Even if labels are moving. characteristic data gathering interval (sec). (b) The firstderivative spectrum of the microwave absorption of the packets of labels. Diagrammatic representation of the effect of isotropic motion on the spectra of isotropicdispersions of spin labels. The values for maximal and minimal splitting(Ti and T’J are respectively smaller and larger than those observed for powder spectra (TI and T. at some time during the data gathering interval. [It should be noted that for an isotropic discorrclation timc of the molecule is I tribution there are. labels which alter their orientation appreciably during this time interval are detectcd via their absorption at an average H . effectively twice as many labels oriented with z l H . . 6d is derived using several simplifications which are describedin the legends to Figs.62 2. at any time. 5 and 6. 6d and 13 should be made.). We then say that the rotational sec. (c) and (d) are analogous to (a) and (b) respectively and illustrate the case when motion is so rapid that the oricntation excursion during sec is sufficiently complete as t o nullify all contributions due to orientation detectable by this method. Thc “strings of beads” represent packets of labels that absorb at approximately equal average values of H . 2. at the orientation 8. as there are labels oriented with zJiH. labeled 0 = +90 to 90.. . The EPR spectrometer has a characteristic time interval for receiving data which can be likened to the shutter speed on a camera.3.) and may be compared with q. relates to these. (a) The tilted Vshaped solid lines to either side are the nuclear hyperfine splittings appropriate to zero or very slow motion (see Figs. not TI^ + TJ as suggested by this diagram which does not take this “weighting” into account. appropriate to their orientation excursion during sec. and the ordinate. a “powder spectrum” will be obtained if the motion is very slow relative to the instrument’s data gathering interval Ho HO * FIG.Effect of Motion The individual spin labels in the powder sample discussed above are essentially motionless. 0 being the angle between the x y plane of nitroxide groups and H . shown in (a) as a function of H .7. to observe similarities and differences between the true and synthetic spectra. . NITROXIDE SPIN LABELS It should be borne in mind that the spectrum shown in Fig. and TL for calculations regarding the motion of spinlabeled molecules. A comparison of Figs.]   . 5 and 6). Because the instrument has a fixed. and were. Therefore the value for the observed splitting in the true case of rapid motion is f(T11+ 2T.2.
). It is sufficient for the present discussion simply to say that the higher the electron density on the nitrogen nucleus.l o p 9 sec). If. a packet of labels might be oriented with z [I H . for example. Shimshick and H . Am.7297 (1971). relative to those obtained from powder spectra. at the beginning of a data gathering interval. with decreasing motion the amplitudes of the high. motion is not sufficiently fast to give rise to a spectrum in which the splittings are completely averaged. M. J. McConnell. 46. McConnell. 93. spin labels are moving so fast that their rotational correlation time is equal to.4. and the nitrogen nucleus’s share of the unpaired electron increases relative to that when the spin label finds itself in an apolar. *’ W.. “hydrophobic” environment. J . Morrisett and C. A.. but yet it is impossible to observe measurable maximum and minimum splittings. this being the weighted average of splittings appropriate to the various orientations of z with respect to H . $(TI1 + 2T.” Frequently. M. Chem. Am.7. . various degrees of mixing of splittings become apparent. Effect of the Electrostatic Environment In environments having high dielectric constants (i. Changes in observed maximum and minimum splittings.and lowfield peaks decrease relative to the center peak. or shorter than. when spinlabel lipid haptens report the “melting” of lipid bilayers. Res.e. For intermediate cases.2.3. Chem. have been used to obtain absolute or relative values for motion. 93.4. Soc. 11). 314(1971). Soc. D. This phenomenon is responsible for the effect discussed in Section 2. Commun.4. Similarly. Wellknown applications include determination of the rotational correlation times of spinlabeled proteins” and the measurement of the “order parameter” of membranes using spinlabeled fatty acids or phospholipids labeled in a fatty acid side chain. Broomfield. sec. This phenomenon has a marked effect on EPR spectra.e. In such cases one can take advantage of the relative amplitudes of the various peaks in order to obtain information regarding motion13. charge separation is favored. isotropic spectrum the amplitudes of all three peaks are almost equal (see Fig. 7 and 11 and also Section 2.2. for a true fast motion.2. polar or “hydrophilic” environments) the polar character of the NO bond is encouraged.)]. l3 I‘ E. Fig. J . 2.321 (1972). the bond lengthens. but will have rotated sufficiently by the end of the interval that the recorded hyperfine splittings of those labels are less than the maximum value observed in the absence of motion. Hubbcll and H. L. [i. labels oriented with z IH o at the beginning or at some other time will give rise during the data gathering interval to splittings great& than the minimum value observed in the absence of motion. 18. J . Such spectra are termed isotropic or “fast tumbling” (see Figs. however. a single value for hyperfine splitting is observed. Biophys. Biochem. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 63 (.
NITROXIDE SPIN LABELS "0 FIG. McConnell.. 6 and 7. Linden.3) This is the basis of a phenomenon described in Section 2.6 and illustrated by Fig. Ll.64 2. The compound TEMPO partitions between aqueous and lipid phases with a coefficient which is a function of the fluidity of the lipid phase. 2271 (1973). Acad. 8. In both environments. (b) Total absorption. A. H. (a) The timeaveraged nuclear hyperfine splitting (see Fig. McConnell.A . of all packets of labels shown in (a) as a function of H. and C. 7c) of TEMPO in the lipid.4. phase. ' ~illustrated ~'~ by Fig. Proc. 70. J. M. or polar. l4 l5 . K. Biochrrnistry 12. Note that the signal h is derived from T E M P O in the hydrophobic phase and the signal pis derived from TEMPO in the polar phase. M . Shimshick and H. C. 8. polar environments increase splittings. Fox. Wright.. apolar environments decrease splittings. derived in a manner analogous to that shown in Figs. F.(c) First derivative spectrum taken from (b). (For further discussion see Chapter 2. the spin labels are in rapid isotropic motion and the amplitude of the firstderivative signals (2h and 2 p ) are approximately proportional to concentration. or hydrophobic. S r i . 17. The highfield lines of these two spectra are sufficiently well resolved that they can be used to calculate the relative amounts of TEMPO E. Diagrammatic representation. S. 2351 (1973). the greater is the influence of the nucleus and therefore the larger the hyperfine splittings. Nor/. It is also the basis of a wellknown method for detecting temperatureinduced phase transitions in lipid bilayers or cell memb r a n e ~ . The observed EPR spectrum is a composite of that generated by TEMPO dissolved in the aqueous (polar) phase and that generated by TEMPO dissolved in the lipid (hydrophobic) phase. phase and the aqueous. of a phenomenon which forms the basis of a wellknown method for determining lipid phase transitions. In other words.
SPINLABEL THEORY : A MATHEMATICAL TREATMENT 65 in the two phases. As is the case when the lines are broadened by a decrease in motion. as it will be in the case of lowmolecularweight spin labels in lowviscosity solvents at concentrations 2 1 mM. the orbitals containing unpaired electrons overlap sufficiently that there is a high probability that the electrons will exchange orientations. however. If the second label is tumbling very rapidly and isotropically around the first. The g shift is down field for apolar environments. thus averaging the effects of the two nuclei. If. this spin exchange has a marked effect on the spectra. An example of this effect is illustrated by Fig.2. the second label is moving slowly.3. Effect of Distribution or Concentration of Spin Labels: DipoleDipole Interactions If a certain spinlabel molecule is close to another but spin exchange does not occur.5. we consider spinexchange broadening first. If labels collide.1.) 2. broadening by spin exchange also results in a decrease in amplitude of the firstderivative spectrum. (See also section 2. 22. All three lines are broadened to a similar extent and eventually a single absorption peak is observed when the concentration is raised to permit such rapid exchange that the effects of the three different nitrogen populations are totally averaged out. or nonisotropically. so that the lowfield lines of the two spectra overlap extensively but the highfield lines are better resolved than they would otherwise be.6.3. The Resonance Condition The term “magnetic resonance spectroscopy” has usually been applied to those forms of spectroscopy i n which the frequency of radiation that is absorbed or emitted is nearly or exactly proportional to the strength of a . again broadening the peaks of the spectrum and decreasing their amplitude because an element of inhomogeneity has been introduced into the magnetic environment. (The resolution is aided by the fact that there is a “g value” change affecting the position of the set of three lines. its magnetic field will affect that of the first spin label.3.2. it may still be affected by this second label in another way.Exchange Broadening There are two ways in which EPR spectra are affected by the distance between individual spin labels. This is covered in more detail in Section 2. 2.2. SpinLabel Theory: A Mathematical Treatment 2. If the collision frequency is sufficiently high.5. The ratio may change dramatically at temperatureinduced phase transitions.3. the direction of its magnetic field is totally averaged and cannot affect the first spin label.12.3.) 2. Effect of Distribution or Concentration of Spin Labels: Spin.
1) where h is Planck’s constant. Zavoisky. Fizrol. Zavoisky.3. and the radiation frequency is in the 900035. NITROXIDE SPIN LABELS laboratory magnetic field acting on the sample.R.S.000 MHz range. In organic free radicalst the orbital magnetic moment is largely quenched and makes only a small contribution to the total magnetic moment.16.2) is often written in terms of the Bohr magneton 8. one giving an orbital magnetic moment. t Except for diatomic CH.3. v is the frequency of the radiation that is absorbed (or emitted).2) The charge on the electron is e (e = 4.10. Equation (2. in 1945.4) ” E.8 x 10. 181. 9.2)in terms of the absolute value of 8. Zb. The quantity g is the “spectroscopic splitting factor”.3.000 G range. (2. (2. In electron magnetic resonance spectroscopy. and c is the velocity of light. (2. .l o em). This magnetic moment is parallel to the vector spin angular momentum Sh. 9. in order to avoid any uncertainty in algebraic sign: p If’ = 9l8lS. (2. An electron can have two kinds of circulating motion.3..66 2. The absorption of radiation by a sample placed in a magnetic field can be discussed in terms of the equation hv = BE. 2a). E. AE is exactly or nearly proportional to the strength of the applied laboratory magnetic field H. and the other giving a spin magnetic moment. and AE is the separation of two energy levels.S. p = g(e/2mc)Sh.3) We shall write Eq.3.3. Here h is Planck’s constant divided by 2n and S is the spin angular momentum in units of h. 211 (1945).0023 when the orbital contribution to the electronic magnetic moment is zero (complete orbital quenching). at least for large fields (see Fig. A circulating motion of the electron gives rise to a magnetic moment. 245 (1945). Electron magnetic resonance spectroscopy is also called paramagnetic resonance spectroscopy.”) An electron is a negatively charged particle. Fizrol. m is the mass of the electron. the applied fields are usually in the 3000. Zh. The electron spin magnetic moment is a vector and is denoted by p. where 8 = eh/2mc. The electronic magnetic moment p and the spin angular momentum Sh are parallel to one another. g = 2. (2. (Electron paramagnetic resonance was discovered by Zavoisky in the U.
= 3 + S .3.. and thus there are two energy levels. (2. A magnetic dipole moment p interacts with an applied field vector H. According to Eq.3. (2. (2.92731 x erg/G. (2.3. as sketched in Fig. Incident radiation of the proper polarization and frequency then stimulates more upward ( S .3. Most organic free radicals have g factors that are approximately equal to 2. = +$). This is electron magnetic resonance.6) If the applied field is in the z direction. From Eq. S . paramagnetic resonance absorption is observed at an applied field H.. then the energy E can be expressed in terms of S. with an energy E. resulting in a net absorption of radiation.3. = 4) and E(S.3. = +$. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 67 The negative sign arises because the charge on the electron e. The conditions for resonance given in Eq..3. The numerical value of is 0. Most commercial paramagnetic resonance spectrometers operate in this frequency region.3. 2.9) (2. of approximately 3370 G.E(S.3. = $) than in the upper energy state (S. where E thus.1.8) hv 9lPIHo. = ++3 S.8) is also often expressed in terms of the magnetogyric ratio y : 2nv or (3 = = yHo (2. = +$) .5) = gl/?lS * H.10) yHo. When a sample containing unpaired electrons is at thermal equilibrium there are more electrons in the lower energy state ( S . the frequency of the radiation v that can produce transitions between two energy levels E(S.8) it follows that for radiation of frequency v = 9.5 kMHz. 2. = +$) is hv = E(S.11) .7) (2. = 1 2) transitions. This frequency region is often termed “ X band” by microwave spectroscopists. = ++)than downward ( S . E = p*Ho. where Y = slPl/h. (2. = = 3). is negative.2. (2.3. microwaves) of the proper frequency and polarization. Transitions between these levels can be induced by radiation (eg.3.
The radiation can thus be thought of as being “stored” in the cavity. and into the cavity.16 cm.68 2. NITROXIDE SPIN LABELS The magnetogyric ratio is the ratio of the magnetic moment of the electron ( g l p l S ) to its “mechanical moment” or spin angular momentum (Sh). whose internal dimensions are usually of the order of magnitude of this wavelength. the frequency of the radiation is normally kept fixed and the resonance absorption detected by changing the strength of the applied field. as discussed below in terms of the Bloch equations (Section 2.3.8)] is met. through an iris.3. In free space. radiation is absorbed by the sample. the resonance cavity can be thought of as functioning in the following way. The resonant frequency v can be regarded as a classical Larmor frequency of the electron magnetic moment. some of the electrons (nl) will be in the lower (“spindown”) energy level. that is. 2. radiation having a frequency v = 9.3.3. Radiation of this wavelength can be conveniently handled by means of microwave tubing and resonance cavities. In this case the radiation is reflected back and forth many times in the cavity before either being absorbed by the sample or the walls.3. the number of photons taken from the microwave field by the paramagnetic sample when the fieldfrequency ratio corresponds to thc resonance condition.2. and the remaining electrons ( t i ? ) will be in the upper energy level: N = t1J + t~?. (2. Paramagnetic resonance absorption is observed when the field of the electromagnet is changed until the resonance condition [Eq. In the simplest possible terms. In the case of the commonly used reflection cavity. or being lost by going out through the iris and back up the waveguide.12) . Note that in an experiment of this type. this absorption is detected by a decrease in power leaving the cavity. In the presence of a uniform magnetic field of strength H . where the electron spins interact with each other only very weakly compared to the thermal energy kT.). Absorption Probabilities and Paramagnetic Relaxation In the above discussion we have considered briefly the fieldfrequency condition for paramagnetic resonance absorption. 3. Let us now discuss the intensity of the resonance absorption. . A paramagnetic sample whose resonance is to be studied is placed in an appropriate holder in the center of the cavity and the cavity placed between the poles of an electromagnet.5 GHz has a wavelength i l = c/v = 3. At resonance. Consider a system of N electrons. microwave energy from a source passes down thc waveguide. This particular type of resonance cavity has a length which is equal to the wavelength of the microwave radiation. (2. The principal features of an EPR spectrometer are shown in Fig.
The probabilities for absorption and stimulated emission are the same. (2. In the same units. This relaxation is often a simple firstorder process.. (2.n f P + n / P .2.3. so that the transition energy measured in wave numbers is 0. That is.3.3. and the second term is the gain of electrons in the upper spin state due to absorption.14) Here nq designates the difference in population of these two states when the electron spin magnetic energy levels are in thermal equilibrium with a “bath” at temperature T. if P is the probability (in sec. Let n = nL . (2.15) The first term in Eq.‘)that an electron in the spindown state absorbs a photon. this is not usually the case because of paramagnetic relaxation. The wavelength of Xband microwave radiation is 3 cm.13) where hv is the transition energy.33 cm’. roomtemperature thermal energy is 200 cm’. The population difference then follows the equation dnldt = 2nP. Fortunately.16) From this equation we see that a system of spins initially in thermal equilibrium at time t = 0 would have an exponentially decreasing population difference if a suitable radiation field were applied at time t = 0: n(t) = n(O)e”’ (2.3.15) is the rate of loss of electrons in the upper spin state due to stimulated emission. equal to g 1 p I H. so .3.n t be the population difference. Thus n? and n l differ only by a few tenths of a percent. Under these conditions a good approximation to the difference in population n is then  nq N N(hv/kT).17) According to this result the spin system saturates with a time constant of ) P and the absorption of radiation decreases at this rate. (2. then P is also the probability that an electron in the upper spin state will be stimulated to emit. The time rate of change of the population of the upper spin state n t is thus dnfldt = .3. (The probabilities of spontaneous emission of radiation can usually be neglected in magnetic resonance experiments.) We shall see later that P depends on the photon density o r energy density in the radiation field.3. (2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 69 The relative numbers of electrons in the lower and upper spin states can be calculated from Boltzmann’s law: nt/nL = ehv‘kT. Paramagnetic relaxation describes the processes whereby the spin magnetic energy (sometimes called “Zeeman energy”) comes to thermal equilibrium with its molecular or crystalline environment (usually called the ‘‘lattice’’ or “bath”).
we have dn/dt = (l/T. (2. the net time dependence of the population difference is governed by the differential equation dn/dt = 2Pn  (l/TJ(n . 9. The oscillatory magnetic component of the microwave radiation field is then in the x direction. and the resonance absorption of radiation does not appreciably disturb the difference in population ofthe two spin states. (I) is 2n times the microwave frequency. the rate of radiationinduced transitions is small compared to the rate of paramagnetic relaxation.19) Under steadystate conditions where dn/dt = 0 we find for the steadystate difference in population nss nss = nq/(l + 2PT). This can also be written in the following form: power absorption = [NP(hv)’/kT]/( 1 + 2PT. Let the strength and direction of this linearly polarized field be represented by HI = H I COS(U +~6).3.18) Here T. NITROXIDE SPIN LABELS that in the absence of an applied radiation field.3.3. As already mentioned. (2. H . In the presence of both the radiation field that induces transitions between the two spin states and paramagnetic relaxation that tends to bring the spin state populations to thermal equilibrium.70 2.20) From this equation one draws a physically obvious conclusion: as long as the incident power (radiation energy density) is low enough.nq). The axis systems in Figs. in fact. 3 and 9 are the same.). The shaded region is. is parallel to the cavity axis z. and 6 is a phase factor that can .)(n .nq). (2. (2. Near the center of the cavity this field is linearly polarized and uniform. but in the presence of a thermal bath with which the spins can exchange energy. The electromagnetic radiation that is stored in this cavity has both electric and magnetic components that oscillate at the resonant frequency of the cavity.22) Here i is a unit vector in the x direction. The lines of force corresponding to one phase of the microwave magnetic field are shown in Fig.3. most paramagnetic resonance experiments are carried out using a resonant microwave cavity.2 1) We now discuss the absorption probability P. (2. is the amplitude of the oscillatory field. the region of the microwave cavity where the aqueous samples are normally placed. is the electron paramagnetic relaxation time.3. The resonance cavity is placed between the pole faces of the electromagnet so that the applied field H. The power absorbed by the sample under steadystate conditions is nssPhv.
.3.3.9. The axissystem is the same as that used in Fig. 21. described by g(v). McLachlan. be set equal to zero. D.) cos(ot + 6).24) An elementary quantummechanical calculation yields the following result for the transition probability for an electron’ *: p = $(?JHJ2g(v). The distribution of energy levels within the sample can arise from local magnetic fields within the sample that are necessarily compounded with the external field and define a distribution of resonance frequencies.3. E Hi.23) Hi = i(2H.3. (2. SPINLABEL THEORY: A MATHEMATICAL TREATMENT 71 z Y FIG. 1967. Carrington and A. “Introduction to Magnetic Resonance. Thesampleis placed in thecentral shaded area. Lines of force of the oscillatory magnetic field associated with the microwave radiation within thecavity. and the microwave radiation has a frequency stability of better than one part in lo’. (2. which is normalized as Jomg(v) dv = 1. (2. The lineshape function g(v) can be thought of as describing the distribution of energy levels between which transitions can take place.3. For convenience we write the amplitude of this field as 2 H .3. instead of Hi : 2H.3. (2. New York. Harper & Row.2. This condition is always valid for the paramagnetic resonance experiments of interest here.25) Here g(v) is the absorption lineshape function.” p. The derivation of Eq. If the energy levels between which A. where typical linewidths are never much less than 1 MHz.25) here since essentially the same result is obtained later from the Bloch equations.25) does involve the assumption that the source of (microwave) radiation has a frequency distribution that is narrow compared to the width of the absorption curve. (2. (2.26) We do not give a quantummechanical derivation of Eq. 3.
(2.3.yM x H.34) Here N is the number of electrons in the sample.31) (2./dt = .72 2. but does not take into account changes in the magnetization due to internal fields in the sample that produce electron relaxation. we obtain pi = ypi x H.3.3. The Bloch Equations Consider an electron i with spin magnetic moment pi and spin angular momentum Mi.27) d (2. 2. dt d dt (2. If we multiply through by the magnetogyric ratio y.30) Equation (2. (2.3. Equations (2.3. dM. the electron magnetization relaxes towards its equilibrium value M o with the relaxation time T. NITROXIDE SPIN LABELS transitions take place have a sufficiently short lifetime.3.29) where M is the total electron magnetic moment M = Cpi. (2.3.33) assume that the components of electron magnetization perpendicular to the steady large field H.28) Then we may sum over all the electrons i in the sample and obtain d M _ .31). A magnetic field H exerts a torque of pi x H on the spin magnetic moment. The equilibrium value of the electron magnetization in the field direction is easily shown to be M = Nh21(I + 1)/3kT. dt (2. relax to their equilibrium value.3.33) d M J d t = .3.3.M.. then the contributions of relaxation to the time rate of change of M are assumed to have the following form: dM. steady applied field.3./Tl. then this can also contribute to the width of the distribution of frequencies given by g(v). zero. with .( B J = ~i x H. According to Eq.29) gives the time rate change of the electron magnetization due to the externally applied fields. If we take the z axis to be in the direction of the large.MJT2.3.32) (2.32) and and (2.3.3./dt = (Mz  Mo)/T.. with a consequent change in the direction of the angular momentum : . (2.
(2. The outofphase component of H l has a negligible effect on the motion of the electron magnetization and can be neglected from Eq.2. (These were originally devised to describe nuclear magnetic resonance.39) (2.35) only the cirularly polarized component that is in phase with the electron magnetization at resonance.3. . If we add together the rate of change of the electron magnetization due to the externally applied fields H [Eq. (2.29) and Fig.3.3. and H i is a (usually weak) oscillatory field perpendicular to H . y. = yH.3. H: also rotates clockwise and is the inphase component.3 1)(2.. The magnetogyric ratio y is negative for electrons. 10. ” ) F. In both microwave electron paramagnetic resonance instruments and radiofrequency nuclear magnetic resonance instruments.[i(cos o r ) . (2. k are unit vectors in the x. H: = H I[i(cos o t ) + j(sin of)]. (2.37) This linearly polarized field can be decomposed into two circularly polarized counterrotating components: Hi = = H: + H. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 73 time constant T.33)] we obtain the Bloch equations. or H:) will be “out of phase” with the precessing magnetization. l9) The Bloch equations can be written in the form of a single vector equation Here i. is the steady large field applied in the z direction.3. (2. one of these components (H: or H. Thus when y is negative. Reo. H. Phys. when viewed from below the xy plane in the positive z direction..38) (2. + Hi. and the other component (H. . When the frequency w is close to the resonance frequency w. Therefore we include in Eq..3. 70.35). the applied oscillatory field (with angular frequency w ) is usually linearly polarized.29)] and due to relaxation [Eqs. Hi = 2H1i(cos wt)..40) H. respectively.36) H. (2.z directions. Let this linearly polarized field be in the x direction and of amplitude 2H.3. j.3. (2. (2. As can be seen from Eq.j(sin wt)]. and H = H.) will be nearly “in phase” with the precessing electron magnetization. Bloch.3.3.3.460(1946). the magnetization (in the absence of any HI field) precesses clockwise about z.3.
Section 2. k’.3. NITROXIDE SPIN LABELS L H .(W. as before. or “slow passage” conditions where duldt = dv/dt = dM.3. and the average power absorbed (averaged over one cycle of the microwave field) is P(0) =   dM dt./dt = 0.w)M/[l yH.u/Tz.42)(2. is.3. ( W ~ w)’ + Y ’ H . (2.44) + ? H I M .3. (2.46) + T. Under steadystate. Let M = i’u + j’v + k’M. .49) . FIG. and M .48) The average power absorbed by the sample is then P(w) = Q ~ H : M . ( = H : when y is negative). v .3. .3. the electron magnetization in the external field direction..M/[l + T:(w~ . [l (2.3.3. Here i’ is a unit vector in the direction of H..( M . (2. : u u = ~H.3.T:<COO . j’ is a unit vector perpendicular to i’ which lies in the xy plane. dvldt = = (wO . Eqs.3.74 2. . The Bloch equations are readily solved by a transformation to the rotating coordinate system i’.Mo)/TI. u is the outofphase electron magnetization. u.44) are readily solved for u. u./dt .41) where u is the inphase electron magnetization.42) (2.W)U (2. the Bloch equations are duldt = (00 . and k’ = k. j’. Illustration of the axis system used in the Bloch equations.3.~ H .w)’ + y2H:TlT2].3.  The instantaneous power absorbed by the sample is H dM/dt. dM. (2. T ~ T ~ ] (2. + T:(co~ ~)’]/[1 + T . In terms of u. and M .0)’ + y2H:T1T2].U/T2.W ) V . and M.3. 10.47) .45) = = M. T2/[l + T:(o  0 0 ) ~ + y2H:T1 T’].43) (2. (2.T.
H I ) when the microwave power is low enough. This hyperfine structure arises from the magnetic interaction between the electron spin magnetic moment and nuclear spin magnetic moments. . (2. (Strictly speaking. The component of the nuclear spin angular momentum in the applied field direction is designated m. P(w).g. .3.3..3. and compare Eqs. in fieldsweep spectra.05 G.2. However. y 2 ~ : ~ . 11 shows the paramagnetic resonance spectrum of 2.) We shall refer to the amplitude of the outofphase component u as the “absorption signal. du/dH..6tetramethyl 14oxopiperidine (TEMPONE) in water. For example.. The nuclear hyperfine interaction splits the paramagnetic resonance into a number of components. .5 1) This result is the same as that obtained by direct quantummechanical calculation of the transition probability.” 2. Most commercial paramagnetic resonance spectrometers are designed so that the recorded signal is proportional to the outofphase rotating magnetic moment u and do not record the power absorbed by the sample. 4). SPINLABEL THEORY: A MATHEMATICAL TREATMENT 75 This absorption curve has a Lorentzian line shape that is independent of the microwave power (e.1 (see Fig.0 0.49) and (2. When I = 1.2.3. (2. These three lines correspond to the three possible orientations of the 14N nucleus in the applied magnetic field. Fig.6. The 14N nucleus has a nuclear spin 1 which is equal to one. Proton nuclear hyperfine splittings are not resolved in the paramagnetic resonance spectra of many aliphatic nitroxide radicals. If we consider the condition for resonance.3. w = w.21) we can obtain an expression for the absorption probability P : P = $y2H:T2. these proton hyperfine interactions often contribute to the observed resonance linewidths.4. Nuclear Hyperfine Structure The most important feature of the paramagnetic resonance spectra of organic free radicals is the rich nuclear hyperfine structure. the quantitized values of rn are m = 1. (2. 0 (TEMPONE) The resonance spectrum consists of three sharp lines separated by 16. ~ 1. the signal usually recorded is the derivative of u.50) The quantity y2H:TlT2 is termed the saturation factor.3.0.
EPR spectrum of a dilute aqueous solution of TEMPONE. The deviation of the freeradical g factor from the freeelectron g factor means that the electron paramagnetic resonance spectrum is displaced from a freeelectron resonance spectrum. Another important feature of the paramagnetic resonance spectra of organic free radicals is the spectroscopic splitting factor g.37 %. (2. 11 is g = 2.76 2. plus local fields Hloc originating within the molecule. NITROXIDE SPIN LABELS FIG. and the separation of these energy levels is ’ ’ hv = 90IPIlHl. This deviation of the g factor from the freespin value is due to a (secondorder) spinorbit interaction.1 % and 0. A free electron with spin ++has two energy levels in an applied field of strength IHI.3.3. the hyperfine field due to the 14N nucleus. The additional weak signals in Fig. For example. The natural abundances of 3C and 5Nare 1. Thus hv = 90IBIIHo + HlocI.53) The local magnetic field acting on an electron in a nitroxide free radical has two components. the spectroscopic splitting factor g for the resonance spectrum of TEMPONE in Fig. See text for discussion.00562 & 0. It is sometimes convenient to consider the effect of the spinorbit interaction (gfactor effect) and the hyperfine interaction on paramagnetic resonance spectra from the following point of view.00002.52) Paramagnetic resonance transitions between these levels then occur at the transition frequency v. respectively. and the spinorbit field due to the (spininduced) orbital motion of the electron itself. 11 are due to a small fraction of free radicals that contain 13Cor ”N nuclei instead of I4N and ”C. (2. The field H acting on the “free” electron in a molecule such as a nitroxide free radical is equal to the externally applied field H. In other words. which is nearly but not exactly equal to the freespin g factor go = 2.0023.11. there is an electronspininduced electron orbital motion that creates a magnetic field which acts back on the electron spin. .
IHLfl and H.3.3. the anisotropic part of the hyperfine interaction contributes to the widths of the resonance lines even when the molecular motion is very fast. Thus Eq.57) In the theory of the effect of molecular motion on the magnetic resonance spectra. (2. In the paramagnetic resonance spectrum exhibited in Fig. That is. as discussed in Section 2. 11./gO)aO. IHLf 1.3. to a good approximation. the hyperfine field acting at the electron due to the nitrogen nucleus. the local hyperfine field acting at the odd electron is +16(f0. Since the second term in this equation is small compared to the first. namely HLf. Thus SPINLABEL THEORY : A MATHEMATICAL TREATMENT 77 (2.3.3. is the local field acting at the electron due to the orbital motion of the electron. in the same direction as H. the three hyperfine lines have essentially the same intensity.05)G. to a good approximation. Since 14N has a nuclear spin I = 1 which can take on three orientations in a magnetic field. where hv = g1B1H0. On the other hand. under these conditions only the isotropic interaction contributes to the splitting. and proportional to H. The anisotropic part of the hyperfine interaction makes a large contribution to the hyperfine splittings in the paramagnetic resonance spectra of oriented nitroxide radicals.lHifl.namely . due to spinorbit interaction that acts on the “free electron” spin is.9).f. at room temperature.3.O.. and for nitroxide free radicals in particular. In general. centered at the field H . (2. (2. = @.has two components: + + f&f = Hhfisotr + Hhfanisotr. it is shown that the anisotropic contribution to the hyperfine interaction is averaged to zero by the rapid tumbling motions of lowmolecularweight free radicals in liquids of low viscosity. .3.1. we see that the paramagnetic resonance spectrum of a nitroxide free radical is a threeline spectrum.3.2. and this isotropic hyperfine field is HAf isotr. Hhf.56) hv = slBlHo + g0lPIH. for organic free radicals in general. . correspond to rn = . the local hyperfine field )Ihf has three possible values. . the effective field H. Here Hhfis the local field acting at the electron due to the magnetic moment of the I4N nucleus and H.Since these values of rn are very nearly equal in probability.55) Here g = go + 6g. The three possible values of HLf. the only effective component of Hhfis the component in the direction of H.0.54) becomes (2. such as water. Also.: H.1.7. with two hyperfine lines centered about this resonance position.54) hv = gOlbllHO f Hhf + Hgl. corresponding to applied fields H. As we shall discuss later (Section 2.1 HAf I.
3. Reading. ‘I ’’ . NITROXIDE SPIN LABELS The theory of the origin of the nuclear hyperfine interaction in organic free radicals has been developed extensively. 1 A in these equations. H.53)]. 1970. The energy of the magnetic dipoledipole interaction is (2.3. and I is the nuclear spin angular momentum in units of ti. Massachusetts.3.IflIgNPNr3(U . As discussed above [cf. Electron Paramagnetic Resonance of Transition Ions. see Goldstein21 The reader can show for himself that by inserting an electronnuclear distance of the order of.” Oxford Univ. London and New York. AddisonWesley.60) where gNis the nuclear y factor. = h’g.” p. see Abragam and Bleaney. Eq. We include a brief outline of this theory here for the sake of completeness and because this theory leads in a straightforward way to the spin Hamiltonian for nitroxide free radicals.3rr/r2).59) The corresponding expression for the nuclear spin magnetic moment is (2. An understanding of this theory is not necessary for most of the biophysical applications of spin labels. 1. (2.62) Here U is a unit dyadic: U = ii + jj + kk.3. = YolPIS. This spin Hamiltonian provides a general and convenient quantitative starting point for the calculation and interpretation of magnetic resonance spectra. say. jjNis the nuclear magneton.61) where T. one obtains hyperfine A. k =YNbNk (2. Abragarn and B.20 Let us first consider the magnetic interaction between a nucleus located at the origin of a Cartesian coordinate system x = y = z = 0 and an electron located at the point r = ix + jy + kz.3.63) For a discussion of dyads and dyadics. and the nucleus with spin magnetic moment A . the freeelectron spin magnetic moment pe is P. 147.78 2. (2. 1950. “Classical Mechanics.3. Press (Clarendon). The energy of this point dipoledipole hyperfine interaction can then be written x d = hS ’ T .3. (2. Goldstein. (2. For an alternative discussion. Bleaney.58) Here r is the distance between the electron with spin magnetic moment p .
This is precisely true in one nitroxide free radical whose structure has been determined.3. C. in “Spin Labeling. (2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 79 interaction energies of the same order of magnitude as the observed hyperfine splittings. nitrogen. and (ii) the electron spin magnetic moment is not concentrated at a fixed point in space. J . = hs * T. Strathder. (2.3. 2. Theory and Applications” (L. LajzerowiczBonneteau.22 For determinations of the structure of other nitroxides.3. BB26. 239. Let m(r) dV be the contribution to the total electron spin magnetic moment arising from the element of volume dV. For quantitative work. Acra Crysfallogr. The spin distribution in nitroxide free radicals can be discussed by reference to Fig. Ac/a Crysrallo.Sect.~~ The “odd” electron is localized in a 2p n atomic orbital centered on the nitrogen atom. .p(r) du.3. ed. 129(1959). the above calculation needs to be improved by taking into account the facts that (i) there is no single distinguishable odd electron in a nitroxide free radical. = J: T. Kruger. McConnelland J .yr. 1.3. The spin distribution doubtless also has some norbital amplitude on the oxygen atom as well.64) It is convenient to define a spin density function p(r) such that25 The spin density function is normalized to 1 : s where p(r)dV = 1. Berliner. . A. Since the odd electron is described by a quantummechanical distribution function. (2.Sect. (2. Boeyens and G. BB26.60) to a distributed dipole interaction as follows. We assume that the oxygen. see Berliner23 and LajzerowiczBonnetea~. (2. I198 (1970). and two bonded carbon atoms lie in a plane.68) J . 24 J . Berliner. p.67) T. J . New York. Phys. pe = Im(r) dV. 1976. M. Academic Press. Mol.2. J .66) One can readily generalize the point dipole interaction in Eq.3. 2. it is clear that the net dipoledipole interaction must take into account this distribution of the spin. but rather is distributed in space according to the molecular electronic wave function. 22 23 2s H .). L.668 (1970). I.
the effective electronspin.3. is that if the spin density in a nitroxide free radical is approximated by a single odd electron in the 2p n orbital sketched in Fig.72) One can make semiquantitative estimates of these dipolar terms using 2p = Slater atomic orbitals. (T& = +76 M H z .3.80 2. and in their effects on the observed resonance spectra. as one can easily show by expanding the vector r in Eq. From the above discussion it follows that (Td)xx = (Td)yy = !dTd)zz. A second point to note regarding the dipolar tensor T. O. ’For ~ an estimate of ( r . and so forth.3. 5. C . In molecules where there is significant contribution of orbital magnetic moment to the nuclear hyperfine interaction. Estimated values for nitrogen are (TJxx = (q)zz 38 MHz. Dousmanis. 1. Mol.)xx= (Td)?). 967 (1955). LonguetHiggins. .} (Td)xx = = 0.3. (2. 27 G. (T&? = (T&.69) (2. 77. Phys. NITROXIDE SPIN LABELS The c in Eq. see D o ~ s m a n i s .. for a 2p norbital spin distribution yields the following result: (2. The spin density distribution function p(r) can be readily evaluated from an approximate molecular electronic wave function. In the case of nitroxide radicals the antisymmetric components in the hyperfine coupling between the electron spin and nuclear spins are negligible in magnitude. Tr{T. becomes infinite near the origin. If z is taken to be the symmetry axis.62) in Cartesian coordinates. nuclearspin dyadic need not be symmetric. conforms to the following trace equation.3 ) derived from selfconsistent field Hartree functions for atomic nitrogen.68) denotes a small region surrounding the nucleus which is excluded from the integration because of the fact that the dipolar interaction formula T. (2.3.An explicit calculation of the dipolar tensor T. as in the case of paramagnetic transitionmetal ions. (2. Carrington and H. is symmetric in the sense that the coefficient of ij is equal to the coefficient of ji.3. Thus the distributed dipole dyadic has this symmetry as well.447 (1962). This difficulty will be considered later in this section. ~ ’ ’’ A. Irrespective of the detailed form of this spin density distribution function. ’ ~ .70) + (Td)yv + (Td)zz This result follows from the fact that the point dipolar interaction itself obeys the same trace equations.71) Here ( r . then (T. C. the dipolar hyperfine tensor T.3. where the average is taken over the radial probability distribution for the odd electron. is the average value of ( r P 3 ) . P\IW R w . (2. The point dipole dyadic T. then the dipolar tensor is axially symmetric.
.2.3. if the oddelectron spin distribution has contributions from s orbitals. Let us assume that the effective A. (2. 36. E. Eq. Ramsey.. Let R. 1956. of the order of interaction between the nucleus and the magnetization inside the sphere with radius R.62). SPINLABEL THEORY : A MATHEMATICAL TREATMENT 81 Since the elements of the dyadic T.” Oxford Univ. d . 1732 (1930). The magnetic interaction of the nucleus with the electron spin magnetization inside the sphere is s i = FN = B (2. does not produce any problem in the integration.3. Now consider the magnetic size of the nucleus is R. This means that R. (T&.68). is only adequate when the two magnetic dipoles are far apart.3. We assume that the spin density p(r) is uniform across the radius R. Z .3. relative to their intrinsic spatial extent. z are a set of principal axes. Fermi. ~ ’ Draw a hypothetical sphere of radius R. ( T d ) y y r and (Td)zz. and (T& in place of(G)xx. Phys. 29 *’ ’’ . must be large compared to the “size” of the nucleus. The axes x. When x. W. This corresponds to the small volume excluded in the distributed dipole integration in Eq. about the I4N nucleus as center.59) or (2. z for which Tdis diagonal are then a set of principal axes. .3. On the other hand. Doerman. 60. If the spin distribution p(r) is due exclusively to spin distributions that can be described in terms of p . f . this dyadic can be written in diagonal form (all nondiagonal elements equal to zero).74) YNPNISB. we shall use the notation (7’Jx.3. Phys. (2. atomic wave functions. Rer.The following semiclassical derivation of the “contact” isotropic interaction has been taken in part from a treatment of this problem by R a m ~ e y . . G. so that the sphere is small enough to have a uniform spin density due to the electron. The problem of calculating the magnetic hyperfine spinspin interaction between a nucleus and an electron in an s orbital was first treated by Fermi” using the Dirac relativistic theory of the electron spin (see also Breit and DoermanZ9). Press (Clarendon). 320 (1930). Breit and F.. . F.68) and consider the small region E that is excluded from the integration. or size. N .3.73) (2. since for these atomic functions the spin density goes to zero at the origin. then it is necessary to consider this small region about the nucleus in detail. “Molecular Beams. We now return to the expression for the dipolar interaction given in Eq. The physical problem concerning integration near the nucleus is that the simple dipoleedipole interaction formula. London and New York. y. then the small region E need not be excluded from the integration because the divergence of T. are symmetric. y. be large enough that the magnetic interaction between the nucleus and the electron magnetization outside the sphere can be treated by the distributed dipole formula. which is the case for nitroxide radicals. (2.
3.3.3. J. 6 .f = hs * T * I.81) (2. The theoretical calculations yield isotropic I4N hyperfine coupling constants from Eq. is large compared to the anisotropy of the dipolar terms. (2.79) Theoretical calculations of sorbital spin density at the 14N nucleus in planar NH. These calculations should be applicable at least approximately to aliphatic nitroxide free radicals. 39). To a good approximation. Hazzard.). p.3.78) where the isotropic hyperfine splitting constant a is a = h’gOIBlgNBN$XdO). G. which have been discussed at length in the literature for various nuclei (see Ref.82) A?. nitroxide radicals are examples of ‘‘zelectron radicals”. Giaconnetti and P. Ma/.80) (2. This leads to the following expression for the isotropic hyperfine interaction : xi = 90 1 P 1 gN PNfXP(op ’ I (2. Rozantzev. Nordio. 32 p. 39.3.3.79) that are equal to +58. NITKOXIDE SPIN LABELS where B is the (uniform) magnetization inside the sphere.UU. (2. As we have stated earlier in this section. The total nuclear hyperfine interaction X h h f is the sum of the anisotropic dipolar interaction between the electron spin and the nucleus when the electron is outside the sphere and the isotropic contact interaction between the electron and the nucleus when the electron spin is inside the sphere:  x h f = x d f xi> (2. New York. which have isotropic hyperfine interactions equal to 45 MHz. E.3. such as TEMPONE in G .3.75) (2. The observed spin density at the nucleus is due to electron spin correlation effects (configuration interaction).” respectively. 301 (1963). the anisotropic dipolar interaction is averaged to zero by rapid isotropic tumbling motions.5 MHz3’ and +40 MHz. 31 32 . This occurs when the tumbling rate. (2.. 1970. Phys. transl. “Free Nitroxyl Radicals” (B.3. Plenum. From classical magnetostatics we know that B = $nm(O) (2.76) = %%3 I B ISP(0). for a single electron the spin density at the 14N nucleus is zero. This is the case for lowmolecularweight nitroxide free radicals in solutions of low viscosity.77) = haS * I. that is. L. T =Td 4. or inverse correlation time. and also in diatomic NO give a spin density p(0) that is positive.82 2.
} = 0 [cf. is (2.:.5. If we combine our previous theoretical estimates for the elements of the dipolar tensor T. it follows that i T r { T ) = a.” The spinorbit interaction can induce a small orbital magnetic moment in the molecule. Thus we expect the total magnetic moment (orbital magnetic moment plus spin magnetic moment) to have the form )Le = IPlS*g.84) 44. but the induced moment need not be in the same direction as S.89) (2.14 MHz. corresponding to an isotropic hyperfine coupling constant a = (2.3.8025 MHz/G) . Additional evidence for the validity of this conclusion will be evident from subsequent discussions. with the experimental value for the isotropic hyperfine coupling constant.86) T. The magnitude of this induced moment is expected to be proportional to the magnitude and sign of S.85) (2..3. These theoretical estimates are compared with experimental values of T in the next section. Eq.3. Note that since Tr{T.90) . Note that Eqs.86) are based on the theoretical evidence that the spin density p(0) at the I5N nucleus is positive.05 G.8 0. Nonlinear polyatomic free radicals have no firstorder electronic orbital angular momentum. + 45 = 7MHz.3.88) The energy of interaction of this magnetic moment with the externally applied field H . the electron orbital motion is “quenched.3. The Spectroscopic Splitting Factor g (2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 83 water. (2.3.3.3.0 0. we obtain T. In such cases the observed (highfield) splittings are equal to the isotropic hyperfine interactions. (2.3. Here the hyperfine splittings are 16.2.3. 38 (2..  76 N + 45 = 121 MHz. and thus a is positive.83) (2. (2. 11.(16.3. that is.85) and (2.87) The theory of the origin of the gfactor term in organic free radicals is more complex than the theory of the nuclear hyperfine interactions and will not be described here except for a few qualitative remarks. as is the case for the spectrum in Fig.3. 2.69)].. = T.0) = (.3.) (2.
. In this discussion of the nuclear hyperfine interaction.1 33 . p. M. Proc. Phys. Transitions between localized atomic 2p orbitals such as the following are “allowed”: p. the orbital contribution to nuclear hyperfine interaction is negligible since Ag/g is 2 3 x Let us now return to the paramagnetic resonance spectrum of TEMPONE in water. McConnell. H . virtual transitions. g can be assumed to be a symmetric tensor. (This is the mixing of lowerenergy occupied molecular orbitals with higherenergy unoccupied orbitals).34 2.6. U.3. Chern. + p. these transitions involve the halfempty 2p . For nitroxide free radicals. E. 11.PNgNI H. p. The g. These qualitative expectations have been verified for a number of organic free radicals. . Acad. The lowestenergy transitions contribute most significantly to the gfactor deviation. we arrive at the following expression for the spin Hamiltonian : &? = 1P 1 s* g ’ H IhS ’ T * I . since the only contribution to gz comes from highenergy pxe p. terms are expected to differ much more from the freeelectron g factor.The Spin Hamiltonian From the above discussion.n orbital. Sci. shown in Fig. (2. p. so we are left with an isotropic.9 1) we recall first that rapid tumbling averages out the anisotropic terms. including nitroxide free radicals. the magnetic interaction between the spinorbitinduced orbital magnetic moment and the nucleus has been neglected. where Ag is of the order of magnitude of the deviation of the g factor from the isotropic g factor.91) We have included the nuclear Zeeman term PNgNI* H. 1018 (1957). . (2.H. see McConnell and Robertson.3. NITROXIDE SPIN LABELS The term X Zis often referred to as the electron Zeeman Hamiltonian.92) 34 H. J ..766(1958)..PNgN1.84 2.33 The deviations of the g tensor from the isotropic freeelectron spin g tensor go U can be understood in terms of spinorbitinduced (virtual) transitions of electrons between different oneelectron orbital states. 61.3. Robertson. 44. + pz. From this discussion it follows that gz must be close to the freeelectron g factor. which is a pz orbital in our axis system.3. To understand this spectrum in terms of the spin Hamiltonian equation (2. A . M.. For an early note discussing these ideas. For molecules such as nitroxide radicals. S. This contribution is expected to be the order of magnitude of Ag/g smaller than electronspin interaction itself. and g. McConnell and R. timeaverage Hamiltonian = I P l g S * H IhaS. Natl.
3. The interaction between an electron spin and an externally applied magnetic field can be thought of classically in terms of a precession of the spin angular momentum about the field direction.97) A fieldsweep spectrum thus consists of three signals at applied fields equal to + a)/g I PI. 3000 G and above) the components s h and of the electron and nuclear spins in the applied field direction are good quantum numbers.5 MHz when H o N 3300 G. Thus H o = h(v + uI. (2. For calculations of energy levels in low applied fields. M.2909 (1965) .a)/g 1 p I. McConnell.y.93) In strong applied fields (e. Griffith. D. Priborostr.. 0. (2. This precession frequency is orders of magnitude larger than the precession frequency of the nucleus in the external field. Thus the secular or timeaverage interaction between the electron and nuclear spin is = IPlgHOSh + + jNgNHO1h. P.92) by quantummechanical calculation.35(1962). J. the components of S and I perpendicular to h are “decoupled” from one another since they precess about the external field direction at such different frequencies.3. Thus.3. that satisfy the equation (2.7. H. W.0. Pby. ~ ~ h(v 2.)/glPI. or in the nuclear hyperfine field (haI)/(g [ p). 1. The precession frequency v is h l p l g H o 21 9.36The free radical often takes 35 76 V. and H. The corresponding energy levels and resonance transitions are sh..3. see R o ~ a n t z e v and ~ ~Ryzhkov and S t e p a n o ~ .2. if h is the direction of the external field. M.3. (2. However.3. and h(v .g. 12. 43. = 0. Cornell. Effect of Orientation: SingleCrystal Spectra It is sometimes possible to include a nitroxide free radical in a diamagnetic host single crystal as a substitutional impurity.3.94) I.95) When we apply the selection rules Ash= (an allowed magnetic resonance dipole transition) and Al. ha]. G~Ofiz. Ryzhkov and A. sh = k ) .3. Stepanov. then in a fieldswept spectrum resonance signals are observed at values of the applied field H . SPINLABEL THEORY : A MATHEMATICAL TREATMENT 85 The resonance spectrum can be obtained from the spin Hamiltonian equation (2. 4. These energy levels are only accurate for strong fields. ++ + where v is the fixed microwave frequency.96) hv = IpIgH. hv/g 1 B I.= +1. (2.own schematically in Fig. Chem. this spectrum can also be obtained by means of elementary physical arguments.
02 G when the applied field is parallel to the crystallographic axis b. Paramagnetic resonance spectra of the cholesterol spin label incorporated at a concentration of the order of one percent in single crystals of cholesteryl chloride.4'dimethyloxazolidinederivative of 5acholestan3one (I) when this substance is incorporated as a substitutional impurity (at a concentration of the order of one percent) in single crystals of cholesteryl chloride.1 G when the applied field is perpendicular t o b.91 k 0. The angle 0 = 90" represents a field direction parallel to b. It is then found that the paramagnetic resonance spectrum of the free radical depends strongly on the orientation of the single crystal (and thus the free radical) relative to the direction of the externally applied field. and the magnitudes of the ' 0 IOG FIG. 5a. As one example of this type of experiment. the directions of the principal axes of the g tensor.'* It is seen that the hyperfine splittings depend strongly on the orientation of the applied magnetic field when the field direction lies in the ab plane of the crystal.86 2. Fig.0 k 0. . The maximum hyperfine splitting is 31. I). and to the z axis of the spin label (indicated in Fig. 12. A plot of the hyperfine splittings as a function of the angle 6' between the applied field direction and crystallographic b axis is given in Fig. fixed orientations relative to the axes of the host crystal. NITROXIDE SPIN LABELS on one or more welldefined. The angle 0 = 0" represents a field direction perpendicular to the twofold monoclinic symmetry axis b. the magnitudes of the principal hyperfine interactions. the minimum hyperfine splitting is 6. 12 shows paramagnetic resonance spectra of the Noxyl4'. Plots such as this may be used to determine the directions of the principal axes of the nuclear hyperfine interaction.
The principal axis z is easiest to locate experimentally. x and y . It can be seen from the data for the resonance line positions of the label (I) when the applied field direction is parallel to the principal nuclear hyperfine axis z. In the case of the free radical (I) dissolved in cholesteryl chloride. This maximum field position is an absolute maximum for this direction. the principal axis direction that is simplest to find experimentally is the principal axis corresponding to the largest hyperfine splitting. is observed where the applied field H. can be calculated from the equation gz = hv/ I p I N. These principal axis directions are denoted x and y. We denote this principal axis direction z. strongly immobilized orientation in a single crystal.02 G.2. When the applied field H.3.1 G. 31.0024 f 0. 12 and 5a from an empirical. there is no firstorder change in the hyperfine splitting for any small deviation in the direction of H. but is an absolute maximum and is much larger than the other hyperfine splittings. g. This principal value of g. The other two principal axes are necessarily perpendicular to z . and the direction perpendicular to this gives the third principal axis direction with intermediate nuclear hyperfine splitting. It is found that gz = 2. plots such as those given in Fig. For nitroxide free radicals with a fixed. In other words. and a minimum splitting of 5. one observes a maximum splitting of 6.is the maximum field strength. is parallel to the crystallographic axis b. and then subsequently justify this discussion in terms of the spin Hamiltonian.0001. as well as the corresponding principal axis directions. The corresponding field direction gives the direction of this principal axis. 5a can be used to determine the elements of the spin Hamiltonian discussed in Section 2. and to one another. SPINLABEL THEORY A MATHEMATICAL TREATMENT 87 elements of this tensor. If one plots the resonance position for the central hyperfine line. because the hyperfine splitting is not only an extremum.31 & 0. is rotated in a plane perpendicular to the principal axis z the minimum splitting corresponds to the principal axis direction with the smallest nuclear hyperfine interaction. That is. Therefore we first discuss the significance of data such as are given in Figs. utilitarian point of view. Many biophysical applications of anisotropic paramagnetic resonance spectra do not require a detailed quantitative analysis in terms of a spin Hamiltonian. The other two values of g.83 f 0. The principal axis directions of the g tensor and the components of the g tensor can be found in much the same way. yx and g. When the applied field H.3. In the case of label (I).. the largest hyperfine splitting. from the principal axis direction.1 G.6.where H. can be determined from the maximum and minimum . is parallel to a principal axis of the nuclear hyperfine interaction.91 _+ 0. when the applied field is rotated in the ac plane (percendicular to b and to z). the nuclear hyperfine splitting is an extremum. so these principal axes of the g tensor and T tensor coincide. the highest field position of this resonance defines the minimum principal value of the g tensor.
J . J. ~ ~ '' L. ~ " . Berliner and M c C ~ n n e l have l ~ ~ shown that the active site of the enzyme crchymotrypsin can be acylated in solution.39 according to the following reaction : 0 EOH + 0 __jc t 0 E0C II 0 t Here E0 I designates the enzyme achymotrypsin.0060 +_ 0. J. McConnell. Biochem. R. NITROXIDE SPIN LABELS values of the applied magnetic field that give the central (rn = 0) resonance line when the applied field direction is perpendicular to z. Sci. For example.0090 +_ 0. Chrm. Mol. Acud. Phys.37 Griffith et permitted determination of T and g values for several simple nitroxides. and the OH refers to the hydroxyl group of the activesite serine. and paramagnetic resonance spectra can be observed from these crystals. A . McConnell. Under the experimental conditions employed by Berliner and McConnellJ9 the enzyme deacylates at a negligible rate in the single crystals. H . Null. Berliner. 43. J . J . 3R 3y 4" 4' L. Lihertini and 0. M. Mol. Commun. J. U . Berliner and R. 1359 (1970). L. and also in single crystals. Biophys. . Biol. Res. Proc. S. Berliner and H. J.88 2. J. 55. More extensive studies of this reaction in solution and in single crystals have been carried o ~ t . Griffith. Anisotropic paramagnetic resonance spectra can also be observed when nitroxide spin labels are attached to protein molecules in single crystals. The directions of these principal axes deviate from those of the nuclear hyperfine interaction by a rotation around z of about 10". M.0001 and gy = 2. there is a similar uncertainty in the directions of the x and y principal axes of the g tensor. Bauer and L. 128.651 (1971). L.001. 1 (1979). 165 (1979). such as that of Libertini and Griffith. S. 129. S. 708 (1966). It is found that g. Bauer. Berliner and H . 53. The earliest study of nitroxide free radicals in single crystals was made by This study and others. Biol. = 2. Because the nuclear hyperfine anisotropy is so small in directions perpendicular to z the directions of the principal hyperfine axes x and y can only be determined to within + 5 " .
3.3. then the spin Hamiltonian becomes 8 = IpIShHh* g * h 8= lPIHsh(gz(‘z  + hShm(h.3.3. Thus the nuclear spin magnetic moment &gN I “sees” a magnetic field equal to hS * T/pNgN + H. which corresponds to a field of the order of 5. and is small compared to the first term (9500 MHz when H 3200 G). (2.3. Under these conditions the simplified approximation spin Hamiltonian is simply  % = lfllS*g*H +hS.2 x lo4 G acting on an 14N nucleus.) 2 2 l/2 .100) The second term is of the order of the nuclear hyperfine interaction. If rn is the quantized component of the nuclear spin in the local field direction.2. (2.4 [cf.102) T:C: YxC: + T. Instead.103) . * (2. we give simple physical arguments using (2. + + gyCz) + hShm(T:c: * * (2. 2 = [PIS g * H  + h S .the electron spin is quantized in the external field direction. To a good approximation the electron spin is quantized in a direction defined by the first term since g is very close to go U .3. T T h)”’. The smallest value of IT I is .3. Eq. defined by the unit vector h:  2@ = I ShHh* g * h + hS . T .3.101) The 14N nuclear spin “sees” a local field parallel to h S T.I.T.16 MHz.C. in terms of the spin Hamiltonian derived in Section 2.91)].3.the secular component of this field is hShh * T.3.98) The energy levels giving rise to the angledependent spectra shown in (I) can be determined using the spin Hamiltonian by a straightforward quantummechanical calculation.T 1 (2. at least as a first approximation. 7 100 MHz. The last two terms in the spin Hamiltonian are linear in the nuclear spin. Note that in general the hyperfine field is not in the same direction as the externally applied field. Applied fields H of the order to 3300 G can then be neglected compared to this hyperfine field. (2.99) where the first term arises from the hyperfine field due to the unpaired electron and the second term arises from the externally applied field. SPINLABEL THEORY : A MATHEMATICALTREATMENT 89 We now return to a brief discussion of the resonance spectra of oriented spin labels in single crystals.98)that lead to a good approximation to the observed energy levels and spectra. and that the direction of the hyperfine field depends on the direction of the electron spin angular momentum S. (2.I + PNgNI H.
c. 9 = g. For brevity.1. 13.2. = +_$. The paramagnetic resonance spectrum of an isotropic distribution of strongly immobilized nitroxide free radicals is illustrated in Fig. Such spectra can arise. c.90 2. and q. (2.3.3.c.2 and illustrated by Fig. sin 8 sin (2. In terms of the polar and azimuthal angles 8 and (6.8.. In isotropic distributions of nitroxide radicals. + 9. some radicals have orientations for which the principal axis z is perpendicular. slowly tumbling proteins in solution.Z + g. 2. to the applied field direction.104) (2. M v .3.3. sin 9 cos (6. these direction cosines are c.3. NITROXIDE SPIN LABELS Here c z . x. + 1.c.107) and T = ( T . AS.3.Tm)/gIPI ) (2. let 4. = = = cos 8. These radicals will then exhibit hyperfine splittings that range between T.3.108) Again using the selection rule for allowed electron spin magnetic dipolar transitions in strong applied fields. we find that observed resonance transitions are observed at applied fields H for which hv = l/?lgH + hTm.105) (2. or from labels rigidly attached to large. 6.106) c.A m = 0. or nearly perpendicular. are the direction cosines of the field in the z. (2.110) 0. Certain qualitative features of this spectrum are easily understood and have been covered by Section 2. with the triplet hyperfine pattern corresponding to the x direction being centered about an applied field direction determined by gx and the triplet hyperfine pattern corresponding .109) The applied fields giving resonance transitions are therefore HYm) = ( where m = . from labels attached to proteins in powdered crystals..Isotropic Distributions of Strongly immobilized Labels: Powder Spectra In many biophysical applications of spin labels one encounters resonance spectra arising from isotropic distributions of strongly immobilized labels.3. y principal axis system of the spin Hamiltonian. c. Z C+ ~ T:c~ + (2. for example. ex.
and when the resonance linewidths are narrow. g . however. the separation of the peaks of these two outer “absorption curves” is to a very good approximation exactly equal to T.2. strongly Immobilized in phosphatidylcholine bilayers. SPINLABEL THEORY: A MATHEMATICAL TREATMENT 91 FIG. 11 z extremum in the hyperfine splitting pattern shown in Fig. We shall therefore discuss these outer signals in some detail. T.. inner hyperfine structure is well resolved when there is axial symmetry. to the y direction being determined by g y . Since the resonance position does not change rapidly with the angle 8 between the applied field H. and q. It is therefore not surprising that the “perpendicular” hyperfine structure is not well resolved. Let the paramagnetic resonance absorption spectrum due to the hyperfine state rn be a. The apparent relatively high intensity of the outer hyperfine signals in Fig. Centered about each of these field positions are hyperfine splitting of T. The outer hyperfine signals in isotropic distributions have been very useful in many biophysical application of spin labels. At X band these two fields are separated by 3 G. (2. where  5 =H = Hr(8. An interesting and very useful feature of these outer hyperfine extrema is their shape. As we show below. As we see below.3. These six resonance signals are then overlapped with the rn = 0 component of the hyperfine triplet corresponding to the principal z direction. = gy.3. 13. = T. 13 reflects the fact that these signals correspond to the H.. for the special case that the applied field H. is exactly parallel to 2. Paramagnetic resonance spectrum of a fatty acid spin label. The paramagnetic resonance spectrum seen in Fig. whose formula is shown.({). to a good approximation the outer signals of this derivativecurve spectrum have the shape of the ubsorption curve itself.11) . 13 is the derivative of the absorption spectrum of an isotropic distribution of radical orientations. and z when 8 is near 90°. a relatively large number of molecules contribute resonance absorption at positions of extrema. 5a. #).’’ Moreover.
which shows the resonance line positions HY(0._. Hyperfine splittings as a function of position of orientcd spin labels.8 for further discussion.cos) for m = k 1.1 12) The corresponding contribution to the derivativecurve spectrum is (2.3.3.O" ? 8 70".(i . In the lower half of this figure is given a rough plot of the total range of Hr(8."(O.(O.114) This approximation is very important and needs to be considered in detail. 4) for the cholesterol spin label (2. 4) for any possible value of 4. The quantitative aspects of these approximations are illustrated in Fig. 4) is the field position of the resonance line for hyperfine component m when the applied field direction in the principal hyperfine axis system is described by the usual polar and aximuthal angles 8. the $ dependence of the line position is neglected. See Section 2.O) + &(I . 0) + ~. This range is due to Ho FIG 14.(o. First. (2. 14. except that in the upper part of Fig. 4): H. The data in Fig. The contribution of hyperfine component m to the total absorption spectrum is Am(H) = S.4. NITROXIDE SPIN LABELS and HT(6.(e.H.cos 6). 14 are the same as the data in Fig.3. . 5a..1). (2.3.1 13) This derivativecurve lineshape expression can now be greatly simplified by using the following approximation to the resonance line position H. and the 6 dependence is taken to vary as cos 8.. 4) N. 14 a plot is given of H.92 2.3. 8=n/2 a m ( < ) sin 6 do. Two approximations are in fact involved.
(2.114).7 we saw that the paramagnetic resonance spectrum of a nitroxide free radical depends on its orientation in space (relative to the 42 43 D./m)a.(H . M. 25. From this calculation it is clear that from the resonance spectrum of an isotropic distribution of strongly immobilized nitroxide spin labels one can deduce the hyperfine splitting parameter T.O)) + .3. . From these plots it will be seen that the approximation in Eq. with the understanding that only the derivativecurve spectrum arising from radicals for which 6 ' 7 45" will be accurate.1 G (highfield line). .3.1 are very nearly equal.Effects of Isotropic Motion on Spectra The paramagnetic resonance spectrum of TEMPO shown in Fig. 1 (1974). . The function a. which is centered at the position it would have in a perfectly oriented crystal for which the applied field H. and 1. 0.3. McConnell. Chem.4.113) is readily carried out.115) Here + . M a p . using the approximation in Eq.3.. J .114) is invalid in this region. . = 47.O)) is of course just the absorption curve. as discussed in Section 2.2.3. the integrated intensities for the three hyperfine components are expected to be equal. with the result dAm/BH = (/Z. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 93 deviations of the spin Hamiltonian from axial symmetry.Hr(0. (2.3. Gaffney and H. 13 that the lowfield signal is positive since 1 ' is positive. D. 11 exhibits three sharp hyperfine components. (2. Phys. (2. . 4 3 2.470 (1974) B.6 G (lowfield line).. Reson.113). and that the highfield signal is negative since 1. = 18. M .Hr(0.is negative. The approximation in Eq. McConnell. (2.3.3. The g factor gz can be determined from the center point of these outer extrema. The observed separation of the outer hyperfine signals is equal to 2T.' . The indicated integration in Eq. 4 2 . Lett. In the plot in the upper half of Fig.3. . is parallel to the principal axis z.9. Further quantitative description of the strongly immobilized spectra may be obtained using computer calculations. Note in Fig.3.114) can now be inserted in Eq. . Since the probabilities for the three 14N nuclear spin quantum states rn = 1. In Section 2. But the derivativecurve peak heights are evidently not equal. J . which is dropped because Eq. 16.3. Thomas and H. indicates the term arising from the 6' = 0 lower limit of the integration. This difference in the linewidths of the three hyperfine components can easily be shown to be related to the motional freedom of the nitroxide free radical. (2.14) is an adequate representation of the resonance line positions in the range 45" 2 6' 2 90" to an accuracy of the order of the linewidth itself (35 G). (2. 14.(H .
Shimshick. Pkyv. 4.3. and the smallest linewidth is usually found for the central hyperfine line (m = 0).(ui d c T 2 1 + uj) .yM x H . T I (2.l). E. . 563 (1956). the position of the central line (rn = 0) changes least with angle of rotation. considered in Section 2. Understandably.Mo)k + DV’M. Phvs.94 2. Here M is the spin magnetization per unit volume (or per unit area on a sphere. See Berliner’ and additional references contained therein. Rev. For rotational diffusion the magnetization is represented as a function of 8 and Cp. This is because the axes of the hyperfine and gfactor dyadics T and g are molecule fixed.. That is. of S and I in the field direction are “good quantum numbers. NITROXIDE SPIN LABELS direction of an applied magnetic field). Chrm. 5a). C . J . Torrey. On the other hand. when the rapid. and H. The theory of hyperfine multiplet line shapes has been developed extensively. this rotational averaging is never perfect. Cp.1) line changes most with angle (see Fig.91) becomes time dependent. The term DV2M gives the change in this magnetization per unit time due to diffusion of spin magnetization into and out of this unit volume. McConnell. 115 (1972).3. isotropic diffusion rotation of the spin label becomes fast enough. rather than the instantaneous spacing. in the presence of rapid motion the components Sh and I . M . . the microwaves are absorbed at the average spacing of the hyperfine energy levels.( M . modified to include the effect of diffusion : dM 1 _ . This gives rise to the sharp threeline spectra seen in Fig. Here M ( 0 .116) This modified Bloch equation was first introduced by T ~ r r e to y~ describe ~ the effects of diffusion in a liquid on nuclear magnetic resonance spectra.” We now turn to a quantitative treatment of paramagnetic resonance line shapes of nitroxide spin labels for the case of slow isotropic rotation. t). That is. M = M(8. there ensues the phenomenon of motional averaging. the largest residual linewidths even with very fast isotropic motion are found for the highfield line (rn = . H. In the presence of the rapid motion considered here. whereas the spin vectors S and I are effectively fixed in the laboratory frame. t ) sin 0 de d 4 44 45 R . C .7. and the position of the highfield (m = . McCalley. Lett. 11 rather than the orientationdependent spectra. then. Of the three resonance lines. see below) and D is the diffusion constant.3. The basic idea is that in the presence of molecular motion the spin Hamiltonian (2. or strongly immobilized spectra. 104.44 The simplest approach is to use the Bloch equations. 13.
3. = ( n . (2. M. N 27t sin 8. . SPINLABEL THEORY : A MATHEMATICAL TREATMENT 95 gives the spin paramagnetism due to radicals so oriented that the large applied field lies between 8 and 8 + d8 and between 4 and 4 + d 4 in the principal axis system of the spin Hamiltonian of these radicals. Divide a unit sphere into N zones between O = 0 and 8 = in.117) From differential calculus the quantity V2M(8) can be written as a limit..3.121) 8. (2. V2M(8) = lim {(1/A2 sin 8)[2 sin 8 cos iAM(8) A0 + sin(8 + $A)M(8 + A) + sin($ . . = / On i A12 2nr sin BM(8) do.116) is (2. For this special case V2 in Eq.iA)M(O . only molecules with principal axis directions 0 I 8 I in need be considered explicitly since the overall spectrum ofthis set of molecules is identical to the overall spectrum of the molecules for which in I 8 In. (2.).3.) The polar angle 8. For the simple case of a spin Hamiltonian with axial symmetry. we need only consider the dependence of M on 8. n.3.120) and the zones are numbered n = 1. t). (2.2. A = n/2N.A)]} (2. This approximation is described below.M(8.119) (When the direction 8 = 0 is taken to be the direction of the applied strong magnetic field H.so that the angular width A of each of the zones is the same.3.*)A.3. M = M(8. corresponding to the center of each zone is 8.A12 For small A (and a unit sphere.118) The form of this equation suggests how the change of magnetization due to rotational diffusion can be approximated by discrete jumps of the magnetization between adjacent angular zones on the surface of a unit sphere. 2. (2.122) .3. .3. r = I). The total electron magnetization in each zone is M. .
u. Under steadystate conditions. k A). 1 I ii 5 N .123) (2. and k. n + 1<N. and the quantities n(n _+ 1 . where M. we need only consider the angle 8.+. relative to the principal hyperfine axis of the nitroxide spin Hamiltonian. as assumed for the present calculation.3. n = N..u..]M.T ( M n Z.3. +~ (n 1 + n)M..i T 2  1 1 + u. yeH:. (2.125) (2. and on the orientation of the strong applied field H.1.128) .3. = (D/A2)cos+A.3. (2. the angle describing the zone in which the principal axis of the radical lies. The quantity t. x H . k +A)/sin(0.: __ = m l dt yM. + n(n + 1 + n)M. one obtains the following differential equation for M.. t. the principal axis corresponding to the largest 14N hyperfine splitting. the Bloch equations are 0= k2'3 +  . in Eq. + n(n + 1 + n)u. in the limit of no saturation. We now must consider the fact that the paramagnetic resonance spectrum of a nitroxide free radical depends on the nitrogen nuclear spin quantum number m.3. the angle between H.' gives the rate of spin magnetization leaving the nth zone due to rotational diffusion..3.n ) give the corresponding expressions for the rates of magnetization entering the nth zone from the adjacent zones.123) is a suitable form for computer program calculations.124) Here n(n k 1 + n ) = (D/A2)sin(Q. j) . The modified Bloch equations (2.M:)k I  M. is the magnetization in the nth zone. and in a coordinate system rotating at the microwave frequency.1 + n)u.3.+ 1 + n(n . When this spin Hamiltonian has axial symmetry.3. When this substitution is made. NITROXIDE SPIN LABELS We may now substitute 0 = 8 . these equations have the same general form as do the Bloch equations when modified to include chemical exchange effects on nuclear resonance spectra.+.3.1 + +.126) = (D/A2) cos A/cos $A. 1 for 1 In. 1. Equation (2.123) include the effect of the anisotropic hyperfine interaction by regarding the effective field acting on the free electron as depending on d.116) and replace M(8.127) + n(n + 1 + n)u. (2.) by [1/(27c sin O.+.96 2.. (2. (2. + n(rz .(u.. f 1 .
. B ~ o ~ ~ JJ . A novel technique. A convenient approximate expression for Wes(n.110): HreS(n. 480 (1977). 149 (1976) 46 ” . termed saturation transfer spectroscopy. rn) is the resonance field for an electron in zone n.I. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 97 In these equations H is separated into its two components.k 2H. . (2. S. The data follow the Debye equation to within experimental accuracy.47 .g L . Hyde. In cases where the same proteins have been studied using fluorescence spectroscopy the results of the two methods are in excellent agreement. ~ ~ and Dalton48to determine very slow J. i cos(2nvJ. subject to a nuclear hyperfine field due to a nitrogen nucleus with spin component m. Thomas. and m = 1. 1.Hres(n.3. ~ that ~ this approximation transitions are induced. Dalton. A h . + T: sin’ + (2. has been developed by H ~ d eThomas.0. = + (2nv/I Y e I )C 1 + (Ag/g 11) sin’ On] rn(Ti cos’ 8. A set of reference curves for obtaining correlation times 7’ from the inward shifts due to slow motion of the outer hyperfine extrema was determined and compared with authentic spectra. Shimshick and McConnell’ have used a particular extrapolation method in which the field shifts are plotted versus (T/v)”~. In the case of globular proteins we expect from the Debye equation z2 = 4nqa3/3kT (2. The Bloch equation calculations assume that the rotational motion is sufficiently slow that it is adiabaticthe local field acting at the nitrogen nucleus changes direction so slowly that no nuclear (or electron) spin . Rimn.129) where Ag = gl. . This time is of the order O f 5 x l o p 7sec. 8. so this approximation introduces no significant error for approximating rotational correlation times in the range 2 x 3x sec. With a knowledge of the strongly immobilized hypcrfine separation. 24.2. m).44 using a computer and employing a 3” angular mesh ( N = 30 zones) and a onegauss field mesh. McCalley et ~ 1 estimate breaks down when the rotational motion moves the hyperfine field through an angular displacement of the order of a radian in a time of the order of the reciprocal of the anisotropy of the hyperfine interaction. p. where Hres(n. x . R. A general theoretical treatment of the effects of slow motion on spinlabel paramagnetic resonance spectra has been given by Freed (see Ref. 53). rn) can be obtained directly from Eq.3. The above set of 2N simultaneous equations was solved by McCalley et a1. Methods Enzymol. H = H. 439 (1978). Mrryn. D. the plot of correlation time versus q/T was obtained.3.130) that the derived correlation times should depend linearly on the viscosity.3. D. 48 L. Hh = H . 49.
hh. + T.98 2. (2. (2. T x x + Yi.. since T.lh(Y.3.Frequency Amplitude Order Parameters Highfrequency anisotropic motion can arise. and (iii) the spin angular momentum vectors S and I are often fixed in the laboratory reference frame because they are quantized in the direction of the strong applied field. the spin Hamiltonian Z ' [cf.If the rapid molecular motion is isotropic. (S and I may sometimes be fixed to the laboratory reference frame simply because of conservation of angular momentum. Thus. = yhz(t) are the timedependent direction cosines of the field direction h in the principal axis system x.93).3. + T. z of the hyperfine Hamiltonian Z ' h f . we obtain the simple isotropic hyperfine spin Hamiltonian discussed above in connection with Eq.. namely x. to a good approximation in high fields. Eq. Y h y = yh. and I ... y .3.131). 2. Thomas and M ~ C o n n e l have l ~ ~ given a comparatively elementary treatment of this problem using the Bloch equations. (2.133) .3. (2.91)] becomes time dependent. = yiz = 4. (2.(t).3. (ii) the principal axes of these dyadics are fixed in the freeradical molecule. If the rapid molecular motion is not isotropic in the z . Ty. This is because (i) the dyadics g and T are anisotropic. Since the components of S and I perpendicular to h precess at very different frequencies. %hf(t) =h S ' T ' I. are good quantum numbers. their interaction averages to zero.y. the observed interaction energy is the time average of Zhf. Eq. In the presence of molecular motion. for example.) Consider the timedependent hyperfine interaction Zhr(f). If the molecular motion is sufficiently rapid.f = hS.10. the hyperfine energy is Here yhx = yhx(t).. when a spin label is attached to a macromolecule through one or more bonds about which rapid rotation or rapid partial rotation (oscillation) can take place.131) Imagine that a magnetic field is applied in the direction h. + Kz).3. with correlation times as long as a millisecond.f : z. This highfrequency anisotropic motion is also often found in membranes or in liquid crystals when molecules with anisotropic shapes are bound in anisotropic liquid environments by noncovalent bonds. Both the experimental and theoretical aspects of this method are too involved to permit discussion here. & = yi. NITROXIDE SPIN LABELS molecular motions. where a is the isotropic hyperfine coupling constant. = 3a. the field being so strong that both S. Line Shapes with Fast Anisotropic Motion and Partial Orientation.
 (2.3.3. are time averages of the direction cosines of z’ in the x.1). Eq. Then from Eq.141) . Thus there are three equations (one each for z’.3. z‘. y ) such as . so do the principal axes of g‘ and T’.134).yX 2 fz   + y..3.. k’. The nine quantities $. The residual or effective hyperfine interaction can be represented by the hyperfine dyadic T :   X h f = hs * T’ * 1.g. Since the principal axes of g and T nearly coincide for nitroxide free radicals. and yfCy and yf. and Tk S y..138) and also three equations (one each for z .137) where yf. (2.134) A quantitative relation between the elements of T’ and T is easily obtained. it is conventional to use a set of equivalent numbers.3.  SPINLABEL THEORY : A MATHEMATICAL TREATMENT 99 sense that yix = y t y = yiz = $.137)]. (2. z. defined by the equations sij= +(3y$ . the order parameters S.. y‘. x. + yf.139) The effective Hamiltonian is The elements of g’ are related to the elements of g by equations similar to those relating the elements of T’ and T [e.. We obtain simply (2. y. = 1. j’.3.. and let the strong applied field be in the direction of k’.. Let the principal axes of T’ be i’.3. . i = x‘. y‘) such as (2.3. are not all independent of one another. y . z principal axis system of T..2. (2. j = x. Instead of using the direction cosines %to describe the motional averaging of one axis system relative to the other.. then there is a residual hyperfine interaction Xhf which is necessarily anisotropic. x’. (2. Similar equations hold for the elements Tbfy.
(gyx .gpy)(2Sk'i + Sk'k) . and g:. T.. .3. From observed values of T'. In this work it was found 1 : Sk!j 1 : +SkCk.Szry. + SzcY= 1 . in principle. The relevant equations for this case are given below T i s z= . and g.3. and particularly 1 T. 2 gzz .3. motional averaging very often leads to effective Hamiltonians X ' which account for observed spectra to within the experimental accuracy. (2..) + SzfzT. In the frequently studied liquidcrystallike systems (phospholipid bilayers and biological membranes). From the foregoing equations one can obtain the following convenient set of equations: ~ ~ Yzz s.100 2.3. 39) have the equivalent forms c Sij i j = 0. The other order are obtained from the above order parameters parameters Syfx.. determine all the elements of the orderparameter matrix. (2.. the second member of the righthand side of Eq.. ~ = S..147) These equations have been used by Gaffney and M ~ C o n n e lfor l ~ determina~ tions of order parameters Sk*kand Sksl(and hence S.SZ>=...... experimentally that Skfi In view of the fact that T.Sz. For example.. .3.3.. Let Ti1and T . Tyy. + i(S.145) for SZCz and SzCx . Sr!y. ~ (1 S. enable one to solve Eqs..J(TX... a determination of TL. two independent order parameters can be determined....144) A similar equation holds for g:.and S..dgxx + gyy) + gyy) si 1 ~~ (2. . and SyCZ and Eqs.. + .These two quantities then permit the calculation of S.143)..3.&IXA .138)and (2..144) and (2. (2.T.I are small for iiitroxide free radicals.143) 1sij= 0.. and Szfysince S z j . (2... 42) (2.142) and (2.. where the effective Hamiltonian has axial symmetry and the symmetry axis is k'. g'. and g:.3.) be the elements of T' (and g'). .S. observed values of T i . (2.j) for phospholipid spin labels incorporated into phospholipid bilayers. NITROXIDE SPIN LABELS In terms of these order parameters the sum rules in Eqs.8.3. and g one can.: The principal elements of T' and g' can often be determined from experimental spectra using methods analogous to those described in Section 2.3. (g. yields the values of S... In such cases.Tyy)..3.. In a similar way.
(c) spectra calculated using Lorentzian line shapes.3. The value of g.148).(CH.CH. (2.3.). are obtained from the observed spectra as indicated.’ /NYo 0 0 ‘(cH.).3.146) can often be neglected for fatty acid and phospholipid labels in bilayers and membranes. i t C CH. (b) spectra calculated using Gaussian line shapes.. Copyright by the American Chemical Society. 0P0CH. (Reprinted with permission from Hubbell and McConnell. and z is the principal axis of the largest hyperfine splitting (norbital axis).NMe o Me I 0 A I Me Valucs of T.93.cocH I1 CH. Am. is obtained . let us illustrate the development up to this point with some early spectra and calculations by Hubbell and McConnell.148) When “order parameters” for nitroxide spin labels are referred to in the literature without further definition. SOC.15. (center point of the inner hyperfine extrema). 314 (1971). Chem.0CR I II I ii CH. SPINLABEL THEORY A MATHEMATICAL TREATMENT 101 A $n FIG. Before discussing rapid. J .. they generally signify the order parameter S. where k is the axially symmetric symmetry axis. Applied field is 3300 G... leading to the convenient expression sk*k = (T. anisotropic molecular motion in more detail.2.l  Ti)/[Kz . given in Eq. (a) Observed spectrum.. at room temperature. Paramagnetic resonance spectra of a phospholipid spin label in an aqueous dispersion of egg lecithincholesterol (2: 1 mole ratio).) A  (2. as in the first approximation to g.’2 Figure 15 shows the paramagnetic resonance spectra of the following phospholipid spin label incorporated in lipid bilayers of egg lecithin and cholesterol (2: 1 mole ratio).3. First approximations to Ti are obtained as indicated.%Txx + Tyy)l* (2.
C. J . Libertini.3. J. The physical origins are entirely different : the isotropic hyperfine interaction is a magnetic interaction. 4183 (1977). C. 83. Am. l(1979). Reson. H. 93.11.4. J . Griffith. C.H. using either isotropic Lorentzian or isotropic Gaussian line shapes. NITROXIDE SPIN LABELS from the center point of the outer hyperfine extrema. The second is the (electron spin)(electron spin) magnetic dipolar interaction. Jost. W. Chrm. 379 (1979). Lin and J . Freed. L. There are two interactions that affect the resonance spectra. P. 15. Freed. Effect of ElectronElectron SpinSpin Interaction: Dipolar Interactions in Biradicals with Fixed Distances between Spins The paramagnetic resonance spectra of organic free radicals are sometimes strongly affected by interactions between unpaired electrons.J. H . analogous to the (electron spin)(nuclear spin) magnetic dipolar interaction responsible for the anisotropic hyperfine interaction discussed in Section 2. Mugn.102 2. F. Biochem.460(1974) J . P. Chem. 5 1 I.” and Libertini er 2. A. J . and 0.49 See also Lin and Freed. ’* ’’ . Cun. Burke. The origin of this interaction is discussed in textbooks on quantum mechanics and is not discussed further here. see Freed. For a guide to more general treatments of relaxation and resonance spectra. It will be seen in Fig. J.2808 (1971). J . This process was then repeated until the best overall agreement between observed and calculated spectra was obtained.3. Phys. Keana and R. The first is the coulombexchange (or simply “exchange”) interaction. Sac. I5 that the comparison between observed and calculated spectra is good. Smith. Chem. and the isotropic electron spin exchange is coulombic. Dinerstein. The exchange interaction is analogous to the contact hyperfine interaction only in that both are isotropic. but not perfect. J . An extreme case of a strong dipolar interaction is found in the case of a nitroxide biradical prepared by Keana and Dinerstein’ : 4y J. Phys. These parameters were then used as the first step in the computation of spectra.” Smith. 66. W. 57.
3.3.  q).3. where s = s + s’.2.’[ve P: .W2]. (2.3. in which case the total electron spin S is a good quantum number.152) P e = solPls.4. Two unpaired electrons have a magnetic dipolar spinspin interaction with one another that is analogous to the electronnuclear spinspin dipolar hyperfine interaction discussed in Section 2.3. (2. The paramagnetic resonance spectrum has been observed in solution.3. Xd= DS: (2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 103 The two unpaired electrons in this molecule are quite close to one another.3(pe * r)(&  .3. the dipolar or “fine structure” contribution spin Hamiltonian can be written + E(S: .2 A apart. A particularly interesting example can be found in the work of Marsh ~ . Molecular models suggest that the electrons are 3. 54) (2.. P L &d = solPls’? = hS * T ’ S’. Although the nuclear hyperfine structure in these resonance spectra is not well resolved.3.156) The observed spectra can be accounted for with D = 777 MHz and E N 0.3.3.57) and (2.153) In this case.S. and in oriented multilayers. Dipolar interactions between the electron spins on different spin labels are often manifest as broadenings of the paramagnetic resonance lines. (2.150) (2. in rigid glasses.6 x IOR ’) is R = 4. Keana and Dinerstein have analyzed the observed spectra under the assumption that the colombexchange interaction is large compared to the nuclear hyperfine interaction.7 A.149) (2. large splittings due to the electronelectron dipolar interaction are evident in the observed spectra. For the case of two electrons. the equations analogous to the dipolar equations (2. In some cases the relative distance and homogeneous orientations of pairs of spin labels are such that (electron spin)(electron spin) dipolar finestructure splittings due to spins on different spinlabel molecules can be clearly resolved.61) are x d = r .3.151) (2.’).85.3. The corresponding distance estimated for two point dipoles ( D = 80. the uncertainty reflecting various plausible lowenergy conformations of the piperdine ring. E = +(T. 55) where D = ST.
Broplzys. where paramagnetic spin labels are normally far apart and noninteracting. Acud. NITKOXIDE SPIN LAHELS and Smith.104 2. and have in fact been studied in somc detail for nitroxide free radicals. In the frequently encountered case where the electron paramagnetic relaxation rate of the metal ion is large compared to the dipolar interaction itself (in frequency units). who have observed dipolar splittings due to neighboring pairs of cholestane spin labels of (I) when incorporated into phosphatidylcholinecholesterol bilayers. with the dipolar interaction only producing a relatively small broadening of the lines with increasing radical 54 I ) . but where they occasionally diffuse together and undergo strong spinexchange and/or dipolar interactions. Nurl.3. Smith. as first described by Taylor et and by Leigh. 54”. or liquidlike systems. Leigh. this interaction may manifest itself in a most unusual way. Phys.3 cos2 8.Marsh and I . fluctuating magnetic field. C‘hem. P. Such effects have often bcen observed for various free radicals in solutions. to the external magnetic field.54 Dipolar interactions in general have been discussed so extensively in the literature that there is no need to present a more elaborate discussion here. S. C. Sci. Proc. A . U. Sometinies the major spectral effects are due to the spinexchange interaction. and M. except for those (nitroxide spin)(metal ion spin) pairs whose orientational vectors make angles close to the magic angle. the resonance signal from the nitroxide group is essentially obliterated. S. The net effect of this dipolar interaction is found both experimentally and in theoretically simulated spectra to produce an apparent reduction in the amplitude of the spinlabel signal.1 2. A c ~ u 298 (1973). 64. J. In the presence of an intense. bound spin labels can exhibit a “strongly immobilized. Leigh. proportional to I . 2. Marsh and Smith have used such data to study the “condensing effect ”ofthe cholesterol bilayer structure. 55 . J. with comparatively little change in the characteristic shape of the strongly immobilized spectrum. S.” powdertype spectrum since the rotational correlation times of the enzymes are large compared to the reciprocal of the hyperfine splittings. 52. vanishes. Cohn.5h For many enzymes.219 (1969) 5 b J . Spinspin dipolar interactions between spin labels and paramagnetic metal ions have often been observed in studies of metalcontaining enzymes in solution. 2608 (1970).Taylor. For such molecules the dominant part of the dipolar interaction. in frequency units. Another important situation arises in liquids. Biochim. J . S. Effect of ElectronElectron SpinSpin Interaction: SpinSpin Interaction Determined by Translational Motion and Resulting from Colliding Spins In the foregoing discussion we have considered the magnetic resonance spectra of interacting pairs of paramagnetic centers held a fixed distance apart.
the spinexchange interaction becomes very strong . When the radicals come closer to one another.3. = 0 and I. strong relative to the nuclear hyperfine interaction and electronelectron dipolar energies. 2 10 A) the spinexchange interaction can be assumed to be negligible. We can now understand the effect of spin exchange on the paramagnetic resonance spectra of nitroxide free radicals. = .1 states. gI/3IH0h' + a. = . For example. In the presence of slow exchange this then leads to a simple lifetime broadening of the resonance lines. Consider that the highfrequency (or lowfield) resonance signal is being observed. and in the absence of spin exchange. because the overlap of electron wave functions is required for the effect. when the radicals in solution are far apart (i.(t)cc(2) b2(t)P(2). This then corresponds to an interchange of spin angular momentum between the two radicals. Since efectrons are.... a solution containing nitroxide free radicals contains three groups of electron spins. In the presence of the exchange interaction. that is.u. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 105 concentration. This can be demonstrated in a formal quantummechanical way through the use of Dirac spinexchange identity: (2. is an operator that permutes the labels of the electrons. = 0 and I. identical to one another. k . Thus. suppose that the spin wave function for the odd electron on the first radical is (Dl(l)= al(t)a(l) + bl(t)/3(1). and that the spin wave function for the odd electron on the second radical is Q2(2) = a. The Hamiltonian operator k J S i S 2 can then give rise to transitions between the states Q1(1)(D2(2) and (D1(2)Q2(1).h' .1) = 0. In terms of the Bloch equations we see that magnetizations in the rotating frame u(1) and u ( 1 ) are different from zero under these conditions.157) In this equation P. g I P I H . An example of broadening as a result of increasing headgroup spinlabeled lipid concentration in the plane of a lipid bilayer membranes is illustrated by Fig. it is more accurate to say that the spinexchange interaction produces an exchange of the electron spin momentum (or magnetization) on the two radicals.1 states with no loss of magnitude or phase.I) = u( . those for which the nitrogen nuclear spins are I . of course. In an applied field of strength H.' . = 1.1. 22. .3. say to within the van der Waals radius. The spinexchange interaction between nitroxide free radicals can be thought of as equivalent to a physical exchange of two electrons. and in the presence of rapid exchange (compared to a in + . the spinexchange interaction between nitroxide radicals depends strongly on the distance between the NO groups. whereas u(0) = u(0) = u( .e. the II( I) and u(1) magnetization is transferred to the I . and this loss of magnetization is replaced by the incoming zero magnetization of the I . Just as is the case for chemical bonds. 0. The corresponding precession frequencies are v = gIPIH.2. one on each radical.
22) the weak broadening of the resonance lines can be attributed to a lifetimelimited state of the radical. Fig. It is assumed that T B T.158) The lifetimes T and T . and N . assumed to be strong.we see that a collision will be strong if T . (2. only result in a net spin exchange.be the probability per unit time that a nitroxide radical has a “collision” with a second nitroxide radical. Here a “collision” means that the associated exchange interaction is of the order of magnitude of the hyperfine interaction. Let T . are related by the equation T/Tp = NS/zN. and if J T . Let T be the “lifetime” of a nitroxide radical between collisions.)P. In dilute solutions of nitroxide free radicals (cf. or greater. Thus the linewidth enhancement due to spinexchange collisions is  ’. is the average time that the radical stays in its site.’ . If J T .5.159) where N . In a strong collision the spin magnetization has.. 1/T2 = + * 1 / ~= 3.’) to a narrowing of the resonance to a single line. Thus after a strong collision the spin magnetization has a 50: 50 chance of having the spin magnetization of the colliding partner. we must decide first whether these collisions are strong or weak. (2. NITROXIDE SPIN LABELS sec. Of the collisions that take place at a rate of T . be the duration of the collision. be the broadening of any given resonance line due to spinexchange collisions.3. That is.160) . In considering the effect of collisions on the paramagnetic resonance spectra. we have D = :(l/z. The number of new neighbors encountered with each step is z.’ is its hopping frequency. 2 lo’ sec. Since nitroxide groups that are in direct van der Waals contact with one another doubtless have an exchange interaction J which is at least of the order of 10” sec.3. If D is the diffusion constant for the free radical in the solvent of interest.106 2. so that the molecular environment of each radical has a relatively welldefined structure. of course depend on the detailed molecular properties of the liquid.3. let T . and T. In this quasicrystalline model T .. Let us now consider this line broadening in more quantitative detail. If the liquid is “ideal” the lifetimes T and T . B I the collision is strong. 6 I the collision is weak. For our present purposes it is sufficient to imagine that the liquid has a quasicrystalline structure. so to speak. Let T . no recollection of which radical it was on before the collision. (2. are the concentrations of solvent and radical in the solution. only 3 of these are between radicals with different components of the nitrogen nuclear spin angular momentum. Of these collisions. and thus only 3 of all collisions can be effective in line broadening due to spin exchange.
3. Adams. J .)(~D/A~). These investigators find for the enhanced linewidt h 1/T2 = 3.3.3. Miller et al. Equation (2. Chem. A. then Eq. Richards. and P. 44. and w 3 are the angular resonance frequencies of the three lines: (W] . 191 (1969).165) (2. M. (2.ZN. (2.3. Chem.162) and make the plausible assumptions that z 6 and A 3.166) and (W1 (4na)2. ’’ A .). w 2 .3. Phys. Miller. R . .3.7 x 109(NR/N.162) In this limit the enhancement of the linewidth is seen to be proportional to the freeradical concentration.164) (2.w2)2 = ( W j w3)3 =  w2)2 = (2742 (2.)6D/A2. In the above discussion. Hudson and G.50 have carried out a study of the paramagnetic resonance spectra of ditbutyl nitroxide in the solvent dimethylformamide.3. These authors found that in the slowexchange region the hyperfine linewidths are proportional to the radical concentration.161) Therefore. Luckhurst. N . The case of rapid spin exchange between radicals corresponds to an exchange rate that is large compared to the hyperfine splitting a.” the distance moved in each step.8 x cm2/sec.3. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 107 where A is the “jump distance.167) N If we return to Eq. for a threeline spectrum. in the limit of infrequent but strong spinexchange collisions. Thus l/Tz = ta%.5 x lo’.3. 69.2. (2. (2. (2. the strongly narrowed residual linewidth is of the form5’ 1/T2 = &T[(Wl  W2)’ + (W2  ~ 3 ) ’ +( ~ 3 01)~]. Rer. (2.3.163) where wl.3. we have considered the effect on paramagnetic resonance line shapes of spinexchange collisions between spin labels for two . the the enhancement of the linewidth is 1/T2 = ~(. 58 T./N. It can be shown that. K.167) leads to an experimental diffusion constant of D 3.4022 (1966).159)can then be written T’ = (zNR/N.
be the three corresponding angular resonance frequencies in a frequencysweep spectrum. modified to include the effect of spinexchange collisions.3.168) In these equations T..(m). Mu. Ann.170) where K is the frequency of the collisions. NITROXIDE SPIN LABELS limiting cases.” the probability per unit time that an electron spin moment will undergo a transition. Acad. are (2. one can use the parameter z . McConnell. due to spinexchange collisions. such that it experiences a new nuclear hyperfine field. (where m # m’). One case corresponds to strong but very frequency collisions. that is. and let w. the probability per unit time that one spin label will encounter another in a “strong” collision.3. McConnell. Let urn and u. 222.56 (1973). and the spinexchange interaction affects each of these states equally... and H. so l/T = iK. random exchange collisions on urnand v. Assume for the moment that a nitroxide spin label gives three Lorentzian resonance lines with corresponding transverse relaxation times T. 1. Scandella. be the outofphase and inphase electron spin magnetizations. M. : ~ to measure the firstorder decay of ~ electron spin magnetization from each of the states m. where the nitrogen nuclear spin quantum numbers m are m = 1.108 2. Y. We have already used modified BIoch equations to treat the effect of slow motion on spinlabel spectra. is the probability per unit time that electron spin magnetization with Larmor frequency om will undergo a transition (due to spinexchange collisions) to a state where the Larmor frequency is w. Devaux. 0. N . Devaux and H. Sci. Reson. From our earlier discussion we see that only 4of all the molecular collisions are effective for spin transfer. M. The quantity l / can be referred to conveniently as the “spin transfer rate. ’’ P. J . Thus we give here only a very brief discussion of the Bloch equations t o treat the spinexchange problem. J .474(1973) .qn. C. Since the populations of the three m states are essentially the same. Spectra in the intermediate range can be calculated using the Bloch equations.’ = 2 ~ . The Bloch equations that allow for the effects of strong. (2. 9. The Bloch equations with spinexchange terms have been used to determine the rates of lateral diffusion of lipid spin labels in phospholipid bilayerss9 and in biological membranes6’ ’’P.
Am.cocH 0 I CH. The incorporation of this label up to concentrations of the order of a few percent of all the phospholipids has a relatively small effect on the rate of C a 2 +ion uptake by these vesicles. A .N~ H. BrQletand H. I1 0 b other phospholipids. 94.10). Many elegant studies of paramagnetic enhanced nuclear relaxation (a field pioneered by M. Cohn) have been carried out to provide significant structural and kinetic information on enzymes and other macromolecular systems. McConnell. the resonance signals are nearly isotropic and sharp (cf. Nail.C. and thus.13. For example. Soc. "' H .4. S. in an isotropic sample. Kornberg and McConnel162 and Bralet and McConnel16' used spinlabelphospholipidenhanced nuclear relaxation of nonlabeled lipids to study lateral diffusion of phospholipids in model membranes. . Sci. A similar approach was used by Trauble and Sackmann in an early study of the lateral diffusion of phospholipid spin labels in model bilayer membranes6' An alternative approach to this measurement of lateral diffusion using spinlabeled lipids is discussed later (Section 2. Section 2. Nail.2977(1976). The lateral diffusion coefficients so obtained were of the order of D 'v 2 x cm2/sec.3. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 109 For example.).C' rt '(cH. M .) 2. 1.CH. and McConnells9 incorporated the phospholipid spin label (1. I II 0 CH. Sackmann. Kornhergand H. Through comparisons of observed phospholipid spinlabel spectra in the membranes as a function of label concentration and calculated spinlabel spectra as a function of concentration it was possible to deduce the rate of lateral diffusion of the above phospholipid spin label in these various membranes. A . and also into membranes derived from the sarcoplasmic reticulum of rabbit skeletal muscle.4.). M .3. 63 P. Clzem. Scandella. 68.2.CH.4499 (1972). Proc. 13.OC(CH. U. Acad. in the presence of ATP. Proc. Tduhleand E. D.14) in phospholipid bilayers of egg lecithin and O. McConnell..N(cH. Devaux.oPcH.60This particular label was chosen since the paramagnetic group is nearest the most flexible end of the chain. Acud..3.).2564(1971). U. Sci. '' R . S. nitroxide spin labels introduced into biological systems can have large effects in enhancing nuclear relaxation. compared to relaxation due to the interactions of the nuclei among themselves. Enhancement of Nuclear Relaxation by Spin Labels Because the spin magnetic moment of the electron is so much larger than the magnetic moments of nuclei.
we consider that the examples presented in this section serve to illustrate the various basic types of information that may be obtained by . or control. The sccond important point is that.” When used in that way it is important that the experimental design and interpretation take account of possible inappropriate perturbation of the system by the presence of these rather large reporters. to our knowledge. Background Our interest in spinlabel lipid haptens centers on the control of immune responses to haptenbearing synthetic lipid membranes by physical factors pertaining to such membranes.4.4. We would like to make two important points regarding the use of stable nitroxides as antigenic determinants. For example. The first is that this concept represents a significant departure from that which led to their more common use as molecular labels or probes of systems in which they have no part except as “reporters. The Use of Spin Labels as Antigenic Determinants Capable of Reporting Their Physical State 2. is a particular immune response helped or hindered by rapid motion of antigenic determinants in the plane of the membrane? Is there an optimal spacing for such determinants and an optimal distance they should extend from the surface of the membrane? Do different immune responses have different requirements with regard to the physical state of membranebound determinants? With answers to such questions we hope to be in a position to elucidate molecular mechanisms controlling immune function at the level of the cell membrane. these studies represent the first attempt to use antigenic determinants capable of directly reporting their physical state to answer very important questions regarding the control of immune function by physical factors concerning antigen presentation.1.4. For leading references.110 2. When spin labels are used as antigenic determinants they are an essential participunt in the action: their function is to perturb. However. but we have not yet employed spin labels in these endeavors. the reader is referred to the books by Berliner’ and Dwek. We have commenced work in other areas of immune function. Specific examples of immune responses and our experimental approach towards their elucidation are referred to in Section 2. NITROXIDE SPIN LABELS Available space makes it impossible to summarize this large body of work here. the system in whatever way is natural. These examples only concern antibodydependent efferent responses.” 2.8.
When haptens are mixed with other suitable amphipathic lipids.COOCH CH.. CHJCH.). 0 I II CH. These include the lipid haptens in the manner illustrated by Fig.COHN b V All are soluble in organic solvents such as ethanol and chloroform.COOCH I I II I CH. CH. Among these examples are several that serve to illustrate a characteristic of spin labels which has been underexploited.2. below. but form ordered aggregates in aqueous dispersion.). New York. o I1 CH3(~H.).. Pascher. (Part 11 is devoted to lipid bilayers.). .. 1977.4.64 Further details regarding particular types of lipid structures are given.CO.cH.2.(cH.OPOC%C%N A 111 CH.. G .0. M. pp.. Plenum. in “Structure in Biological Membranes” (S. I oco(cH. and introduced to an aqueous medium by an appropriate method. and H.NHCO(CH.0CO(CH.)..CN 0 H b IV C%(CH~). where relevant. Such techniques are particularly useful for studying systems composed of several compartments that differ in the ease with which they are penetrated by spin labels and/or reagents. M.cH. 16.NCH.CH.4. The structures of these are shown below.OPO(CH. whether they be used as bystander reporters or as central components of the system of interest. Abrahamsson and I. K. such as phosphatidylcholine.). their susceptibility to agents which chemically (or photochemically) destroy their paramagnetic properties. Humphries. McConnell. Brdlet.OPOCH. 321329. eds. continuous lipid bilayer structures are formed. SpinLabel Lipid Haptens Three different spinlabel lipid haptens have been used successfully to study the physical properties of membranes and certain relatively simple immune responses to membranes.CO& I CH. 2.). USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 111 using spin labels.) O4 P..CH.).
Therefore the membranes of similar vesicles lacking watersoluble spin labels but including compound Ill in the manner illustrated by Fig. Biochemistry 10. McConnell. M. The rate at which equilibrium is approached. ' ~ ) 2. . M. Diagrammatic reprcsentation or a lipid biliiyer containing a spinlabel lipid hupten. Thc experimental procedurc used to detcrmine thc rate of flipflop involved preparation of small ( 250 A diameter) singlccompartmented egg phosphatidylcholine vesicles by sonication.I12 2. Dcvaux and H. A typical result was that the asymmetry decayed with a halftime of 6. signals received on the external side are believed to be modulated by the membrane to give appropriate messages to the cell. This phenomenon has important consequences with respect to maintenance of the asymmetry of biological membranes. at different temperatures.5 hr at 30°C.2. (From BrClet p t ~ 1 . by sodium ascorbate (see Section 2. spin labels being exclusively on the internal face of the vesicle membranes.)  2. Determination of the Rate of InsideOutside Transitions of Phospholipids in Vesicle Membranes Compound I l l was used by Kornberg and M ~ C o n n e l to l ~demonstrate ~ that the rate of insideoutside transitions (flipflop) of phospholipids in vesicle membranes is slow. Rothman and J . J . 1 1 I 1 (1971). D. (See Ref. SOC. NITROXIDE SPlN I.16. Science ( Wushington. '' P.94. At 0 ° C such vesicles protect trapped watersoluble spin labels from chemical reduction. Chem. McConnell.Label Lipid Haptens in Membranes The first measurements of the rate of lateral diffusion of spinlabeled lipids in membranes were made by Devaux and McConncllh7and Trguble and ''J. R. was used to determine rate constants and activation energies for the process. D. 743 (1977). 66.4.ABELS FIG. Using other lipids. I ). E. Lenard. Kornbcrg and H.66 the major structural feature responsible for the vectorial nature of membrane function. We feel safe in assuming that asymmetry is of fundamental importance for membranedependent cellular immune function .4475 (1972). Determination of Lateral Diffusion of Spin.4. Am.) 195.4.C. 16 become asymmetric when treated with ascorbate at 0"C.3. even slower rates for flipflop have been reported since these measurements were made. and consequential loss of paramagnetic properties.
4has therefore been used by Schwartz and McConnell to determine these quantitiese9 '* 6y J. McConnell. (An important early clue as to the rapid lateral f lipids in vesicles was provided by Kornberg and McConnell. Pror. A technique similar to that discussed in Section 2. S.2. It is frequently important to know this quantity.6' Devaux and McConnell created a nonequilibrium distribution of labeled and nonlabeled lipids in multibilayer membranes.62 diffusion o who studied the dipolar effect of spin labels on the proton nuclear resonance spectra of nonlabeled lipids to infer the rapid lateral motion o f lipids. M. A. for work involving immune responses to spinlabel lipid haptens. . For example. Sheats and H . A . their external surface area cannot be determined by simple calculation. 75.4.4661 (1978) M. The product is stable and nonparamagnetic. U . Schwartz and H . M . Sequential exposures of partially masked multilayer samples to light from an argon ion laser (351 1 and 3534 A) are followed by determination of residual spinlabel signal by EPR spectroscopy and computer integration. Nail. Sci. 68). 2. at 48"C. Determination of the Surface Area of Lipid Membranes As continuous lipid bilayer preparations are rarely unilamellar. then followed the time dependence of the spinspinmediated line broadening to deduce the lateral diffusion coefficient.4. to know the number of such molecules accessible to the exterior of lipid structures. McConnell.4. hv 314 + R Co"(CN)~ + R For this work the EPR spectrometer is interfaced with a computer programmed for double integration of spectra. Diffusion constants calculated from such data are similar to those obtained using other methods (cited in Ref.) The lateral diffusion of spinlabeled lipid haptens in hydrated planar phospholipids multilayers has been measured by Sheats and McConnell using low probe concentrations and a photochemical reaction to generate an initial concentration gradient. 837 (1978). Acud. R.68 The equation for this reaction is given below. Biorhumistry 17.5. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 113 Sackmann. or.9 x lo* cm2/sec. the diffusion coefficient for dipalmitoyl phosphatidylcholine is found to be 9.
6. is stable in light and air for several days. Humphries and H. W. when spin labels are in rapid isotropic motion their hyperfine splittings are averaged. 16. This ion pentrates phospholipid bilayers very slowly. M. Reference to Figs. McConnell. is relatively high (due to superimposition of the contributions from many labels). and is highly specific for photolysis of nitroxides even in the presence of air and phospholipids. This latter rate is thought to result from slow leakage of radicals through the membrane. U . As described in Sections 2.2. Biochem.and highfield lines.70 or with spinlabeled bacterial flagellin. Hafeman. we hope.0 and 5. Therefore the amplitude of the peaks (or lines). Biophys. 86. Humphries and H. J. Biophys. Commun. Rey and H. 6 and 7 will. M. Res.275 (1976). McConnell. NITROXIDE SPIN LABELS The number of externally exposed spinlabel haptens is a measure of the external surface area of membrane structures containing a low concentration of such molecules. can be correctly predicted to produce a ’’ G. For the commonly used multicompartmented “liposomes” (prepared by handshaking dried lipid films with aqueous solutions after hydration for a suitable length of time and at an appropriate temperature) the external signal is between 5. The rate of reaction is first rapid and then slow. McConnell. J . Biophys. This figure is also obtained when such bilayers include up to 50 mole % cholesterol. reduction of spinlabel EPR signal intensity is. M. and H. 74. to prepare IgG. particularly the low. 7 2 P. 73. Antibodies Specific for SpinLabel Determinants Antibodies specific for TEMPO have been prepared by immunizing rabbits either with spinlabeled hemocyanin. In order to determine the proportion of external labels. . 522 (1979).3 and 2. S. therefore. Sci. McConnell. G .4. Photochemical reduction of exposed haptens is induced by an argon ion laser in the presence of excess 4(N. 7 3 D. M. M. resulting as it does in the representation of very many different splittings.7 They have been purified with respect to immunoglobulin class and specificity for spin label^. K.Ntrimethylamino) benzyl pentacyanocobaltate ions.N.6. M. Res. to prepare IgM.5 % for all preparations of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. ” G . suffice to convince the reader that immobilization of spin labels. measured at various times after illumination. Acud.^'^ We are currently producing monoclonal antibodies with specificity for TEMPO.114 2.248 (1976). 3537 (1977). Proc. A . Commun. These properties make it more suitable for this application than the ion used by Sheats and McConnell.3. K. Nutl. Parce. Biorhem. 2.68 The reaction proceeds via an alkyl free radical split off from the pentacyanocobaltate complex.
The vesicles were prepared by dialysis of a detergent solution of lipids. particularly the high. (From Brclet et a / . watersoluble species.4. relative to that previously obtained from samples giving rise to powder spectra. 17.4. with or without specificity for TEMPO. An unexpected result is that the affinity of such antibodies for the reduced form of a TEMPO derivative is very close to that for the TEMPO derivative itself. with TEMPObearing bilayers. which shows the effect of mixing immunoglobulins. in contrast to the usual maximum 2Tl. As the signal intensity of the “rapid motion” spectral component is proportional to the concentration of unbound spin labels. This would increase the unpaired electron density on the nitrogen atom. this principle has been used to determine the affinity of antibodies specific for TEMPO. EPR spectra of large singlecompartmented dimyristoylphosphatidylcholine vesicles containing 2.’* Univalent haptens are employed for such measurements.” the EPR spectrum changes dramatically. 6 4 ) . either presented at the surface of a membranes or as constituents of a lowmolecularweight. it probably accounts for the fact that antiTEMPO activity can be obtained. of approximately 69 G ) . When specific antibodies bind to TEMPO groups. there is a high probability that the spinlabel immunogen is reduced in v i m before stimulating the antibody response.2. Although this cross reactivity surprises us. such as a hydrogen bond to the oxygen atom. F 20 G FIG. These effects are illustrated well by Fig.17.5 ”/. and would lead to increased splittings as discussed in Section 2.It is probable that the charge separation in the NO bond is enhanced by an electrostatic interaction within the antibody combining site. of spinlabel hapten 111 together with IgC prepared from rabbit antiTEMPO hemocyanin serum () or IgG prepared from rabbit antihuman erythrocyte serum (). The new spectrum is a typical “powder spectrum.2.” except that the hyperfine splittings are remarkably broad (2Tll = 7879 G. indicating a marked reduction in motion. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 115 decrease in the amplitude of the lines.and lowfield lines.
2. integrated. All four of these types of variation are also accompanied by changes in the ability of such membranes to bind TEMPOspecific antibodies and to interact with complement in an antibodydependent manner. (iii) the cholesterol content of those bilayers. Brulet and H. The EPR spectra shown in Fig. have all been shown to affect the EPR spectra of spinlabel haptenbearing model membranes. Antibody binding has also been shown to be positively correlated with the length of the hydrophilic moiety for the case of these three haptens (in bilayers of phosphatidylcholine or phosphatidylcholine/cholesterol).5 mole 7. ofedch of the three haptens (Structures IllV). BiorherniJtry 16. (ii) the fluidity of their host bilayers. McConnell. 18. I FIG. Dipalmitoylphosphatidylclioline bilayers containing 0. Spectra were recorded at 32°C and stored. and (iv) the twodimensional concentration of haptens in bilayers. normalized.116 2. the relative heights of the spectral lines indicate incrcased motion of the nitroxide head group as the length of the hydrophilic moiety of the molecule is increased. NITROXIDE SPIN LABELS 2.74 74 P. M. see Section 2. . and replotted by means of a Digital P D P 8/e computer interfaced with a Varian E l 2 EPR spectrometer. 1209 (1977).4. Physical Factors Affecting the Binding of SpinLabel Specific Antibodies to HaptenBearing Bilayers Changes in (i) the length of the hydrophilic moiety of spinlabel haptens. For these three labels. Our view is that such events are likely to parallel physiologically important events concerning antibodydependent immune effector mechanisms such as complementmediated cell lysis and certain types of cellmediated lysis. 18 indicate that the mobility of the head group increases with the length of the hydrophilic moiety for the case of membranebound spinlabel haptens 111V.4. 20G.7.
The principal phase transition temperature of dimyristoylphosphatidylcholine is 23°C. (It should be noted that melting the bilayers both sharpens the spectrum. Allison. Humphries. Electron paramagnetic resonance spectra of dimyristoylphosphatidylcholine liposomes containing 0. Gregoriadis and A. together with a plot of temperature versus the ratio of the amplitude of the lowfield line to that of the center line. Wiley. in “Liposomes in Biological Systems” (P. K . This effect can be readily observed when membranes and antibodies are mixed at a temperature below the principal phase transition temperature and then warmed slowly through it. M . by increasing the rate of motion. by increasing the collision rate of spinlabel haptens. melting is accompanied by an appreciable increase in broadening.2. the dominant effect is that due to increasing the rate of motion. In this instance. M. ’ ~ ) Figure 19 illustrates the spectral consequences of melting bilayers containing a spinlabel lipid hapten.” The predictable increase in mobility of the head group is indicated.5 mole % spinlabel hapten IV. Figure 20 shows the binding of IgG antibodies to liposomes composed of two G. (From H ~ m p h r i e s . When haptens are already sufficiently concentrated to show significant spinexchange broadening in the solid state. 1980. because the hapten is present at a low concentration in thc membrane.4. 10 2 0 30 TEMPERATURE (“C) ’ FIG. eds. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 117 “ w V n LL . a marked increase in agglutination commences at the phase transition (G. pp. 345375. Agglutination of haptenated liposomes or vesicles by amounts of IgM or IgG antibodies which do not saturate the available binding sites is strongly favored by the fluid state of the membranes. New York.). unpublished observation). ’’ . 19. and broadens it. Humphries. K.
dimyristoylphosphatidylcholine. 5 %of the total hapten on these liposomes is on the outermost membrane surface. as a function of antibody dilution. lipsomes were removed by ultracentrifugation. Hapten was 1 mole %of total lipid. The observed spectral changes indicate that the rate of motion of the head group is increased by increasing the cholesterol content.) different phosphatidylcholines at a temperature which is above the principal thermotropic phase transition temperature of one lipid but not of the other. Since ca. The cholesterol content of phosphatidylcholine membranes has a marked effect on the spectra of spinlabel haptens included with these lipids. M. J . but that the differences in absolute amounts bound to the two types of liposomes become less significant as antibody/lipid decreases.4M total phospholipid. After 15 min incubation at 3 7 T . . the total exposed hapten concentration was of the order of lO’M. Immunol. containing spinlabel hapten I V . Binding of rabbit antinitroxide IgC to dimyristoylphosphatidylcholine (0)or dipalmitoylphosphatidylcholine( 0 ) liposomes. G. FIG.20. Levine. and residual antibody was assayed by a WassermanLevine complement fixation assay76 in which the antigen was liposomes consisting of cholesterol.118 2. ’‘ E. This is illustrated by Fig. K. Humphries. Wasserman and L. In both cases it is apparent that the percentage of antibody bound increases as the ratio of antibody/liposomes decreases. NlTROXIDE SPIN LABELS RELATIVE A N T I B O D Y CONC. (Previously unpublished data. It is also evident that “fluid” liposomes bind more antibody than “solid” liposomes under these experimental conditions. The 1 : 1 specific antibody concentration was estimatcd to be of the order of lO’M and the liposomes were present at a concentration of 2 x 10.290 (1961). and spinlabel hapten I V in the molar ratio 50:45:5. 21. 87.
21. A. at 2050 mole %. and in each case increased mobility of the spinlabel head group. and replotted by means of a Digital PDP 8/e computer interfaced with a Varian El2 EPR spectrometer.  J. ~ ~ We have discussed three examples of physical changes which control antibody binding. Biophys. appears to favor binding. M. Biochirn. and H. In addition to its positive or negative effect on lateral mobility of membrane r n o l e ~ u l e s .95 (1980). M. A . I . A fourth type of physical change which affects antibody binding is the twodimensional concentration of haptens in the membranes. ’In ~ brief. Owicki and H.2. Biophys. ’* 77 . ~ ~EPR *~ spectra indicate that cholesterol. Acad. Rubenstein. integrated. C. 76.4. This phenomenon has been studied recently by Rubenstein ef ~ 1 . M. L. EPR spectra of liposomes composed of dipalmitoylphosphatidylcholine and the various molar proportions of cholesterol. U. Owicki. Smith. Acra599. . Similar spectral changes are observed when dimyristoylphosphatidylcholine bilayers are used. Biochemistry 19. Proc. Spectra were stored. R. Antibody binding is favored by inclusion of cholesterol at 2050 mole % in such b i l a y e r ~ . 7 9 B. The broadening of the lines is typical of spin exchange. possibly because of the increased rotational freedom resulting from a decrease in their interaction with phosphatidylcholine head groups. McConnell. Narl. B. Copeland and H. and this result is consistent with a homogeneous distribution of spin labels in the plane of the membranes. The temperature was 35°C. J. it is evident that the effect of cholesterol on the physical state of lipid haptens in phosphatidylcholine bilayers is complex. S. normalized. as indicated by EPR spectroscopy. Figure 22 shows the spectral changes observed when the distance between adjacent haptens is decreased. 383 (1980). J. McConnell. R. indicated on the figure. L. in the range 050 mole %. 15 (1979). McConnell. C. McConnell. 30. and H. Sci. Liposomes contained 3 mole % spinlabel hapten IV. Rubenstein. R. USE OF SPIN LABELS AS ANTIGENICDETERMINANTS 119 FIG.569 (1980). M. permits increased mobility of spinlabel hapten head groups. J.
Also shown is a plot of the amplitude of the center line (in arbitrary units) versus the reciprocal of the mole fraction of hapten present in the bilayers. monomolecular dispersion of this hapten in cholcsteroldipalmitoylphosphatidylcholine bilayers of the chosen composition. A systematic study of the effect of hapten density on IgG binding has not yet been attempted. EPR spectra of liposomes at 32°C containing rqualumounts of hapten at the various molar percentages (with respect to total lipid) indicated. total lipid per milliliter of liposome suspension was varied). However. Samples were prepared as spccified but such that all had the same ourrull concentration of nitroxide ( i c . because of the heterogeneous nature of lipid suspensions. Spectra were obtained with a Varian E l 2 EPR spcctrometcr interfaced with a Digital P D P 8/e computer. spectra were integrated and normalized to the same spin concentration and then plotted at these new normalized values in order to measure comparable spectra parameters.") Binding of spinlabelspecific IgM is favored by decreasing the distance between adjacent haptens. The data indicate a random. Even so.22. Spectral consequence of decreasing the spacing of spinlabel hapten IV in dipalmitoylphosphatidylcholine membranes with 50 mole cholesterol. This latter parameter is proportional to the averagc area of membrane available to each hapten. even for the case of membranes for which the rate of lateral diffusion is very high. NITROXIDE SPIN LABELS 0 5 10 10 0 1 OF IIAPTFN FIG.120 2. some early work using a monoclonal antibody suggested that for some IgG molecules. hapten density may be important even in the range where the distance between adjacent haptens is less than ." This presumably relates to the ease with which multipoint binding is maintained. (From Humphries and McConnell.
4. For a discussion see Ref. Oxidation of the reduced form of a suitable watersoluble nitroxide (i. It is our suspicion that the ease with which antibodies bind to single sites on membranes may. H. Hydrogen exchange between an appropriate nitroxidereduced nitroxide couple can only occur when neither species is bound to antibodies. This topic is controversial. in the immunological sense. Metzger. 18. one which does not cross react. Of central importance is the question of the significance of multipoint versus singlesite binding of antibodies to membranes. Schwartz. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 121 that between the two binding sites of an IgG molecule. Immunol. as they are released from the protection of TEMPOspecific antibodies. has been used in some preliminary studies designed to measure antibody off rates from membranes. To what extent. Chew. McConnell. Antibodies are polyvalent. antibody conformational changes are induced by binding to antigens and are required for various functions such as activation of complement. SUC.4. 82 .2. When antibodies bind to membranes their concentration (or time spent) in the region of the membrane increases and their orientation in that region is no longer isotropic. W . This increase in concentration and anisotropy is favored by multivalent rather than monovalent binding. A. M. Am. A h . J . It would be of interest to measure off rates for antibodies bound to lipid haptens on membranes. Efferent Immune Responses Controlled by Antibodies Bound t o AntigenBearing Membranes Our thesis is that the many responses initiated by the binding of antibodies to cell membranes will be elucidated by the study of analogous systems in which the target membranes are synthetic. and H. but a few points of general interest to biophysical chemists will be mentioned.. and how. A full discussion of this subject is clearly not appropriate for this book. IgG and IgE having two antigenbinding sites and the secreted (serum) form of IgM having ten. We anticipate that the use of spinlabel haptens and monoclonal antibodies will be particularly useful for elucidation of this important point. in some cases. 82.e. 169 (1974).3592 (1979). Schwartz et aLsl have approached this problem by studying selective hydrogen exchange between nitroxides and reduced nitroxides. 2. 101. in addition to concentration and orientation. do these properties of local concentration and orientation of antibodies control antibodytriggered responses? It has been suggested that. with TEMPO and which is a hydrogen donor relative to TEMPO) by lipid haptens. be far less than that with which they bind to the samedeterminants in soluble form. It is apparent that different nitroxide species differ greatly in their affinity for hydrogen atoms. J . Parce. M.8.
Parce. and H. M. and M~Connell. 76. and the induction of phagocytosis. McConnell. As is true for the case of antibody binding all these types of physical variation do.’~ using freezefracture methods. M . Nail. M. T. under certain circumstances.). Proc. W. S . J. G. Parce. 4177 (1979). 179191. Hafeman. PCM 7723586 (H.5387 (1980). McConnell.~ ~ 75 * 8. M . B i d . U . Hafernan. Biochemistry 19. M . . Henry. Parce. 522 (1979). G . affect complement activation. Tom and H . Another related area of study has been the binding of antibodycoated synthetic membrane structures by macrophages and neutrophils. 75. M. More recently Smith et aLM4 have shown that IgG bound to lipid haptens in bilayers diffuses as rapidly as do the unbound lipids. J. Hafeman.). J. Acknowledgments We are greatly indebted to Laurie K. D. and H. pp. 254. Commun. M. Res. G. M.K. superoxide production. S .M. Hafeman. Biochrm. 89 J. Biophys. Smith. McConnell. F. W. J. Natl. Parce. Lewis. J . We have undertaken several studies of the control of antibodydependent complement activation (or depletion of activity) by spinlabel lipid haptenbearing synthetic membranes. A.M. McConnell. 8 3 N. Such IgG appeared to be aggregated by subsequent addition of Clq. 1980. 71 concerns IgM. T. 86. Smith. McConnell. 3933 (1978). A . eds. Sci.M. ’(ii) ~ membrane fl~idity1 .). 5376 (1980). Proc. but Ref. Lewis. NlTROXlDE SPIN LABELS Regarding the physical state of membranebound antibodies. New York. A . Henry. D . To what extent this is due to quantitative differences in antibody binding and to what extent other differences(related to the behavior of bound antibodies or the way in which they are bound) play a part in not always clear and is the subject of continuing investigation. D.122 2. 1768 (1979). M. Esser. McConnell.). 8689. R. in “Liposomes and lmmunobiology” (B.McC. G . 8 4 L. W. Doepel for skillful assistance in preparation of this manuscript. ” J .H.and H. Lewis. and H. Biochemistry 19.~’ In most cases IgG antibodies have been used. Six.McC. B. 8 5 A . and 1R23 A114813 (G. McConnell. Am. Parce. . Bartholomew. have observed no significant aggregation of IgG bound to haptenated bilayers. This work has been supported by National Institutes of Health Grants No. Chem. Bh D.5~(iii) cholesterol ~ o n t e n t . 5 R 0 1 AI13587(H. Sci. and a respiratory burst which are frequently a consequence of such binding. and H. J . ~ ’and ” ~ (iv) twodimensional hapten c~ncentration. and H. Acad. I/. W. Acad. These have concerned the effect of changing (i) the length of the hydrophilic moiety of the h a ~ t e n .and by National Science Foundation Grant No. and H. Elsevier. T. See Refs.
The Raman spectrum is a record of vibrational modes for all groups of atoms in the scattering molecules whose concerted atomic motions result in a change of polarizability.1 M or the maximum soluble. this method permits selective study of the neighborhood of biological chromophores. Typical concentrations of biomolecules studied in solution are. The Raman spectrum is obtained by spectral analysis of the light scattered from a sample illuminated by a laser source. I23 METHODS OF EXPERIMENTAL PHYSICS.1. is a weak Raman scatterer although highly opaque over much of the infrared spectrum. when sufficiently powerful laser sources ( > 100 mW for conventional scanning. Inc All rights of reproduction in m y rorm reserved ISBN 0124759629 . the spectral record has the form of the intensity I of scattered light versus the displacement v.3. 20 Copyright 0 1982 by Acddcmic Press. Both conventional and resonance Raman biological studies burgeoned in the 1970s. The intensities of vibrational modes which are vibronically coupled to electronic transitions may be enhanced by illumination at frequencies within the electronic absorption band. surpasses its elder in immense versatility and applicability to studies of biological molecules. whether in crystalline array or aqueous phase or biological cell fraction. Introduction Raman vibrational spectroscopy. and the sample usually survives its examination unaltered and intact. The high intensity of such resonanceenhanced bands makes possible the study of much lower concentrations. no perturbing molecules need be added to the sample. The information in this record concerning molecular structure and conformation constitutes an abundance of riches. in conventional (nonresonance) Raman spectroscopy. typically being of the order of several microliters. These advantages over infrared derive largely from the fact that water. and the fact that molecules can be studied in any state. sample size is economical. this figure may drop to several micromolars in resonance Raman work. in technological development the younger sibling of infrared spectroscopy. which in many cases better represent the biological norm. Furthermore. < 100 mW for resonance) became readily available. of the frequency of scattered light from the incident laser frequency vL. 0. Bunow 3. RAMAN SPECTROSCOPY By Margaret R. the natural biological solvent. VOL. As in infrared spectroscopy.
which is usually reported in wave numbers (cm. The process just described is known as Raman Stokes scattering. The very near neighbors are Brillouin bands resulting from the interaction of light with coherent fluctuations in the sample medium. The interaction of many photons with the sample molecules produces the complete Raman vibrational spectrum. Finally. RAMAN SPECTROSCOPY This article reviews the principles of measurement of Raman scattering and then presents a guide to the application of conventional and resonance Raman spectral analysis to several classes of biological molecules. N .excurses to a “virtual” (nonstationary) state.6 lines for an Natom nonlinear molecule only by quantummechanical selection rules and by the detectable intensity of the allowed lines. most of the scattered light is unshifted in frequency. The emphasis is placed on the strategic choice of problem and its handling in light of the body of spectroscopic data already gathered for biophysical applications. upon emitting a photon of energy h(vL . limited to fewer than 3N . that is. are found at both higher and lower frequencies about the central Rayleigh band. In the Raman phenomenon.. one incident. and exits to a second intermediate state by creating a vibrational quantum hv. illuminates a sample. In addition. the molecule in the ground state interacts with an incident photon of energy hvL. An antiStokes spectrum is seen at frequencies greater than v. and the Raman spectrum consists of bands shifted to frequencies lower than those of the incident line. the molecule returns to its ground electronic state but to the first excited vibrational level rather than to the ground vibrational state. which closely follow motion of the oscillating atoms of the sample molecules... many weak bands. Upon spectral analysis. 1). that is. the array of vibrational bands composing the Raman spectrum is displaced by from 3 to 3500 cm’ from the central band. These displacements result from the interaction of light with the changing electrical field of the electronic clouds.v. this is produced by incident photons interacting with the smaller number of molecules which populate the vibrational levels above the groundstate level in proportion to the Boltzmann factor ehvv’kT (Fig.124 3. and one emitted. representing inelastic scattering. 3. elastically scattered.2. The process is essentially instantaneous ( 10. (see Fig. The spectrum is very complex.l) of displacement.l 5 sec) and involves two photons.). but displaced from vL by the vibrational frequency v. usually in the visible range. The emitted line thus occurs at a frequency close to v L . 1). Origin of the Raman Spectrum If a beam of light at visible frequency v .
. bandwidth. is the equilibrium position of the vibrational coordinate e in the ground electronic state. When vL falls within or near molecular absorption bands. The atoms vibrate with small changes in atomatom distances and angles about their equilibrium positions. Characteristic vibrational frequencies can be calculated by normal coordinate analysis of the set of atoms of a molecule or chemical grouping connected by “springs” for which the force constants are related to bond strength. These quantities are defined and interpreted as follows. Transitions involving a jump of more than one vibrational level are also weak.3. and depolarization ratio. The vibrational frequencies are clearly sensitive to alterations of normal coordinates or of bond strengths.1. antiStokes. This spectrum is too weak to scan more than 500 wave numbers (cm’) from v L .3. 1). This sensitivity is the foundation for Raman spectral investigations not only of molecular conformation but also of its mutability in differing environments. intensity. and q. and sets of vibrational bands of the proper symmetry and overlap relative to the electronic transition may become resonance enhanced (Fig. the molecule may transit to an excited electronic state. wherehv. 3..3. ANALYSIS OF THE RAMAN SPECTRUM W 125 E electronic state ‘he qk FIG. . Analysis of the Raman Spectrum Each Raman vibrational band is characterized by its frequency vv. and resonance Raman effects. Relationship between Stokes... is the energy difference between the first excited and lowest vibrational levels of the ground electronic state.
as well as proportional to the concentration c of scatterers.3) The indicesj . and the fourth power of the frequency of scattered light.which is symmetric in the conventional vibrational Raman effect. . composes the derived polarizability tensor.3. Totally symmetric vibrations.6 molecular vibrations which may contribute to the Raman spectrum. y.). the incident light intensity I . The a. The matrix of these coefficients. and the [ ( d a / d q i ) ] . contribute only diagonal elements to the polarizability tensor.3. qi term expresses the dependence of the induced dipole moment on the 3 N . Nontotallysymmetric vibrations have nonzero offdiagonal elements. one can count and classify by symmetry the number of Raman lines expected for a molecule. and inversions which transform the molecule into itself. k run over x. It follows that measurement of the polarization of the scattered light for each Raman line gives direct . where the coefficients a j k for the ith vibration are evaluated by quantum mechanical theory.1) azz The molecular polarizability a can be expanded in terms of linear deviations from the equilibrium coordinates of the molecule (3.3. RAMAN SPECTROSCOPY The induced dipole moment P of a molecule is related to the electric field vector E of the impinging light by the polarizability tensor a: F] f. term gives rise to Rayleigh scattering.: 5:. These coefficients are evaluated in terms of the probabilities of transitions between the initial and final states and contain information on the magnitude and direction of the induced dipole moment. for which the periodic fluctuation in polarizability has the full symmetry of the molecular skeleton. The intensity of a Raman line scattered by randomly oriented molecules can now be written as dependent upon the sum of the products of cofficients M j k . The symmetry of small molecules and of chemical groupings in large molecules can be characterized in terms of the set of symmetry operations consisting of rotations. as in classical scattering processes: (3.!):" = Mzx Mzy (3. and z.2) i where q iis a normal coordinate describing a molecular vibration and higher (anharmonic) terms are neglected. reflections. Using grouptheoretical rules.126 3.
collection optics. . analyzer.3. The scattered light is analyzed by transmitting light with the field vector first parallel. focusing optics.75 for totally symmetric vibrations. FO. Claessen. These results are refined by calculation. Infrared activity requires that a vibration change one of the three components of the dipole moment of a molecule. and helps one to deduce the assignment of the associated vibration. is the depolarization ratio p . polarization scrambler. multiple reflectibns of incident and scattered light in turbid solutions destroy the polarization.8 (1969). the incident laser beam is polarized out of the plane of the paper ( 0 ) .75 for nontotallysymmetric vibrations. 2) eliminates unequal grating response to different polarizations. H. H. 2. CO. dispersion or interference filter of laser output. and p = 0. a measure of the rotation of the electric field vector of the scattered light. Schematic diagram of instrumentation for measurement of conventional or resonance Raman scattering: F. then normal to this plane. J . this measurement gives 0 < p < 0. If a sample is a single crystal or oriented polymer. may be measured in one of several related scattering geometries. 23. Shamir. Typically. In practice most mode assignments in biophysical studies are made from comparative scans of model compounds or by reference to tables of chemical group frequencies compiled for Raman and infrared work. Appl. S. Raman and infrared vibrational spectra are largely complementary. A. the ratio of intensities thus recorded for each vibration is the depolarization ratio p. information on the symmetry. Selig. For molecules oriented randomly in solution. ANALYSIS OF THE RAMAN SPECTRUM 127 FIG.2. measured in the respective spectra. and then normal to the plane so defined (0). Spectrusc. The depolarization ratio. the exact value reflecting the symmetry group. it is possible to choose the orientations of the sample axes with respect to the incident beam so as to measure individual elements of the tensor a and assign vibrational modes more precisely. The ratio of peak intensities for each vibration. the Raman spectrum is recorded first with the analyzer rotated in the plane defined by the incident and scattered beams (). and J. The ratio p can be measured accurately only for liquids or clear solutions.' The polarization scrambler placed between the analyzer and slit (Fig.3. H. the electric field vector of the polarized laser beam is chosen to be normal to the plane containing incident and observed scattering directions. In Fig.
2. eds. RAMAN SPECTROSCOPY in general.” Wiley. l o S.4.). New York. p. Vol. New York. When a mode has both Raman and infrared activity.T. 35 of “Chemical Analysis” (P. B. Part 3b. CRC Crit. R. 33. in “Mcthods in Experimental Physics: Molecular Physics” (D. Part 2. G.128 3. H. in “ Raman Spectroscopy” (H. Vol. New York. and review articles4’ may be consulted for Several classical details of theory. Cross. the mixing of electronic and vibrational states allows preresonance or resonance enhancement of the intensities of Raman G. or environment.’~~’ ’ 3. Tang and A. Vol. Plenum. Academic Press. The fourth useful spectroscopic variable is the linewidth. it can be shown that Ramanand infraredactive modes are mutually exclusive. E.). “ Molecular Vibrations. and P. 1 1 1. 1974. Jr. A variety of introductions to theory and modern instrumentation are a~ailable. Marx. Freeman. Rev. Clark and R.” Vol. 1. C. London. 205. eds. highly polar chemical groups have strong infrared bands while chemical groups of low polarity and high polarizability are strongly Raman active. 1972. J.). 3 .229 (1977). Yu. J. “ Infrared and Raman Spectra of Polyatomic Molecules. ” N. 6 . 85.). B. Heyden. Placzek. 2nd ed. “Applications of Laser Raman Spectroscopy. allowing use of infrared spectroscopic compilations. 127. Observation of line splitting for dissolved or crystalline samples can be interpreted in terms of inequivalence of molecular orientations. C. ed. p. C. Durig and W. Wiley (Interscience). Leipzig. bonding. p. 1955. Weissberger and B. Akad. Raman spectra of crystalline substances at low temperatures exhibit sharp lines. J . C. as the symmetry of the molecule is lowered.. New York. Williams. 1934. Szymanski. ’ J.. more bands are found to be allowed in both types of spectra. K. in “Handbuch der Radiologie” (E. P. 1977. Decius. New York. 1945. J. 3. ed. 1971. 1970. Hester.~. Kolthoff. C. New York. in “Advances in Infrared and Raman Spectroscopy” (R. E. Vol. Elving and I. Rossiter. Stoicheff. ’ ‘ .). 1962. Resonance Raman Scattering When a molecule is illuminated by light at frequency vL near or beneath an electronic absorption band. p. “Laser Raman Spectroscopy.. A. Wiley. 1. Tobin.). New York. in “Physical Methods of Chemistry” (A. Herzberg. reflecting the increased molecular conformational freedom and range of solutesolvent interactions. If a molecule has a center of symmetry. I . p. Vol.” Van NostrandReinhold. M.” McGrawHill. Biochem. Harris. W. Albrecht. the frequencies in both spectroscopic appearances coincide. p. These lines are broadened when the molecule is placed in solution. Verlagsges. ed. 4. Shepherd. Wilson. eds. M.
Biophys. T.286 (1974). Chem. Miyazawa. F.465 (1976). G.'2'6 3.12 The polarizability can now be expressed. a useful rule for identifying vibrational modes involved in Aterm resonance was promulgated by Hirakawa and Tsuboi" : when the laser frequency vL approaches an absorption band.3.I3 as a function of two types of terms. M. 6 . Mol. 273 (1977).1.6). Phys. the vibrational modes most strongly enhanced are those whose normal coordinates lie along the direction describing the distortion of the molecule upon transition to the excited electronic state. 501 (1977). G. Phys. Annu. (Chem.4. Sac. Vibrational modes associated with chargetransfer transitions of ligandmetal complexes in proteins are similarly enhanced (Section 3.7. Spiro and B. Gaber. lnagaki. 34. Mol. 28.14318 The magnitude of Aterm scattering is a function of the square of the transition moment of the electronic state in resonance such that it becomes proportional to the square of the extinction maximum of the absorption band. 100 (1974). Examples occurring in biologically interesting polyenes and porphyrins are given below (Sections 3.6. An analogous summation is made for Bterm scattering. The reader is referred to reviews of the theory. and T. Reu.7. Biochem. Accordingly. This type is usually dominant in resonance Raman scattering. Spiro and P. 27. ATerm Scattering Aterm scattering involves vibrational interaction with a single excited electronic state. Annu. '' A. D.7).) 188.359 (1975). Higher terms of smaller magnitude can usually be neglected. Stein. but it is nonzero only for totally symmetrical vibrational modes. l 6 J. 1476 (1961). A. Aterm scattering depends upon FranckCondon overlaps. L. j 4 T.7. Tsuboi.4. Rev. B. according to Albrecht's formulation. Science (Washington. Bioeng.9 and 3. J . Phys. Rev. Warshel. Johnson and W. 46. except that two excited states are included in each term. called A and B terms. Behringer. Vibrational stretching modes aligned with bond weakening such as that associated with nn* transitions receive strong Aterm resonance enhancement. RESONANCE RAMAN SCATTERING 129 vibrational modes associated with the chromophore. Rer. Peticolas. C. The preresonance and resonance regimes are theoretically distinguishable. Tasumi. l3 . which require shifts in the excitedstate equilibrium coordinates or changes in the shape of the excitedstate potential well relative to the ground state. Chem.C.50. A.13"9 B. Specfrosc. 553 (1977). London) 2. The total Aterm scattering for each vibrational mode is found by summing over terms describing all contributing excited states. P. Specfrosc. Annu. Annu. J. Albrecht. Hirakawa and M. Chem. Y.
C.130 3. Chem. As a result.3. 4438 (1971). BTerm Scattering Bterm scattering involves vibronic mixing of two excited states. Peticolas. a is antisymmetric. RAMAN SPECTROSCOPY 3. J . Bterm scattering may fall into any of the above categories. SOC. for each vibrational mode against the variable The intensity profile plots are corrected for the usual v4 dependence of scattering [see Eq. The excitation profile is a plot of the measured intensity. and the mode is depolarized . *’ T. 92. 56. 23 A. A. J . Proc. and the polarization is termed inverse. and B. The two types can also be distinguished. and normalized with respect to a nonresonant reference mode. (iv) p = 00. Phys. R. the depolarization ratio p may assume a value in the following ranges: (i) 0 < p < 0. and the polarization is termed anomalous. G. C.24925 ” T. 2622 (1972).and Bterm scattering can hence essentially be distinguished by the values of the measured depolarization ratios. Albrecht and M. . A. 69. and the Raman mode is polarized. Transitions that have become allowed in the absorption spectrum by vibronic mixing are generally reflected in the excitation profiles of the contributing vibrational modes in the resonance Raman spectrum. Phys. G . Raman Spectrosc. 2 4 L. C.’ 3. the polarizability tensor is no longer necessarily symmetric. These plots ’9’ ’’ vL. The magnitude of Bterm scattering depends on the product of the transition dipole moments of the two electronic states being mixed. (3.3824 (1970). B. Aton. less easily. and of the depolarization ratio.6 Vibrational modes that have rotational symmetry are particularly effective in vibronic mixing in producing anomalously or inversely polarized bands.4. Phys. Johnson. the vibration is totally symmetric. as noted previously.3)] and for instrumental spectral response. A . Strekas. a is unsymmetric. Callender. H . G. R. Chem. Len. requires total symmetry of the mode. ” B. vibrations with Bterm activity are allowed any symmetry contained in the direct product of the representations of the two electronic transitions. and T. L. Am. Narl. The mixing of inplane vibrational modes with nn* transitions in porphyrins provided the first example of inverse polarization in vibrational Raman scattering. 1 . (ii) p = 0. A . Spiro and T. J . Acad. Li. by the shape of the excitation profiles in the region of preresonance enhancement.75. and D. Sci. G. J. Doukas. Packer.303 (1977). Gill. A. Hutley.4.3. Chem. the vibration is nontotallysymmetric.2. Spiro. Aterm scattering.75. Chem. Kilponen. C.248 (1978). Rimai. The Polarizability Tensor When vibronic mixing with electronic states is admitted. S. 55. Honig. 197 (1973). Strekas. B.75 < p < 00. 19. L. and W. Nafie. (iii) 0.
A continuouswave (CW) laser beam is focused on a sample. INSTRUMENTATION 131 contain information on the relation of the resonance scattering to the absorption spectrum.428 (1976).3. G. J. the data can be recorded in digital form or integrated for recording on an xt recorder. 3n .1 and 363. Biophys. two weak argon laser lines at 351. Appl. Acta 416.0 (blue) and 514. and the light scattered at 90” is collected and directed into the slit of a double monochromator. Frequencydoubled pulsed argon laser lines lie at even shorter wavelengths. 12. The spectrally analyzed signal is detected at the photomultiplier cathode and amplified. The gratings of the scanning monochromaters are preferably driven by a stepping motor to allow flexibility for computer interfacing. 30. Lockwood. 7. 387 (1973). a range of longerwavelength lines may be supplied by adding a dye laser pumped by an argon laser or flash lamp.14. Substitution of a krypton ion laser supplies several lines toward the red. Spectrosc. Nakamura and S. 169 (1975).8 nm are widely used. I . Spiro. W. 29 T. 2). Its strongest lasing lines lie at 488. the strongest at 647. Weak signals are optimally processed by pulse counting circuitry. E. Kuriakose. Edgell.5 nm (green). 2. Ultraviolet work requires quartz optics and 26 ’’ T. . Appl. J.5. 1073 (1978).2 ’” 3. F. and P.53(1974). For nearultraviolet excitation. currently available holographic gratings have impressively high straylight rejection.5. Opt. Biochim. Spiro. the heliumneon laser supplies a 632. The CW argon ion laser is the workhorse of Raman spectroscopy.8nm line. E. Highintensity signals are processed by direct current amplification or synchronous detection and can be recorded on an xt recorder or converted to digital form for computer processing. Strekas and T. 1 . J . J . T. Raman Spectrosc. Alternatively. as well as on vibrational symmetry and its alteration by environment. W. Ruman Spectrosc. G . Schrnidlin. instrumentation The basic Raman experimental configuration provides for spectral analysis of scattered light (Fig. K . Arthurand D. Lurix. J. Schwartz. The double monochromator arrangement nearly eliminates the intense scattering of laser light unshifted in frequency. C .28 The weakness of many bands in the typically complex Raman spectra of large biological molecules makes the addition of computercontrolled data acquisition and data manipulation facilities very de~irable.’~’~’ The basic Raman instrumentation can be purchased commercially as a “package” which allows easy interchange of laser source.1 nm.
1.3. 3 5 T. Strekas. Spectrosc. sample concentration c. Johnston. Optimization and Standardization of Signal Attainment of a good signaltonoise ratio in conventional Raman spectroscopy requires optimization of the factors vL and Zo of Eq. using a variety of sample chamber^. and photodecomposition. (3. Acud. 609 (1971). 1621 (1976).’0. S . Adams. Moulton. the sample concentration is adjusted in successive trials so that absorbance does not surpass ~cattering. B. Spccrrosc. A . The signal generally increases nearly tenfold (for equal laser intensities) as the laser line is moved from the red to the blue. l(1976). and K.3. These effects are reduced by moving the sample rapidly in the beam path. Absorbing (colored) samples suffer heating in the focused laser beam. J. p.132 3..5.32Absorbing samples in solution can be pumped in a closed circuit through a capillary segment or through a jet orifice past the laser beam. photoreaction. To optimize resonance Raman signals. ed. New York. 25. Sample Handling The standard sample container is a meltingpoint capillary holding either a solid or liquid sample. J.or airdriven) spinning plate. Levin. 1974. Turk. A.~’ 3 1 1. (3. Solids are usually pressed into an annular groove on a (motor. resonance Raman studies as a rule require precautions against heating. The electronics for gating of pulsed signals and other modifications are generally acquired separately from “packaged” instrumentation. A. and T. Appl. R. R.33s34 Other precautions include beam defocusing and use of lower laser power. a result of the vL4 scattering dependence [Eq. 32 W .5. Academic Press. Sci. H. 3. Kiefer and H. Small glass or quartz cuvettes are also used. W.3)] multiplied by the photomultiplier and grating responses. Nutl. 73. Crouch. Proc. Stryer. Bcrnstcin. Spiro.^. Suspended samples (such as subcellular fractions or crystals in mother liquor) can be centrifuged to concentrate the sediment. Jr. Doukas. or humidity. Appl. C. . 3. Mathies. and D. D. and L. controlled atmosphere. 45. G. 28. Nakanishi. U. or may be held in vacuum. Oseroff. Solid samples should be well tamped to maximize scattering and conduction of heat away from the sample area heated by the laser beam. Stationary samples may be thermostatted at temperatures from that of liquid nitrogen to l W C . in “Human Responses to Environmental Odors” (A. Biochemistry 15. H.). and an adequate. usually maximal.2. W.324 (1974). RAMAN SPECTROSCOPY may require exchange of gratings for those more efficient toward the ultraviolet.3). 3 3 R. Callcnder.^^ The molecular order and spectral reproducibility of solids is improved by annealing the sample by repeated immersion of the sealed capillary in liquid nitrogen.
twochannel accumulation of the two signals scattered from a rotating. . 21. lipids by silica gel chromatography. H. computer averaging of multiple fast scans is preferable to slow scanning. Spectra accumulated under varying conditions may be computer subtracted to yield difference spectra and thus detect small spectral changes or cancel solvent or fluorescence background. in “Advances in Infrared and Raman Spectroscopy” (R.68a (1978). and background fluorescence make small intensity changes hard to quantitate. A.5. multiple spectral accumulation plus subtraction of fluorescence background will result in goodquality spectra.3. The background fluctuation is partly due to convective motion of photoquenched and unquenched molecules in and out of the beam. 1. 1977. twocompartment cell36. Hester. Heyden. chemically noninteractive intensity standard such as the 983cm’ band of sulfate or 610cm’ band of cacodylate. rapid. 3. Management of Fluorescence Biological molecules tend to exhibit a fluorescence background. The fluorescence can be substantially reduced by a combination of the following procedures.3. usually spectral intensity changes are measured with respect either to a spectral band of the sample assumed to be invariant or to an added. Rousseau. INSTRUMENTATION 133 Slow drifts in incident power. If the Raman signal can be detected above the fluorescence background in a single scan.~’ Absolute intensity measurements are uncommon in biological studies. and J. M. Vol. p. For this reason.).5. Clark and R. 30. 36 W. Photoquenching by exposing the sample for a period of hours to as intense a laser beam as it can tolerate. Kiefer. sample turbidity.such an arrangement has been used to compare cytochrome ~pectra. Optimization of signal/fluorescence. Proteins may be purified by ionexchange chromatogra. eds. J . which overwhelms the weak Raman signal. London. Moving the laser line to the red to skirt the absorption bands associated with the fluorescence. Because the fluorescence background fluctuates. J . 3. repeated scanning is preferred. 2. Dissolved samples may be passed through activated charcoal or alumina. Purification by repeated recrystallization or fractionation. a narrowwalled capillary may reduce this circulation. 3 7 D. E. J. Difference spectra may be obtained directly by gated. The sample should be kept cool. L. 3.phy or gel electrophoresis. due sometimes to intrinsic chromophores and more often to impurities. then a smallpore filter. Biophys. Shelnutt. Friedman.or alternatively as far to the blue as possible to skirt the longerwave length fluorescence envelope frequently improves the spectrum dramatically.
. Thus highly polarizable groups such as C=C. Of all these methods. Strategy of Raman Spectroscopic Applications in Biology Raman modes strong enough to be followed as indicators of molecular conformation usually belong to highly polarizable chemical groups or to groups which exist in high concentration in a small number of conformations. photoquenching is also routinely utilized. ~ ~ 6. 5. Even momentary occlusion of the beam reverses the photoquenching effect. which must have close access to the fluorescent impurity sites. by chemical modification. the signaltonoise ratio has been improved less than tenfold.6. the backbone configuration of biological polymers that undertake a regular conformation is apparent from the typical Raman modes. and depolarization ratio of a Raman mode may change in response to a change in molecular conformation.9). The identity of the mode can at the same time verified by its frequency shift upon such treatment. Spectrosc.’. either it or the interfering modes may be shifted into an emptier spectral “window” by selective deuteration or other isotopic substitution of the molecule.and SS. those requiring modified instrumentation are expensive and time consuming. When a conformational marker mode happens to be juxtaposed with a conglomerate of other modes. the free iodine level must be kept low by addition of a reducing agent. RAMAN SPECTROSCOPY most easily by directing a stream of chilled air or nitrogen on it. F. Short group frequency tables are found in introductory texts. 7. Sample purification is in nearly all circumstances the critical procedure. Funfschilling and D. assuming the background is not a function of time or small shifts in laser f r e q u e n ~ y . for biological molecules) by gated detection of the (nearly instantaneous) Raman signal scattered by illumination with nanosecond or shorter laser pulses.134 3. 38 J. useful Raman bands. 30. Iodide ion is frequently used for watersoluble samples. Modulation spectroscopy. Williams.443 (1976). Appl. Coherent antiStokes resonance scattering (see Chapter 3. 4.’ The use of vidicon or photodiode array detectors should yield further improvement. intensity. 3. Since the frequency.’’ Similarly.groups give strong. Using nanosecond pulses generated by a modelocked argon ion laser. or by the use of deuterium oxide instead of water as solvent. typically. Energy transfer to an acceptor molecule. Rejection of fluorescence (of 110 nsec lifetime.
6) Porphyrin symmetry.7. and Structure of cartilage sidegroup configuration Possible monitor of helixcoil transitions in plant polysaccharides Proteins (Sections 3. bonding scheme sites Structure of membranebound Ca2+ATPase. Helix tilt Structure of rRNA in ribosomesb Polysaccharides (Section 3.7. chlorophyll Metal ion situation. Electron transport or conformation. bonding 0. cakequestrind Location of tryptophan residues in calsequestrind Hemoglobin structure and dynamics (see text) Resonanceenhanced modes involving liganded metal ion Resonanceenhanced modes involving liganded metal ions ~ ~ O2 binding in hemocyanin' (continued) . sugar chain. its perturbability Miscibility of different lecithins" Nucleic acids (Section 3. 111.5) Polypeptide conformation Conformation. base stacking Conformation of nucleic acids in nuclei. side groups of homopolymers and copolymers Amide I. ribosomes.7.7.carrying mechanisms in energetics cytochromes.7) Structure of redox or transport Metalligand geometry.4. hemoglobin. substrate. viruses. 3.7.7.2) Base pairing.g. ahelical content) noncrystalline proteins in native environment Sidechain configuration and Interaction of local region of environment protein with solvent. Applications of Raman Spectroscopic Analysis to Biomolecules Useful vibrational modes Structural information Biological insight Example Stretching modes of polymethylene chains Preresonanceenhanced ring modes of bases Phosphate diester stretching modes Modes associated with sugar linkages. backbone CC stretching modes Conventional or preresonanceenhanced modes associated with side groups Resonanceenhanced vibrations in plane of porphyrin ring Chain conformation and interchain interaction Lipids (Section 3. and other agents Porphyrins and hemoproteins (Section 3.7..1) Membrane fluidity. bonding scheme Nonheme metalloproteins (Section 3. etc.TABLE I. especially of (e.3) Structure of chondroitin sulfate' Sugar linkage.
. nonresonance modes nonresonance modes for slower processes ( r > 10 sec) Spectral information from resonance CARS similar to that from resonance Raman. Freedman. R. Bansil. R. L. R. 192 (1978). G . Carey.8) Kinetics of hemoproteins. J. K. Biochim. Biophys. Young. enzymes. M. Dutta. Schneider. Loehr. Natl. Chem. Acta 541.9) Configuration of polyene. 108a (1979). Biophys. Am. potential for kinetic studies Flavin adenine dinucleotideglucose oxidase binding” R. Salares. V. Osterhout. Hamilton. Proc. 1. 224a (1979). Nestor. Biophys. Acta 506. of environment membrane fluidity Kinetics (Chapter 3. R.7. M. J . R. P. . and T. ‘T. Biochim. R. Lippert. and D. J. Lindsay. Phelps. P. M. polarity Polyenes as photopigments. U. Bernstein. 1081 (1978). and G. I. and P. G . Schultz. R. Thomas. Biochemistry 17. J . Maisano. and T. 535 (1978). S. J. G . S. kinetics of papainlabeled substrate complex’ Photopigments of lobster carapaces Dependent on choice of vibrational modes Photocycle of bacteriorhodopsin (see text) Nonlinear phenomena (Chapter 3. photoreceptors. 4751 (1977). Loehr. E. J. and H. A. Sci. V. I. Acad. Yannas. Stanley. R.25.9) Dependent upon molecule and Same insights as for resonance chromophore under study Raman studies. and H.8) Configuration at site of bound Mechanisms of enzyme activity chromophore Biological polyenes (Section 3. H. 25. Carriere. conformational transitions in biopolymers Chemistry.. M. Spiro. J. 4146 (1977). Mendelsohn and J. Biochemistry 16.7. Jr. SOC. 98. Biophys. N. 74. B.or resonanceenhanced stretching modes of polyene backbone Resonanceenhanced modes for most kinetics (T < 10 sec). but fluorescence free Extrinsic chromophores in proteins (Section 3. Prescott. J. J. Carey. B.TABLE 1 (confinued) Useful vibrational modes Structural information Biological insight Example Resonanceenhanced modes involving labeled substrate or active site of enzyme Preresonance.2809 (1976). D.
7. Acta 489. Levin.3. 4 3 K. 4 2 N. 11501050 cm. R. and/or intermolecular order. local electrical field and symmetry. which have a distribution of backbone configurations . Biochim. and h1090/h1130 and h1090/h1060 are most commonly used to monitor changes in interchain (6 R. 1572 (1971). analogously. L.I). A . Lipids 10. Phys. W. Acad.1. Raman spectral correlations are imperfect for heteropolymers. Biophys. Vibrations associated with the chains are listed in Table 11. The spectral pattern in the skeletal optical region can be correlated with the populations of trans and gauche conformers of the The profile of the CH stretching region. Chem. G . In Chapter 3. W. is a function of lipid flUidity. A summary of these Raman spectral applications is given in Table I. 177 (1977). CONFORMATIONAL STUDIES 137 bond energy and polarizability. along with the direction of change of frequency and peak height with increasing conformational disorder. Biochemistry 16. 1316 (1967). Conformation of Lipids in Biological Membranes The simplicity and redundancy of the nonpolar lipid chains endows the lipid array of natural and model membranes with a simple. 44 M. Snyder.7. S. 30002800 cm. The sensitivity of the Raman profile to conformation in each case delimits the types of biological questions that may be posed using this technique. Phys. Yellin and I. the ordered secondary structures. Biochim.”4244 Peak height ratios h 2 8 8 0 / h 2 8 . ahelix and &structure.and CH stretching modes. Raman bands reflecting hydrocarbon chain order can be roughly classified as those reflecting primarily only intrachain conformation (skeletal optical modes associated with CC stretching. Chem. Biophys. are readily recognized in the Raman spectrum. Peticolas. however. Bunow and I. and intrachain plus interchain interactions (methylene CH bending modes at 1450 cm.I).7. Conformational Studies 3. Levin. strong Raman spectrum which is exquisitely conformation sensitive. 47. 3.7. Natl. the correlation of changes in Raman spectra with conformation has required a great deal of comparative spectral observation and computation. L. W. 4 1 N. U. As might be expected. such as globular proteins. 642 (1977). 165 (1973). The reader is alerted that some of the assignments in Table I1 supersede several erroneous ones found in the literature. Levin. J . Sci. 39 40 . 5 0 . J. Acta 487.388 (1977). Yellin and I. Larsson. the information available from Raman spectroscopy is surveyed for several categories of biological molecules. Lippert and W. h2930/h2850. Proc. 68.
456 (1976). W. J . Levin. Levin. modes) a Frequencies and behavior of perdeuterated chains are in parentheses. ' B. and W. Peticolas. I J .842 (1979). R. S. These two assignments may be reversed [see M.388 (1977). W. P. Yager. Biophys. W. R. L. W. J . Gaber. sym. Sunder. Peticolas. Phys. M. J. R. 191 (1977)].C. Biochim. mode too weak to monitor. and 1. R. C. Bunow and I. Chem. Jr. M. 8 (1972).2070 (2126. Phys. Biophys. Biophys. Biophys. infrared active CH asym stretch CH sym stretch C H 2 bending deformations C H 2 twisting deformations + (+I + (0) + (+I  + () (+) CC skeletal stretching modesb. Asym.457. Biophys. R. Acta489. symmetric. Biochim. Acfa388. Bunow and I .2075) CH asym stretch CH sym stretch.8. 22. Chem. Biophys. Biochim. W.h 1130 (1249) 1090 (?) 1060 (1145) Alltrans conformers Gauche conformers Alltrans conformers . Hill and I. W. Acfa 282. Mendelsohn. J . R. Levin. (1976).()  + () Terminal methyl group CH stretching m o d e s h ~ d ~ e ~ ' ~ J 2960 (2217) 2935. Acta 489. Selected ConformationSensitive Modes of Hydrocarbon Chains of Lipids" Change with increasing lipid disorder 5 (cm') Assignment Methylene modes te Frequency Peak height 2925 (2200)f 2880 2850 1450 1295 (2180)f (2103) (985960) (940920) CH asym stretch. Spiker. Acta433. Biochim. w. and 1. Biophys. Acta 487. 361 (1975). and H. split by Fermi resonance " w (w) w (0) w (w) w (1 Reference" moded3' 0 (0) 0 (+) 720 (720) Choline group sym stretch (plus CD. Bernstein. R. Levin. Levin. Lippert and W.. Biochim. I. Spiker. 191 (1977). Bunow and I . Lipids 17. L. Levin.3. L. Jr. 191 (1978). Biochim. asymmetric. RAMAN SPECTROSCOPY TABLE 11. P. 70.
each containing two fatty chains and one sugar residue (galactose in cerebroside and inositol in phosphatidylinositol) attached to the head group.4951 and bandwidth^. W. P. and stabilization by intermolecular hydrogen bonding in the head group of the cerebroside (observable in the amide I region) which is absent in phosphatidylinositol. ~~. W. 191 (1977). r e s p e ~ t i v e l y. Bunow and I. A broad dispersion band is seen in the present case.48 Several frequency shifts42. Biophys. Bernstein.560(1976). Mendelsohn. Levin. M. Biochim. Mendelsohn. Spiker. Biophys. Normalized lateral and intrachain “order parameters ” have been introduced to interpret the peak height ratios of Table I for phospholipid arrays. J . The high degree of chain order in the cerebrosides (note the relatively narrower peaks). Acta443. Acta455. A more refractory problem arises in considering the CH stretching region. are equally good monitors of fluidity. Biochim. Sunder. and head group bulk. The peak height h288. Acta 290. Jr. 22. Biochim. R. P. Levin.’). L. Levin. W. Biochim. R. 329 (1075). Biochim. Acta433. Acta 465. which peak at 2850 cm.457 (1976).^^^^^ also associated with the hydrocarbon chains. M. 5 3 R. Peticolas.260 (1977). (Fermi resonance interaction of a fundamental with an overtone or combination mode of compatible symmetry and similar energy usually results in mode splitting.~~~~ peak heights may be normalized to the height of a stable band. P. 49 R. Figure 3 illustrates the sensitivity o f the Raman spectrum to the conformation of the alkyl chains in two different. Acta 464. Biochim.~’ or to a mode of an external standard. Spiker and I. and hydrogen bonding capability. ” B. J. Sunder. and H. The conformation and packing of lipids in the membrane bilayer is influenced by chain length and unsaturation.613 (1976). Biophys. 48 R. L.choline mode in fully hydrated le~ithin.202 (1978). Caber and W. Biophys.methylene deformation modes. C.191 (1978).7. 15 (1972). Bernstein. Acta489. such as the 720cm.with overtone and combination modes of the 1450cm. Biochim. fully hydrated glycolipids. Biochim. charge. Acta 413. of the lateral order parameter rests on a background produced by Fermi resonance of the methylene CH symmetric stretching modes. Peticolas. S.3. Mendelsohn. 5 1 B.alternatively. Yager. Biophys. and reference modes which themselves do not vary significantly.47 These are not absolutely defined or necessarily linear with increasing chain disorder unless additional care is taken to choose welldefined reference states for the solid state. Biophys. W. Biophys. 45 46 . J. 5 2 R. S. Biophys. CONFORMATIONAL STUDIES 139 plus intrachain order and intrachain order. Levin. Biophys. and W. and I . and H.the presence of one very long (24 carbon) acyl chain. Bunow and I. C. Caber. relative to phosphatidylinositol reflects the behavior of the almost totally saturated chains (compare the intensities of C=C bands at 1665 cm.) The Fermi resonance background has substantially different contributions ’ ’ ’. R.
15°C. as well as temperature dependence within these states. Spectrochim. 3. The band heights may be compared to the parameters of Table 1: note the peak height ratios of methylene CH asymmetric stretching modes at 2935 cm' and at 2880 cm to the symmetric stretching modes at 2850 cm'. R. S. in the lipid gel and liquidcrystalline states. which broaden upon increasing chain disorder. from the gauche/trans peak height ratio (hl. Vibrational Raman spectra of two different glycolipids illustrating spectral sensitivity to conformation of alkyl chains. Top. gel phase. 395 (1978). with respect to the high gauche conformer (9) population of PI (top spectrum) versus the high trans conformer population (t) for cerebrosides (bottom spectrum). nor can it easily be d e c o n ~ o l u t e dThe . Levin.Acfa. V tcrn ' i FIG. Bunow and I. ' RAMAN SPECTROSCOPY I I I Methylene CH stretch: asym /\ syrn J I // 2 3oM) 2800 . unpublished). plant phosphatidyl inositol (PI). liquid crystalline phase. Hsu. brain cerebrosides.140 1 8 1 3. L. Since the 2880cm' peak height is the sum of methylene CH asymmetric stretching modes.. the 2880cm. " 1600 . Snyder. ~ ~ peak height ratios in the 30002800 cm. W. 1 1400 1200 loo0 . Krimm. Modes in the CC stretching region (11501050 cm') can be treated in a more analytical manner. Part A MA. The more disordered PI shows broader bands (M. and S. and the band profiles in the skeletal optical region (11501050 cmI). . 15°C. A fairly conservative approach allows the direct computation.region serve actually as phenomenological parameters of lipid order. G. of the 54 R. Bottom.' peak height itself cannot be expected to act as a linear parameter of intrachain order.90/h 130) and an estimate of the gauche/trans conformer energy difference. plus the variable background.
32. Biochemistry 17.41 Using these two sets of peak height ratios for the 30002800 and 11501050 cm. Lehmann. and choline group vibrational frequencies to hydration of lecithin5’ have been studied. Lippert and W. E. B. Koyama. L. Acta 464. Biochim.3. 8 (1972). D. Bunow and I. combined with Raman spectral parameters of chain packing. Ovchinnikov. Larsson and R.@ Since the frequency of the 1130 cm’ alltrans skeletal mode is sensitive to chain length for short chains. J. 161 (1978). Toda. Wallach. S.7. Chem. Lipids 19. Kyogoku. Biophys. and Y. J . Raven. Wildermuth. Peticolas. Yellin and I . Curatolo.490 (1977). Weber. 4 ~ and *~ the ~~ increased ~ ~ . Biophys. R. G . S. 55 56 ’’’ .62 N. and R. W. 1007(1980). Tosteson. phosphate. H. Acta 282.442 (1977). ~ ~disorder imposed by high curvature in small lipid vesicles.616 (1976). W. D. Yu. Rand. R. Biochim. In this case. 4 ~ antibiotic^.regions. Levin. 6 2 W. Phys.). Biophys. M. The hydrationinduced frequency shifts of the amide I and C=C vibrational modes of the headgroup region of different cerebrosides. P. W. which may be interpreted to indicate the formation of two populations of lipid. in “Membrane Transport Processes” (D. Levin.60 ~ and has been studied by Raman spectroscopy. F. P. P. D. Acta 468. Verma and D. Levin.62 The lipid may melt in a biphasic manner. and W. C. L. 6 3 M. 5 8 Y. Biochim. Conformation of the glycerophosphocholine head group of lecithin5* and sensitivity of glyceryl. 6o S . 6 1 K. Yager. 5 9 E.^^. allow one to distinguish different molecular packings in hydrated cerebroside bi1a~ets. Vol. Peticolas. ~ ~ ~ Modes associated with the polar head groups of lipids are also observable. 1978. Biophys. 1. Gaber. The depth of penetration of the added component can be judged by the degree of disruption of order in the original and sometimes by the observation in the Raman spectrum of a second peak around 2880 cm’. Macco. New York. 1802 (1978). F. Shipley. Wiedekamm. Verma.74 (1977). H. G. L. Sakura. G. Latorre. Acta 426. one can follow the main gel to liquidcrystalline phase transition in saturated the pretransition in saturated l e ~ i t h i n s . Small. and D.’~~ The response of lipid lamellar fluidity to the incorporation of additional components including c h o l e s t e r 0 1 . 21.180°C. M. Biochim. CONFORMATIONAL STUDIES 141 number of gauche conformers formed at a given temperature in lecithins with saturated chains. J. Brdiczka. Biophys. Bamberg. W. 5 7 J. Wallach. P. that segregated by the added compound and that of independent bulk lipid. Bunow and I. A. eds. 2. P. p. the reference state (alltrans chains) was defined as that at . F. Biophys. and R. it has been possible to monitor the melting of chain segments on each side of the double bond in unsaturated phospholipid^^^ and in the unsaturated acyl chains of c e r e b r o ~ i d e s .^' * ~ ~ ~ ~ ~ ~ ~ polypeptides. Acta 326. Biophys. Biochim.245 (1973).
Biochim. Phys. Cancer Res. A . Milanovich.other modes of the perdeuterated chains also lie in spectrally “clean” region^^'. Biophys. sarco~~. 192 (1978). B. Biophys. Robbins. This effect may be detected by testing for the dependence of spectral profile on laser beam power. S.633 (1975). Mendelsohn and J. and A. P. P. are usually compared for different samples of reconstituted model membranes. Acra 419. The methylene CD stretching modes for this species can then be observed free of interference in the 23002050 cmregion50. Y. differential behavior compared to that of the bulk lipid. Proc.. of red blood ~ e l l s . and R. 7 1 R. SchmidtUllrich. and H. Maisano. 70 S.142 3. Verma. H. 477 (1976). Biophys. Tu.67and thymocytes68has been assessed by Raman spectroscopy. SchmidtUllrich. Increased fluidity of membranes of transformed and lectintreated cells has been r e p ~ r t e d . F. Wallach. one lipid species may be enriched in or substituted by its analog containing perdeuterated chains. E. R. Biophys. 243 (1976). Nail. F. H. Biochim. 73. 6 6 F. J .^^ (Table I). Carew. Kaufman. Chem. and D. 37. Sci. Wallach. Acra 506.g. Baskin.51. ‘* S . P. Verma. Wallach. 3558 (1976). ~ Su ~ pplemental ~l analysis of resonanceenhanced Raman spectra of lipidembedded polyene probes has confirmed the interpretation of the behavior of lipid chains. constructed from Raman spectra accumulated at a number of temperatures.9). R. Yeh. T. P. J . and corrections then made. Milanovich. H. P. 64 ‘’ S. S. RAMAN SPECTROSCOPY In these studies thermal phase transition curves. local heating in the laser beam of the compacted. U. Shore. Acra 394. SchmidtUllrich.64 . Acta 426.7. Biochim. Acad. W. Biochim. polyenes act as rather useful probes of fluidity (see Section 3. e. In order to monitor separately the conformation of a lipid species in a multicomponent membrane containing either other lipids or a large amount of protein. 106a (1976). Verma and D. F.~~ plasmic reticulum. Using this approach.53. F. C.3490 (1977). invalidating the thermal comparisons. Wallach. Thompson. P. one should be able to identify a species of deuterated lipid bound to intrinsic membrane proteins by its isolable. H. or independent determinations of lipid phase transition temperatures may be made using a calorimeter or a microscope equipped with heating stage. Harney. Stanley. The fluidity of natural membranes. H. P. R. W. D. fatty samples may vary from sample to sample. 79 (1976). R. Biophys. 6 9 E. and R. Verma. Harvey. 6. Lipids 17. C. 67 F. S. W. If the lipid or added components are absorbing (as reflected also in a high fluorescence level). and D. . 16.
3.7.
CONFORMATIONAL STUDIES
143
3.7.2. Conformation of Nucleic Acids Nucleic acids have proven to be superb polymers for Raman spectroscopic studies. The conformational states of these polymers are represented by the spectral profiles of three classes of vibrational modes: numerous modes assigned to inplane ring stretching of the nucleic acid bases, stretching modes of the carbonyl groups involved in hydrogen bonding and base pairing, and phosphate diester stretching modes associated with the sugarphosphate backbone (see Table 111). Spectra superior to those from proteins and lipids are obtained with only about 15 mg/ml of polynucleotides.
TABLE I l l . Spectral Regions Containing Conformational Markers for Hydrated Nucleic Acids
5 (cm')
Assignment C0 stretching, base carbonyls Inplane ring modes of the different bases 0P0 symmetric stretch 0P0 diester symmetric stretch A form of helix Disordered backbone B form of helix Inplane ring modes for the different bases
Conformational sensitivity''
(1 6841620)d
H bonding or base pairing;
also base stacking Base pairing and/or stacking Reference mode Helical conformation of backbone and its disruption"
15791 186 11001091 8 14795
8 14807 795 7x7 786656
Base stacking
G. J. Thomas, Jr., Appl. Spectrosc. 30,483 (1976). S . C. Erfurth and W. L. Peticolas, Biopolymers 14, 247 (1975). M. C. Chen, R. Giege, R. C. Lord, and A. Rich, Biochemistry 14,4385 (1975). Parentheses indicate frequencies observed in D,O. ' S . C. Erfurth, P. J. Bond, and W. L. Peticolas, Biopolymers 14, 1245 (1975).
Hypochromic and hyperchromic effects seen in the ultraviolet absorption spectra of polynucleotides are largely reproduced in the behavior of the inplane ring vibrational modes in the Raman spectrum; as would be suspected, some inplane modes in the Raman spectrum obtained with visible irradiation are enhanced by preresonance effects involving the 260nm absorption
" S . C.
73
74
75
76
Erfurth and W. L. Peticolas, Biopolymers 14,247 (1975). P. C. Painter and J. L. Keonig, Biopolymers 15, 241 (1976). M. Pezolet, T.J. Yu, and W. L. Peticolas, J . Raman Spectrox. 3, 55 (1975). M. Tsuboi, A. Y. Hirakawa, and Y. Nishimura, J . Raman Spectrosc.2,609 (1974). D. C. Blazej and W. L. Peticolas, Proc. Matl. Acad. Sci. U. S. A . 74,2639 (1977).
144
3.
RAMAN SPECTROSCOPY
Denaturation of deoxyribonucleic acid and ribonucleic acid (DNA and RNA), with co’ncurrent loss of base stacking and base pairing interactions, is reflected primarily in large Raman spectral intensity changes, as well as in several frequency shifts of vibrational modes assigned to the different bases; these have been tabulated along with other mode parameters for DNA7*and RNA.7780 The intensities and frequencies of the carbonyl modes of the bases respond to rupture of hydrogen bonds occurring in the premelting and melting n DNA and in the melting of RNA; these modes participate phenomena i to some extent in the hyper/hypochromic shifts involving the bases; that is, they also reflect the degree of base stacking.72 The carbonyl modes are observed for nuclerc acids in deuterium oxide, so that the interfering background from water modes at 1650 cm is removed. The consequent protondeuteron exchange of the nucleic acids has been carefully evaluated.77 The conformation of the sugarphosphate backbone has been very successfully correlated by experiment and calculation with the frequency of the phosphate diester symmetric stretching mode (807787 cm I). The frequency of this vibration is sensitive to the torsional angles about the P  0 bonds as well as to ribose ;it is an extremely valuable conformational marker for both DNA and RNA. It has been used to categorize conformations of RNAs and DNAs in solution in terms of the A and B helices (at 75 and 98% relative humidity, respectively) of DNA fibers.84 Overall, Raman spectroscopy is the best technique for determining secondary structure of RNAs and DNAs in association with virus proteins or with histones. Reference spectral states for fully stacked and paired polynucleic acids are supplied by pure doublestranded DNA or highly basepaired RNA. The wholly denatured state is modeled by thermally denatured nucleic acids. Decrements in base stacking, hydrogen bonding, and changes in backbone configuration upon association with protein .can then be followed by monitoring the score of Raman modes described above, with the proper normalizations for base composition. Raman spectral
’
” G.
78
J. Thomas, Jr., Appl. Spectrosc. 30, 483 (1976). K. A. Hartman, R. C. Lord, and G. J. Thomas, Jr., in “PhysicoChemical Properties of
Nucleic Acids” (J. Duchesne, ed.), Vol. 2, p. 1. Academic Press, New York, 1973. G . J. Thomas, Jr. and Y. Kyogoku, in “Practical Spectroscopy” (E. G. Brame, Jr., and J. G . Grasseli, eds.), Vol. 1, Part C, p. 717. Dekker, New York, 1977. 8o R. C. Lord and G . J . Thomas, Jr., Spectrochim. Acta, Part A 23A.2551 (1967). E. B. Brown and W. L. Peticolas, Biopolymers 14, 1259 (1975). 8 2 S. C. Erfurth, J. K. Kiser, and W. L. Peticolas, Proc. Nutl. Acad. Sci. U. S. A . 69, 938 (1972). 8 3 G. Forrest and R. C. Lord, J . Raman Spectrosc. 6, 32 (1977). 8 4 S. C. Erfurth, P. J. Bond, and W. L. Peticolas, Biopolymers 14, 1245 (1975).
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145
studies have thus shown, for example, the apparent preservation of Bhelical structure upon binding of DNA to polyLlysine and polyLarginine, contrasted to the introduction of some disorder into some Atype RNAs upon similar binding77235 ; noncooperative thermal rupture of base pairs, as contrasted to cooperative unstacking of bases in the RNA of MS2 virus, and stabilization of the RNA by the presence of MS2 coat proteinB6;and ~ * * ~ specific guaninearginine the state of DNA in c h r ~ m a t i n , ~including attachment.” The major conformations of histones or virus proteins can be simultaneously determined in association with the nucleic acids, as well as Raman studies of details of DNA structure, such as the effects of cross linking” or a l k y l a t i ~ n are ~ ~ also highly rewarding. Recent Raman studies of nucleicacids using ultraviolet excitation74376393*94 demonstrate that these resonanceenhanced spectra can now be isolated where the nucleic acids occur as a minor (<lo %) ‘biologicalcomponent.
3.7.3. Raman Spectral Analysis of Polysaccharides
Polysaccharides that lack repeating structure, such as the branched oligomers attached to glycoproteins, are poor candidates for Raman analysis: their spectra are weak but complex. For homopolymers such as cellulose95 and a m y l o ~ eand , ~ ~copolymers such as hyaluronic acid and chondroitin sulfates,97one can determine the configuration of the glycosidic linkages97 and of side groups such as the sulfate groups in chondroitin sulfate97 and differentiate polymorphic forms, as has been done for a m y l o ~ e . ~ ~
B. Prescott, C. H. Chou, and G. J. Thomas, Jr., J. Phys. Chem. 80, 1164 (1976). G. J . Thomas, Jr., B. Prescott, P. E. McDonaldOrdzie, and K. A. Hartman, J. Mol. Biol. 102, 103 (1976). G . J. Thomas, Jr., B. Prescott, and D. E. Olins, Science (Wushingron, D.C.) 197, 385 (1977). 8 8 D. C. Goodwin and J. Brahms, Nucleic Acids Res. 5, 835 (1978). S. Mansy, S. K. Engstrom, and W. L. Peticolas, Biochem. Biophys. Res. Commun. 68, 1242 (1976). 90 J.G. Guillot, M. Pezolet, and D. Pallotta, Biochim. Biophys. Acfa 491,423 (1977). 9 1 R. Herbeck, T.J. Yu, and W. L. Peticolas, Biochemistry 15,2656 (1976). 9 2 S. Mansy and W. L. Peticolas, Biochemistry 15,2650(1976). Y . Nishimura, A. H . Hirakawa, M. Tsuboi, and S. Nishimura, Nature (London) 260, 173 (1976). 94 L. Chinsky, P. Y . Turpin, M. Duquesne, and J. Brahms, Biochem. Biophys. Res. Commun. . . 75, 766 ( 1 977). 9 5 J. J. Cael, K. H. Gardener, J. L. Koenig, and J . Blackwell, J. Chem. Phys. 62, 1145 (1975). 96 J. J. Cael, J. L. Koenig, and J. Blackwell, Biopolymers 14, 1885 (1975). 97 R. Bansil, I. V. Yannas, and H. E. Stanley, Biochim. Biophys. Acra541,535 (1978).,
85
86
’’
\
146
3.
RAMAN SPECTROSCOPY
3.7.4.Raman Vibrational Analysis of Protein Conformation
The polypeptide backbone of proteins is revealed in the Raman spectrum as a superposition of vibrations from each planar peptide unit. The strongest of these vibrational bands are the amide I (primarily C=O stretching plus CN stretching) and amide I11 (CN stretching plus NH inplane bending) modes. Extensive model studies and normalcoordinate calculations on homopolypeptides and simple copolymers have established the frequencysecondary structure correlations shown in Table IV. These have been refined by studies of natural proteins, which as heteropolymers have lower symmetry, diverse sidechain populations, and different chargedresidue distributions, thus imposing further spectral qualifications. The reader is referred to several review articles.' The uniformity of the backbone CCN conformation and the welldefined intrachain and interchain hydrogen bonding patterns in the a helix and antiparallel j sheet, respectively, result in fairly sharp amide I and amide 111 bands. The threefold helix assumed by polyglycine I1 has also been analyzed. O 2 The socalled randomcoil or disordered structure encompasses a variety of conformations and hydrogen bonding and solvent ,I for rotation contacts. A general correlation between the torsional angle $ about the C"C peptide bond and the amide 111 band frequency has been proposed"' ; this correlation must be modified by considering additional factors including the angle of rotation about the C"N bond and the range of hydrogen bonding strength^.'^'*'^^ The resulting amide I and 111 bands are relatively broad, and the frequency for the amide I11 band in particular is relatively variable; the amide I band frequency is suggested to be a more dependable marker for the disordered state.Io3 The amide I and amide 111 bands are used to estimate the amounts of each type of secondary structure in a protein. The amide I band overlaps a broad water band which must be ~ u b t r a c t e d ,or, '~~ more commonly, spectra of proteins in solution must also be obtained in D 2 0 . Upon hydrogendeuterium exchange on the amide nitrogen, the amide I band shifts to slightly lower frequencies; the amide 111 band shifts to 1110950 cm', a region which contains a substantial number of other vibrational modes. The observed translocation of peaks upon deuteration helps confirm their
8398101
B. G. Frushour and J . L. Koenig, in "Advances in Infrared and Raman Spectroscopy" (R. J. H. Clark and R. E. Hestor, ed.), Vol. 1, p. 35. Heyden, London, 1975. 99 N.T. Yu,CRC C r i f . Rev. Ezochem. 4, 229 (1977). l o o H. E. Van Wart and H. A. Scheraga, Methods Enzymol. 49G, 67 (1977). l o ' R. C. Lord, Appl. Specfrosc. 31, 187 (1977). l o 2 E. W. Small, B. Fanconi, and W. L. Peticolas, J . Chem. Phys. 52, 4369 (1970). '03 S. L. Hsu, W. H. Moore, and S. Krimm, Eiopolymers 15, 1513 (1976). I o 4 T. W. Barrett, W. L. Peticolas, and R. R. Robson, Eiophys. J . 23,349 (1978).
3.7.
CONFORMATIONAL STUDIES
147
TABLE IV. Selected ConformationSensitive Modes of Proteins
v (cm')
16701655 (16601632)/ 16901685, 16451640 (variable) 1670 (1660) sharp 1664 (1657) broad 1655 (1632) 13151235 13151275 weak 1305 12541240broad 12391235 sharp 995 (1010) 940 (950)
Assignment
Conformational sensitivity
Polypeptide backbone". Amide I band: Secondary structure: turn ("U turn"). Antiparallel fl sheet Disordered a helix Secondary structure: u helix fl turn Disordered Antiparallel p sheet Antiparallel p sheet a helixg
__
Amide 111 band :
CC stretch CC (CCN) stretch
Amino acid side groups 2580 2560 1410 1360 850, 830
S  H stretch
COO symmetric stretch Tryptophan ring Tyrosine doublet
745700,670630 5405 10
CS stretch, methionine SS stretch, cystine
Secondary, tertiary structure : Free SH, its accessibilityh Intensity increases with ionizatione Intensity decreases with denaturation'.' Relative peak heights reflect ionization, H bonding strength, indirectly reflect environmentk Frequency ranges for gauche, trans conformers, respectively' Frequency sensitive to bond conformationb*"'
T. G . Spiro and B. P. Caber, Annu. Rev. Biorhem. 46, 553 (1977). N.T. Yu, CRC Crir. Rev. Biochem. 4, 3 (1977). T.J. Yu, J . L. Lippert, and W. L.Peticolas, Biopolymers 12, 2161 (1973). S. L. Hsu, W. H. Moore, and S. Krimm, Biopolymers 15, 1513 (1976). J . Bandekar and S . Krimm, Proc. Nutl. Acad. Sci. U.S . A . 76, 774 (1979).
Parentheses indicate frequencies observed in D,O solution. B. G . Frushour and J. L. Koenig, Biopolymers 13, 1809 (1974). N.T. Yu and E. J. East, J . Biol. Chem. 250, 2196 (1975). ' M. C. Chen, R. C. Lord, and R. Mendelsohn, J . Am. Chem. Soc. %, 3038 (1974). N.T. Yu, J . Am. Chem. Soc. 96,4664 (1974). ' M. N . Siamzawa, R. C. Lord, M. C. Chen, T. Takematsu, I. Harada, H. Matsuura, and T. Shimanouchi, Biochemisfry 14,4870 (1975). N. Nogami, H. Sugeta, and T. Miyazawa, Bull. Chem. SOC. Jpn. 48,2417 (1975). H. Sugeta, Specfrochim. Acfa, Parf A 31A, 1729 (1975).
'
'
148
3.
RAMAN SPECTROSCOPY
assignment. The weak amide I11 band of the a helix at about 1300 cm' is superimposed upon methylene deformation modes; loss of 30% of the band intensity in this region upon deuteration indicates very high helix content.99 To quantify the amounts of each type of structure in the protein it is assumed that the intensity of each vibrational mode employed as a conformational marker is linearly related to the concentration of the conformer. The intensity of the methylene deformation modes at 1450 cm from side chains of the protein is used as an internal reference. Several groups of workers have made such estimate^.'^^'^^ L'ippert and c o  w ~ r k e r s ' ~ ~ estimated fractions of ahelix, psheet, and disordered structure in ten proteins in solution; the estimates fell within the range of results from xray diffraction and circular dichroism studies. The heterogeneity of the natural proteins and the variety of structural segments found therein probably precludes accurate estimation of any but the predominant structure. The amide I, nominally infraredactive amide 11, and amide I11 modes in simple amides are enhanced upon ultraviolet excitation'08; it may be possible to separate these modes in proteins from the rest of the protein spectrum by their excitation profiles. Raman spectroscopy is uniquely qualified for comparative studies of crystalline and solution states of soluble proteins. For small proteins, the introduction of solvent may considerably alter conformational energies so that moderate changes in backbone conformation become apparent. For larger proteins, a number of sidechain vibrational modes act as additional Raman parameters of conformational alteration. These are described below (Section 3.7.5). Unlike circular dichroism and optical rotatory dispersion spectra, Raman spectra of proteins in solution are not affected by solution turbidity. Comparative Raman studies of many proteins in crystalline, lyophilized, and dissolved (native and denatured) states have been p e r f ~ r m e d . ' ~ ~ ~ Structure ~'~' determinations by xray diffraction of crystalline proteins provide the most detailed reference state for these comparisons. Raman spectral mapping of conformational variation in proteins has a broad field of applicability. Isoenzyme conformations, for example, have been distinguished. O9 Such mappings can detect irreversible changes associated with preparative procedures or protein modification for
'
'
Io5
B. G. Frushour and J. L. Koenig, Biopolymers 13, 1809 (1974). M. Pezolet, M. PigeonGosselin, and L. Coulombe, Biochim. Biuphys. Acta 453, 502
(1976).
I07
lo*
J. L. Lippert, D. Tyminski, and P. J. Desmeules, J . Am. Chem. SOC. 98,7075 (1976). I. Harada, Y. Sugawara, H. Matsuura, and T. Shimanouchi, J . Raman Spectrosc. 4, 91 J. Twardowski, Bipolymers 17, 181 (1978).
(1975).
lo9
3.7.
CONFORMATIONAL STUDIES
149
cases where the protein cannot be monitored by an assay of biological activity. Few nonresonance Raman studies have attempted to detect overall conformational changes in large proteins associated with biological action ; noteworthy is the report of increased backbone disordering in rabbit antiovalbumin upon precipitation with ovalbumin.' This study is also noteworthy in confirming structure not previously well documented in the protein under study. The biologically active site of small peptide hormones and antibiotics occupies a large proportion of the molecule, so that behavior of the amide I and ester carbonyl stretching modes can be largely associated with the active site. Prototypical Raman studies include those of oxytocin,"""* valinomycin,' 3 , 1 l4 and gramicidin A'.' For large proteins, studies of the active site are best accomplished by exploiting resonance Raman effects that are dealt with below (Section 3.7.8). Raman spectral resolution of the conformation of opsin'16 and red blood cell proteins'17 in the native membranes, of lens protein in the intact ocular lens," and of proteins in singlecatch muscle fibers' demonstrates the capability of the technique for wholetissue analysis. The best objects for this approach are tissues rich in a single protein, so that the Raman spectrum does not comprehend a rather meaningless average of protein conformations.
''
'
3.7.5. Vibrational Markers of Protein SideGroups
The frequency of the stretching mode of the SS linkage in cystine (540485 cm') is a function of the CSSC and the SSCC dihedral angle~.'~*' 1 9 , 1 2 0 Identification of the disulfide band can be verified by chemical modification. Structural heterogeneity can in some cases be inferred from this mode; for example, the distribution of SS frequencies allowed inference of multiple conformers of the nonapeptide lysine vasopressin in
P. C. Painter and J. L. Koenig, Bipolymers 14, 457 (1975). F. R. Maxfield and H. A. Scheraga, Biochemistry 16,4443 (1977). A. T. Tu, J. B. Bjarnason, and V. J. Hruby, Biochim. Biophys. Acta 533,530 (1978). ' I 3 I. M. Asher, K. J. Rothschild and H. E. Stanley, J . Mol. Biol. 89,205 (1974). ' l 4 K. J. Rothschild, 1. M. Asher, H. E. Stanley, and E. Anastassakis, J. Am. Chem. Soc. 99, 2032 (1977). K. J. Rothschild and H. E. Stanley, Science (Washington, D.C.) 185,616 (1974). 'I6 K. Rothschild, J. R. Andrew, W. J. DeGrip, and H. E. Stanley, Science (Washington, D.C.) 191, 1176 (1976). 'I7 J. L. Lippert, L. E. Gorczyca, and G . Meiklejohn, Biochim. Biophys. Actu382,51(1975). 'I8 M. Pezolet, M. PigeonGosselin, and J. P. Caille, Biochim. Biophys. Acta533,263 (1978). 'Iy H. Sugeta, Spectrochim.Acta, Part A 31, 1792 (1975). H. E. Van Wart and H. A. Scheraga, J. Phys. Chem. 80, 1823 (1976).
'lo
'I1
150
3.
RAMAN SPECTROSCOPY
aqueous solution.’ Similarly, the conformation of the thioether side chain of methionine is sensitively mirrored by the CS stretching frequency (745630 cm’).”’ Tyrosine exhibits a Fermi resonance doublet, involving ring modes, at 850 and 830 cm’. The relative heights of peaks within the doublet are sensitive to the state of ionization and the type of hydrogen bonding of the phenolic hydroxyl group ; the peak height ratio may be interpreted, indirectly, in terms of tyrosine residues being buried within or exposed at the aqueous interface of the protein, and may be used to monitor protein unfolding.”’ The intensities of tryptophan ring modes at 1359and 1337cmare indicative of tryptophan environment, diminishing when tryptophans are exposed to ~ o l v e n t . ’ ’ ~The * ~ ~intensities ~ of tryptophan modes preresonance enhanced by excitation at 363.8 nm have been tested as monitors of the tryptophancontaining active site of l y s ~ z y m e . ’ ~ ~
3.7.6. Resonance Raman Studies of Porphyrins and
Hemoproteins
The most popular object of resonance Raman biophysical studies is the porphyrin macrocycle, the ubiquitous chromophoric site of redox and oxygen binding activity. Several reviews of work in this field are availab1e.15~18~27,99~1261’* The resonance Raman studies of hemoproteins described briefly here supply examples of the properties of Aterm and Bterm scattering. The 16atom inner ring of the porphyrin macrocycle has 18 n electrons. The lowest two of the inplane xn* transitions in hemoproteins and metalloporphyrins are split by configuration interaction to form the Soret ( 400 nm) and the o! and fi bands (above 500 nm) where the fi band, a higherenergy sideband to the ci band, results from vibronic mixing between the Soret and o! transitions. Excitation at laser frequencies near the intense Soret band enhances only totally symmetric, or Aterm vibrational mode^.^^.^^." The complete excitation profile of the Soret band has not been obtained
12’
”’ M. N. Siamzawa, R. C. Lord, M. C. Chen, T. Takamatsu, I. Harada, H. Matsuura, and
T. Shimanouchi, Biochemistry 14,4870 (1975). 1 2 3 M. C. Chen, R. C. Lord, and R. Mendelsohn, J . Am. Chem. SOC. 96, 3038 (1974). 1 2 4 N.T. Yu, J . Am. Chem. SOC. 96,4664 (1974). 1 2 ’ K. G. Brown, E. B. Brown, and W. B. Person, J.Am. Chrm. SOC. 99,3128 (1977). 1 2 6 R. H. Felton and N.T. Yu, in “The Porphyrins” (D. Dolphin, ed.), Vol. 3, Part A, p. 347. Academic Press, New York, 1978. 12’ T. G. Spiro, Proc. R. SOC. London, Ser. A 345, 89 (1975). 1 2 8 P. R. Carey, Q. Rev. Biophys. 11, 309 (1978).
N. Nogdrni, H. Sugeta, and T . Miyazawa, Bull. Chem. SOC. Jpn. 48, 2417 (1975).
3.7.
CONFORMATIONAL STUDIES
151
because of the lack, until recently, of a spectrum of nearultraviolet laser frequencies. Partial data are available for ferrihemoglobin22 and cytochrome P450,,, . 2 9 Apparently because of the weakness of the c1 band, Aterm scattering is difficult to observe by excitation at wavelengths near the c1 band. Instead, excitation near the a and p bands results in enhancement of Bterm modes identified by their depolarization ratio^.^^,^^ The vibronic mixing of u and Soret bands which is responsible for the p band gives rise to the Bterm s ~ a t t e r i n g ~(but ~ . ~see ’ also Ref. 130). The excitation profile (mode intensity versus wavelength) for each resonanceenhanced Raman mode of vibrational frequency v, possesses a peak coincident with the uband maximum, and a secondary peak displaced into the p band by v, .27 This behavior is predicted by vibronic theory for simple Bterm ~ c a t t e r i n g . ~ , ’The ~.~~ excitation profile for the depolarization ratio peaks midway between the two intensity profile peaks.2s,’3 1 Far from resonance, the depolarization ratio must approach a value p 5 0.75 characteristic of the nonresonant scattering process. When the Raman scattering tensor is not symmetric, there are actually three tensor invariants: the trace and the symmetric and antisymmetric parts of the a n i ~ o t r o p y . ’ The ~ ~ depolarization ratio p , which supplies just two invariants, is usually measured in resonance Raman work and may be sufficient for mode assignment and for determining the actual preservation of symmetry in the molecule. For example, the observation of several bands showing anomalous polarization where inverse polarization was expected has been interpreted in terms of reduction of porphyrin symmetry by the effects of substituents or by rhombic di~tortion.’~~.’ 34 The alternative interpretation of accidental degeneraciesamong modes of different symmetry, producing “hybrid ” depolarization ratios, was considered unlikely in these cases. It is also possible to measure all three scatteringtensor invariants by means of a circularly polarized laser beam and additional Such measurements for the resonanceenhanced modes of ferrocytochrome c have allowed the conclusion that the porphyrin symmetry is in fact lower than that of point group C4v.133 The lifetimes of resonance Raman transitions can be estimated from excitation profile bandwidths; comparisons of the perturbation of heme
’
P. M. Champion, I. C. Gunsalus, and G. C. Wagner, J. A m . Chem. SOC.100,3743 (1978). Y. Nishimura, A. Y. Hirakawa, and M. Tsuboi, J. Mol. Spectrosc. 68, 333 (1977). 1 3 ’ D. W. Collins, D. B. Fitchen, and A. Lewis, J . Chem. Phys. 59,5714 (1973). 1 3 2 T. G. Spiro and T. C. Strekas, Proc. Natl. Acad. Sci. U . S. A. 69,2622 (1972). 1 3 3 J. Nestor and T. G. Spiro, J. Raman Spectrosc. 1, 539 (1973). 134 R. Mendelsohn, S. Sunder, and H. J. Bernstein, J. Raman Spectrosc. 3, 303 (1975). 1 3 5 M. Pezolet, L. A. Nafie, and W. L. Peticolas, J. Raman Spectrosc. 1 , 4 5 5 (1973).
””
152
3.
RAMAN SPECTROSCOPY
TABLE V. Selected ResonanceEnhanced Modes of Hemoproteins
1 (cm')
Polarization"
Configurational sensitivity
Porphyrin ring modes (CC, CN stretch, methine bridge CH bend) Frequency sensitive to spin and oxidation stateb 16421 60 1 dP 15901570 aP Frequency sensitive to ring centerpyrrole N distance, ring size' 15021466 P Frequency sensitive to oxidation state' 13751344 P Frequency sensitive to oxidation state, ligand electron donating capacityb
567 4974 13
Feaxial ligand stretching modes Fe0 stretch (oxyhemoglobin), bond strengthd P Feaxial ligand (methemoglobin), sensitive to ligand identity'
Here dp denotes depolarized; ap, anomalously polarized; p, polarized. P. M. Champion, I. C. Gunsalus, and G. C . Wagner, J. Am. Chem. SOC.100, 3743 (1978). L. D. Spaulding, R. C . C. Chang, N.T. Yu, and R. H. Felton,J. Am. Chem. SOC. 97, 2517 (1975). H . Brunner, Naturwissenschafen 61, 129 (1974). S. A. Asher, L. E. Vickery, T. M. Schuster, and K. Sauer, Biochemistry 16,5849 (1977).
symmetry in cytochromes c and b5 have been made using such data.'36 In sum, the depolarization ratio and the excitation profile yield evidence on deviations of a molecular configuration from its expected symmetry. Besides confirming aspects of the theory of resonance scattering, resonance Raman studies of porphyrins and hemoproteins provide detailed information on the electronic environment of porphyrin molecules in protein pockets. These pockets consist of one or more ligands to the metal ion of the porphyrin plus a protein enclosure which in some cases is covalently linked to the periphery of the porphyrin. Several bands associated with heme ring inplane vibrations and enhanced as described above act as markers of the oxidation and/or spin state of the heme (see Table V). The lowering of ring stretching frequencies in the Fe(I1) state, compared to Fe(III), has been suggested to be due to greater back donation of electrons from the ion d, orbitals into 7271" antibonding porphyrin orbitals and resultant bond weakening.'5"37~'38Variations in
'36
J. M. Friedman, D. L. Rousseau, and F. Adar, Proc. Natl. Acad. Sci. CJ. S. A. 74, 2607
T. G. Spiro and T. C. Strekas, J . Am. Chem. Soc. %, 338 (1974). T. G. Spiro and J. M. Burke, J. Am. Chem. SOC. 98, 5482 (1976).
(1977).
13'
3.7.
CONFORMATIONAL STUDIES
153
the Fe(I1)ligand bonding scheme affect these f r e q u e n c i e ~ . ' ~Un ~ *usually '~~ low frequencies for band I in cytochrome P45OCAM in both oxidized and reduced states have been taken as evidence for the identity of the strong electrondonating axial ligand of P450 as the mercaptide sulfur of cysteine.'29,'40 Other bands are also sensitive to the identities of the diverse ligands of c y t o ~ h r o m e s , ~while ~ ~ , ' the ~ ~ 1590cm' band has been correlated with ring e x p a n ~ i o n . ' ~ ~ The frequency lowering of these "marker" bands upon conversion of hemes from low to high spin has been interpreted in terms of slight doming of the heme group (downward bending of the pyrrole rings) when the iron atom moves out of the mean heme plane,lz9 and, more importantly," expansion of the heme ring as also reflected in increased ring nitrogeniron distances. ' 5 , '43 The Feaxial ligand stretching modes in hemes are desirable monitors of heme catalytic activity. These stretching modes, lying approximately normal to the set of heme ring nn* transitions, are poorly enhanced by excitation at wavelengths shorter than 500 nm (in or above the a and /? bands), but have been enhanced in methemoglobin derivatives using dye laser excitation at 500650 nm of chargetransfer bands in this region. 144 Analysis of the Raman modes described above benefited greatly from an enormous spectroscopic literature on the structurally diverse heme proteins and crystallizable metal lop rote in^.'^^ The sensitivity of resonance Raman spectroscopy to small structural changes around the heme group equals or surpasses that of xray analysis and of newer approaches such as extended xray absorption finestructure studies'46; the difficult step in resonance Raman analysis is still the precise parametrization of electronic structure in terms of Raman spectral variables.' ' * I 5 A sizable number of Raman investigations are now focused on the problem of the mechanism coupling oxygen binding to subsequent cooperative interplay in the hemoglobin tetramer, that is, how the electronic restructuring of the heme group that follows changes in iron coordination number and spinstate accompanying oxygenation, is sensed by the protein pocket
9
T. Kitagawa, Y . Kyogoku, T. Iizuka, and M. I. Saito, J . Am. Chem. Soc. 98,5169(1976). Y. Ozaki, T. Kitagawa, Y. Kyogoku, H. Shimada, T. Iizuka, and Y. Ishimura, J . Biochem. (Tokyo) 80, 1447 ( 1976). 14' T. Kitagawa, Y. Ozaki, J. Teraoka, Y. Kyogoku, and T. Yamanaka, Biochim. Biophys.
139
140
Act0 494, 100 (1977).
' 4 1
143
T. Kitagawa, Y. Ozaki, Y . Kyogoku, and T. Horio, Biochim. Biophys. Acta 495 (1977). L. D. Spaulding, R. C. C. Chang, N.T. Yu, and R. H. Felton, J . Am. Chem. Soc. 97,
2517 (1975). 144 S. A. Asher, L. E. Vickery, T. M. Schuster, and K. Sauer, Biochemistry 16, 5856(1977). 14' J. L. Hoard, Science (Washinyfon,D.C.) 174, 1295 (1971). 1 4 ' P. Eisenberger and B. M. Kincaid, Science (Washington, D.C.)200, 1441 (1978).
Woodruff and S. T. Is' 1. Farquharson. J. M. in the cubanelike FeS clusters in oxidized ferredoxin is demonstrated more convincingly by observation of splitting of one of the FeS symmetric splitting modes.) 201. inequivalence of FeS (cysteine) linkages. Biophys.15. Loehr. Salmeen. L. Rimai. G. Biochemistry 15. F. Science (Washington. V. Szabo.138*147149Similarly. Bid. K. R. Lovenberg. Resonance Raman Parameters for Nonheme Metalloproteins Chargetransfer transitions of ligandmetal complexes in nonheme ironcontaining proteins and metalloenzymes provide (Aterm) resonanceenhanced views of the active sites of these proteins.660 (1975). 831 (1978). C. H. J . ' . s 4 B. Acad.'" The identification of two FeS (cysteine) symmetric stretching modes where one was expected provides a good example of the utility of comparison of the number of observed modes. Biochemistry 17. Tang. Lutz. Szabo. Chem.154 3. A. and L. L. 62. Antanaitis. I s 3 T. Am. T. Proc.7. Babcock. A. Biochim. S. J .408 (1977). Barron and A.Nutl.' The coordination of 0. H.C."~ 3. D.151 Studies of chlorophyll environment in intact chloroplasts are also in progre~s. and W. characterized by their depolarization ratios. 800 (1978). Commun.' 54 Loss of tetrahedral symmetry is suggested by the nonzero depolarization ratio observed for a totally symmetric mode in the highly symmetric tetrahedral configuration assumed for oxidized r~bredoxin. ' ~ 53 ~ . Spiro. A. E. Vallee and R. D. Moss. Biophys. 378 (1976). Fairhurst and L. T. M . Nagai. H. Prog.150. 34. 15' S. R. Simon. Long. U. M o l . Kilmartin. 1 (1978). H. Stretching modes of the ironsulfur linkages involving sulfide or cysteine bonds to iron can be enhanced by excitation in the visible r e g i ~ n . 97.498 (1968). 93. representative references are given for this wide fie1d. 1 (1975).' 1. Perutz. V. Holm."~ Loss of symmetry. L. Biorhrm. Herskovitz. l S 2 M. the dynamics of heme restructuring upon substrate or oxygen ligation are being tackled for cytochromes. Soc. Biophys. that is. Res. R. J. Mortenson. S. to the number expected according to simple grouptheoretical rules. P. T. SOC. RAMAN SPECTROSCOPY and then by the other. 59.P. Chem. Sci. J. and G. Actu 460. ' ~ . Alkins. Am. Williams. and S. to Fe irons in hemaerythrin and to Cu ions in hemocyanin can be inferred from the 00 stretching frequencies resonance 1 4 ' 1 4 ' '49 W. 1809 (1971).'29~'36~141~'42.It '~ ~ *been ' has hypothesized that distorted geometry of the ironsulfur complexesfor example. Sutcliffe. This splitting is also seen in the spectrum of reduced highpotential iron protein. distant heme groups. the inequivalence in bond lengths in the approximately tetrahedral configuration of iron and sulfur atoms in oxidized rubredoxincan be related to redox potential in these proteins.7.
Several reviews are available. SOC. I ~ ~ The frequency is also sensitive to solvent p ~ l a r i t y . Am."l Heteroatoms bonded in conjugation with the polyene also possess T. 171 R. Thamann.Y. L. 1 5 9 P. J .2195 (1976). SOC. This approach. T. Rimai. R.3. These are enhanced primarily by Atype vibronic mixing with nz* transitions. Petersen. and I. Carriere. M. Carey. 1 6 5 R. 14. 98. Biochemistry 16. L. King. L. Van Wart.744 (1976). E. and with electronic absorption band ~ a v e 1 e n g t h . Zerbi. serve to fingerprint the length and isomeric identity of polyenes.15916s and hence is anticipated to be widely exploited.248 (1978). 15' . L. and B.I ) varies inversely with increasing electron delocalization. Honig. Biochemistry 15.7. Warshel and M . Am. Rimai. Acud. and P.stretching modes. Chem. D. 51. Masetti.. SOC.Silman. polyene length.679 (1976). 102. G. Sufra.7. resonance enhanced by admixture of C=C stretching.S. and H. Gill. R. Honig. Carey. II. CONFORMATIONAL STUDIES 155 enhanced by illumination in the chargetransfer bands of these respiratory pigrnent~. R. Chem. 16' A. yields a wealth of detail on substrateenzyme binding configurations and dynamits.5677 (1974). F. Am. SOC. M. Loehr. M . J. Am. Biochem.267 (1977). Phys. Am. 6. Nutl. B. Carey and H. W. that is. modes between 1400 and 1100 cm'. K.831 (1973). SOC. K. Res. R. R. A. J. G.7. Schneider. Proc. Doukas. R. Aton. Lett. Li. P.6776 (1971).8. Biol. Scheraga. Rumun Spectrosc. 99. McFarland. S. Heyde. N. T.726 (1977).4493 (1973). E. Acc. J . R. Chem. J . 98. and K. Kurtz. " ~ In addition. Res. Jr. M. E. Am. J . H. Biophys. Biochemistry 15. J . Chem. 95. The resonance signal intensity is compatible with kinetic studies. Loehr.3273 (1977). D.1'~18~100~128~160 3. Commun. M. 122 (1978). Lynn. P. R. J . Chem. Schneider. Biophys. J . Schneider. Careyand H. R. R. Chem. Vallee. Kumar. Kilponen. M. and G.'~" 56*1s7Copperligand modes in blue copper proteins have also been analyzed. Heyde. Young. J. Callender. Bioeng. Dellepiane. 1661 68 For alltrans polyenes.2387 (1976). Scheule. Gill. Rev. P. Chem. 128.128 3.9. Sci. Shriver. Callender and B. H. D. 6. R. K. and H. 15 . B. R. Karplus. J . G.4187 (1977).18. 96. G.' 58 The reader is referred to recent reviews. 0. Extrinsic Chrornophores for Resonance Rarnan Studies o f Proteins Vibrations involving the active site of enzymes which contain no intrinsic chromophores can be selectively enhanced upon addition of a chromophoric substrate or inhibitor. 56. U. Chem. A . the C=C stretching frequency (16001500 cm. and T. E. pioneered by Carey and coworkers. Watters. S . and P. G. and L. Carey.ResonanceEnhanced Vibrational Behavior of Biological Polyenes The exceptionally simple resonance and preresonance Raman spectra of extended linear polyenes are dominated by C=C and C. M o f . Annu. Carey and H. 5033 (1976). 93. SOC.33 (1977).100. and D. Schneider. Klotz.
Verma and D.17' These different modes have allowed very fruitful resonance Raman observations of the behavior of the retinylidene Schiff base of rhodopsin and isorhodopsin' 7' (Chapter 3. Res. Terner. A band several hundred cm' wide of the signal dispersed by a monochromator can usually be observed per pulse. ' ~ ~ The . but only surface bound in sterolfree lipid bi1aye1s. Kinetic Studies Raman spectral studies of photoinduced chemical and physical events occurring on the nanosecond time scale can be accomplished using a pulsed laser source. DeKruijff and R. ~ * The *'~~ intensity of the C=C stretching mode of p carotene decreases dramatically when the lipid matrix of the membrane passes. Membranes 2.8. Anal. 48. Nature (London) 265. Wallach. Demel. Bioehem. Acta 339. Cs. 1 7 6 A.156 3. Atkinson. as the lipid bilayer thins and becomes more 3.8).659 (1977). A. B. from the gel to the liquidcrystalline state. 76. Biophys. Commun. H. RAMAN SPECTROSCOPY resonanceenhanced modes. It can be concluded that amphotericin B is well embedded in sterolcontaining. A. Holz. Szalontai. Horvath. Finkelstein and R. Campion. probably by formation of an oligomeric sterolamphotericin B p ~ r e . F. ElSayed. Biochim. 168 (1975). combined with a gated detection system incorporating a vidicon or photodiode array target and optical multichannel analysis.' 73 The latter experiment illustrates that the dynamics of live tissue may be followed using resonance Raman probes."* The mode frequency of pcarotene in sciatic nerve tissue also responds to excitation of the nerve. I. B. Isolated cell membranes contain minute amounts of c a r ~ t e n e . Woodruff and G. and L. 186 (1976).660 (1977). H. The polyenic antibiotic amphoterician B acts only upon cell membranes containing sterol. Chem. Biochim.~~ The spectral responses probably reflect changes in the fit of the polyene among the hydrocarbon chains. Acia401. 1 7 ' A. 17' 173 . J.'47'76'177 Studies on this time scale require a signal S. a nitrogenlaserpumped tunable dye laser.Biophys. and the decrease in dielectric coefficient sensed by the polyene. P. Alltrans polyenes have been shown to act as Raman probes of membrane fluidity when they are mixed with the hydrocarbon chains of lipid membranes. W. H . with increasing temperature. ' ~ ' frequency and intensity of the preresonanceenhanced C=C stretching mode of amphotericin B decrease upon the gel to liquidcrystalline phase transition in lecithincholesterol lamellae but are indifferent to the phase transition in cholesterolfree lamellae. 377 (1973). for example. Biophys.57 (1974). and M. Bagyinka.
Terner. J. 6. ElSayed. A. These pulses have also been produced by electromechanical chopping. J. R. A. J . A. J. Steadystate concentration profiles of a photoinduced process can also be observed with conventional detection methods by means of a photolysis pulse followed after a fixed delay by a probe pulse. '*'M. Laser Spectrosc. and M. Oseroff and R. M. 233. 45. H. 18" . S. A. Modification of the scan drive has been suggested for faster scanning. 128 (1977). A.369 (1977). D. Campion. Terner. A. Nature (London). Lewis.4) or resonance CARS (Chapter 3. Science (Washington.. C. Proc.9) process. and J. A. Acad.L. Mol. A. can be achieved by computercontrolled scanning over a limited spectral region. Natl. Biochemistry 18. Kinetic processes may be triggered. the steadystate concentration profiles are pumped by a CW laser beam and detected either with a conventional photomultiplier tube or with a vidicon and optical multichannel analysis. Nut/.3629 (1979). Raman Spectrosc. consisting of glycoprotein plus retinal bound in Schiff base linkage. A. and M. R.261 (1977). Acad. B. A. Bacteriorhodopsin. Biochemistry 13. 1. ElSayed. of seconds. 527 (1979). 76. 5212 (1 977).New B i d . using a stoppedflow arrangementIE6)or temperaturejump impulses. ElSayed. as in other types of spectroscopy. ElSayed. KINETIC STUDIES 157 intensity characteristic of a resonance Raman (Chapter 3.' 78181 Studies of bacteriorhodopsin kinetics on the microsecond time scale have also been tackled by flowing the sample past the beam at a controlled. Merlin. M. Sombret. U.'" The bacteriorhodopsin studies mentioned above illustrate the dynamic and structural resolution gained by the combination of stationarystate and kinetic resonance Raman studies. Stoeckenius.3. Campion. Terner. J . using a conventional grating instrument. C.8. and J. Campion. 3046 (1979). by concentrationjump (for example. 20. A. Wallart. Terner. R. Burns. A. combined again with vidicon detection. C. S t r u t . Hsieh. Rapid scanning on a slower scale. Biophys. A . 1 8 3 J. variable flow rate. Sci. e. Kinetics of the photochemical bacteriorhodopsin cycle on the microsecond to millisecond time scale have been followed by imaginative substitution for the pulsed laser of the electromechanically chopped beam of a CW laser. and M. M . S. 4243 (1974). D. Terner. enzymesubstrate associationdissociation (time constant z < sec) and substrateinduced conformational changes < z < lo' sec) thus become feasible. C.349 (1978). ElSayed.g.''' but its photosynthetic function as a transmembrane 178 J. A. and M. Burns.C.'~. 74.L. Hsieh. 1 8 ' J. U . Biophys. Proc.' 78.IE2lB4 In this approach. J . 1328 (1977). CrunelleCras and J. '" M. Rerny. Callender. Sci. Osterholt and W . Hsieh. and F. is structurally very similar to the visual pigment rhodopsin"* for which similar studies have been perf~rrned.185 Studies of macromolecular dynamics. Adu.) 195. 149 (1971).L. 26. ElSayed. Terner. A . Marcus and A.
955 (1974). C. the prominent feature in the timeresolved spectrum is the 1530cm' band. 913 (1974). Becker. K590. Using 5nsec laser pulses at 582. R. a wavelength near the 412nm absorption maximum of M412. and M. Section 3. the other intermediates are seen only with kinetic resolution.5 nm. for example. 15. Nutl. 1 9 * D. Lozier. Science (Washington. P. and W. Shank. Sri. visible region absorption identified by the rates o ma~ima. 4462 (1974). resonance enhanced by conjugation with the retinal chain. and T. Biophys. V. The structures of the intermediates of the cycle are not apparent from the nearly structureless absorption bands at room temperature. Osterholt. 194 R. Biophys.5 nm. Aton.'^^*'^^'^^ The intermediate M412. R.7. appears to resemble 1 3cisretinal. and D .6288 (1971). 1279 (1968). the C=C stretching band at 1570 cm' associated with the intermediate M412. and L. and located at 1643 cm' when protonated or 1620 cm' unprotonated. A. B. Nakanishi. Spoonhower. J. and isomeric retinal intermediates have been thus f appearance of new. and W. Am. Lozier. In contrast (Fig. Callender. Ebrey. A . 4 . RAMAN SPECTROSCOPY proton pump is better understood than the visual process. A. the C=C stretching mode assolE9 E.176 The lower frequency of this mode is correlated with the longer absorption maximum of K 5 9 0 (cf. and K. Heyde.'~~ The step at which the Schiff base is deprotonated can be located in the timeresolved resonance Raman excitation profiles. M. In Fig.192 In the resonance Raman spectrum. Acad. A. H. 93. and the 1620cm' band associated with the deprotonated C=N stretching mode are seen to increase dramatically in time when laser excitation is at 476. R.C. Lewis. J . . Lewis.' 79*183. 4a. Proc. A .'~~The ' ~ ' cycle is shown in Fig. Doukas. H. J. Crouch.' 89 The initial rearrangement occurring in the chromophore in picoseconds (isomerization versus electron redistribution) is contr~versial. 71.9) which has been observed within picoseconds of illumination. Chance. Bogomolni. A. U. H. H. A . near the 570nm absorption maximum of R570. J .as a shoulder arising at about 1515cm' on the 1530cm. R. E. SOC. Doukas. 15. C . Gill. researchers have observed the C=C stretching mode of the first intermediate. Ippen. Callender.band of R570. Hess. Porte. M. when laser excitation lies at 514.lg3 Substantial stationarystate concentrations in bacteriorhodopsin preparations are seen only for R570 and M412. Stoeckenius. 4b).1 nm. 2004 (1977). Stoeckenius. 1 9 ' B. Rimai. but retinals can be "fingerprinted" by ~ * matched '~~ to the their resonance or preresonance Raman s p e ~ t r a ' ~and appearances of bands in bacteriorhodopsin preparation^. 1 9 5 A. S . the protonation state of the retinal chromophore is differentiated by the frequency of the Schiff base C=N stretching mode. 1 9 ' B. D. Bogomolni. B.158 3.'82 The kinetics of proton cycling of the retinylidene chromophore have been studied by absorption spectroscopy. R. G. Chem. Biochernisrry 15.) 200. Marcus. Biochemistry 16.1621 (1976).
is tuned through the desired spectral interval. primarily employing the steadystate flow technique with appropriately fast sampling by the laser beam.9. The 1643cm' band visible in Fig.v2. .9. 3. Nonlinear Phenomena Coherent antiStokes Raman scattering (CARS) is a fourphoton process accomplished with two relatively highpowered pulsed laser beams of frequencies v1 and v 2 passing through the sample at a small.v 2 is a vibrational frequency. using excitation at (a) 476. 4. Whenever the difference v1 . Spectra adapted from Campion el al. (c) Bacteriorhodopsin photochemical cycle. as suggested by the data of Fig. In the CARS process. A CARS signal beam emerges at frequency v 3 = 2v.5 nm from an argon ion laser. the subscript on each intermediate is the wavelength (nm) of its visible absorption maximum. phasematching angle. The combined results of these studies establishes M412as the first deprotonated intermediate of the photocycle.5 and (b) 514. a tunable dye laser. NONLINEAR PHENOMENA 159 . showing the isolability of structural information by a combination of excitation profile and time analysis. 4c has been obtained by similar analyses. Resonanceenhanced kinetically resolved Raman spectra of bacteriorhodopsin.3. 4b indicates the protonated state of R570. 17001500 cm'. an antiStokes Raman line is seen.Mk H ' release FIG. 4 . very high conversion efficiencies ( 104106 times those in spontaneous Raman scattering) are obtainable. It is hoped that the differential resonance Raman studies just described. and the resultant strong antiStokes Raman lines occur not only at higher frequencies than . 1 7 9 ciated with R570. presage new applications of resonance Raman techniques to other problems in biological kinetics. the parent compound. Information about the structure of the other intermediates shown in Fig. one of the incident lasers. In order to scan the Raman spectrum.
Nestor. A. Hudson. CARS is exactly the technique needed for vibrational studies of particularly fluorescent biological chromophores. a “singleshot” spectrum may be obtained. Dutta. A . 135 (1977). R. therefore. 73.84 (1978).74. Appl. 3798 (1976). only. Appl. and L. and G.multiple scattering by sample molecules results in loss of incident beam coherence and loss of the CARS signal. and G. 1 9 9 I.4146(1977). B. Hetherington. It should be noted that samples must be nonturbid . W. Acad. G . Proc. 6. 20’ J. depolarization ratios for ferrocytochrome c have also been measured in the CARS configuration. Proc. Gilson. Lett. 7. Spiro.nitrogen NH deformation mode. Toiles. S. Proc. also nonlinear in response to the applied field. D. The tremendous fluorescence rejection and signal strength means that CARS has great potential for biological studies. Sci. ’02 J. and B. R. Greenhalgh. A significant CARS background from glass sample holders has also been reported to be troublesome. W. Natl. K. Annu. Sci. 111. Biophys. assignments were suggested for some of the modes enhanced by vibronic mixing with the first nz* transition in the plane of the flavin. Klauminzer. Black. Spectrosc. McDonald. Acad. J . Hudson. K. S. Nibler. with great potential for kinetic studies. U. However. but essentially equivalent spectral information can be extracted from the CARS spectra. allowing a frequency increment in the 1359cm’ band in the presence of glucose oxidase to be interpreted as a shift in the flavin N. Raman Spectrosc. S. . I. Nestor.27 (1976). 31. G.160 3. Acad. U . K. C. illustrating the similarities in spectral profile to the resonance Raman spectrum and the absence of fluorescence background’”. Klauminzer. Sci. S. S. The inverse Raman spectrum appears as a ’’’ W . Rev. Bioeng. G. Chabay. S. Harvey. and A. 90 (1978). Spiro. Phys.3329 (1976).253 (1977). Chabay. under resonance conditions is a good CARS signal obtained. T.”~ Another Raman process.200Since resonance Raman conditions are precisely those suffering from the worst intrinsic fluorescence problems. Laycock. T. M. RAMAN SPECTROSCOPY most fluorescence but in a beam spatially separated from the incident beams and requiring only a single monochromator for filtering. B. andT. J. Hudson. Nail. 203 P. reflecting hydrogen bonding of FAD to en~yme. J . U . 73. A . B. Klduminzer. Natl. If a broadband dye laser is used. R.’” CARS spectral profiles and depolarization ratios differ somewhat from those obtained in analogous conventional and resonance Raman studies. A.’02 A resonance CARS spectrum has been obtained for ferrocytochrome c. R.202In a resonance CARS study of highly fluorescent flavin adenine dinucleotide (FAD). 961 Both conventional and resonance Raman antiStokes spectra may be obtained in a CARS experimental configuration. is inverse Raman scattering. *On J. J. 28. Cramer. J. Laser Focus 14. the background emission arising from aqueous solvent is competitive with CARS scattering from the solute. Nestor.
Ira W. J . some interesting biological investigations can be envisaged. Vol. Mathieu. Conclusions and Prognostications Raman vibrational spectroscopists who were able in the 1970s to acquire newly developed laser sources had a glorious field day of the sort which a technological revolution customarily brings about. A. taken precedence over applications of the inverse Raman effect.10. ed. Dhamelincourt. in part by using a defocused illuminating beam and a wellcooled microscope stage. The vibrational spectrum again lies at higher frequencies than the fluorescence Exploitation of CARS for biological studies has. Biological applications of resonance Raman spectroscopy. I 1. I . most especially to problems in biochemical kinetics. If specimen damage can be controlled. Inverse Raman effects can occur with other nonlinear processes: negative bands observed in CARS scanning of a vitamin B. sample have been conjectured to be due to inverse Raman effects.1 1. and also to cellular and subcellular systems including semiordered systems oriented with respect to the beam direction. 33 (1975). Heyden. Raman Spectrosc.). P. 3.200 3. a commercial model is available. 1. Delhaye and P. The Raman Microprobe A Raman microscope with simultaneous spatial and spectral resolution has found application in minerologyZo5.. however. 3. 1973. Acknowledgment The author would like to thank Dr. Levin for reading this article and for providing a stimulating research environment. ’04 D.3. Use of the microscopein biology is limited because of damage of the biological specimen by laser beam heating. and then to apply these correlations. CONCLUSIONS AND PROGNOSTICATIONS 161 vibrational absorption spectrum on the antiStokes side when a pulsed laser at fixed frequency and a continuum from a dye laser simultaneously illuminate the sample. Further work remains to be done to quantify the spectral correlations for the behavior of biological molecules. London. in “Advances in Raman Spectroscopy” (J. p. . * 0 5 M. Long. are anticipated to expand in scope.
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For example. Bloembergen. Therefore. Phys. Lett. Javan. New York. they have very broad electronic absorption or emission spectra. * M. Similarly. photon echo. Only two years after the first report on laser oscillation in 1960. F. Bennett. Nature (London) 187. S.1.. Since the environment of biological molecules is usually inhomogeneous and aperiodically organized. H.the early photoproducts of rhodopsin in visual transduction appear in a picosecond regime. All rights of reproduction In any form reserved. spectroscopy of high time resolution is of more importance to biological systems than highspectralresolution spectroscopy. “Nonlinear Optics. eds. Feld and V. “Coherent Nonlinear Optics. J. in photosynthesis. in many cases. A. R.” Benjamin.4. Letokhov. 828 (1962). J. ~addition ~~ to the abovementioned properties. ~ Qswitching techniques involve an artificial impairment of the optical path in a laser with the purpose of delaying the onset of laser oscillations in order * T. Berlin and New York. 163 METHODS OF EXPERIMENTAL PHYSICS. R. New kinds of spectroscopy have been devised taking advantage of these properties. Temporal coherence is used in coherent antiStokes Raman scattering (CARS). Jr. The spatial and temporal coherence of the laser makes it a unique light source for spectroscopy. PICOSECOND LASER SPECTROSCOPY By Takayoshi Kobayashi 4. and also in highresolution spectroscopy. The 1960s was a time of very rapid development in short pulse generation by lasers. W. 33. some kinds of lasers have one more important characteristic for spectroscopic measurementsshort pulse width. N. This property is utilized in hightimeresolution spectroscopy. Inc. Hellwarth.’ a Qswitched laser with a pulse of several tens of nanoseconds was i n ~ e n t e d . 1965. 20 Copyright 0 1982 by Academic Press. and other coherent transient spectroscopies. Rev. Spatial coherence gives a means of producing a high photon density and is used in many fields of nonlinear s p e c t r o ~ c o p yIn . S.” SpringerVerlag. VOL.493 (1960). W. 106 (1961).. ISBN 0124759629 . Herriott. the absorption of a photon by chlorophyll leads to an excited state responsible for electron transport whose lifetime is of the order of 10 psec.2 coherent Stokes Raman scattering (CSRS). McClung and R . which is the main subject of this chapter. 1980. Appl. and D. Phys. Maiman. 6. Introduction The discovery of the laser’ has led to the development of many new fields in spectroscopy.
(London). and DNA are also being studied by many research groups. in the transmission of information and in the conversion of light energy to chemical energy. J . J. Appl. To be efficient in the application of light energy in the visible region. photosynthetic applications are most prevalent. N. Phys. Phys. or photochemical transient species with rise times or lifetimes in the picosecond regime.8. and the S . Soc. W. Left. i( s 1 R. Chem.47 x 10’) v i a x E ~ V (sec). 174 (1966). 270 (1965). G.) in the visible region. G . PICOSECOND LASER SPECTROSCOPY to increase the Q value of the laser resonator and to obtain a very short (a few tens of nanoseconds) highpower pulse called a “giant pulse. The chromophores have an electronic structure similar to that of many organic dye molecules.. These improvements led to the application of picosecond laser spectroscopy to biophysical situations. R. chlorophyllprotein complexes in photosynthetic pigments. J. the techniques of picosecond laser pulse generation. Mocker and R. J. Kasha.6 Before the appearance of modelocked lasers. DeMaria.390 (1926). 95 (1968). W. ’ . The lightabsorbing substances are rhodopsin. Norrish and G.658 (1949). The chromophores in rhodopsin and bacteriorhodopsin are protonated Schiff bases of retinal and those in chlorophyllprotein complexes are chlorophylls. A. The advent of the modelocked solidstate laser7 enables one to study photophysical. 994 (1945). Nature (London) 164. Phys. Other important biological systems such as rhodopsin. Life utilizes solar energy in two ways.67. J. where solar energy transmitted to the earth is most intense. hemoglobin. Heynau. and time resolution were improved by many physicists and chemical physicists. Lewis and M. A 308.284 (1950). i. photobiological. Among the biophysical studies using picosecond laser spectroscopy. detection. Chem. 47. The relationship between the S1 t So radiative lifetime t . and xanthenes. * F. A 200. Novak and M. and bacteriorhodopsin in proton pumping pigments.164 4. from a few tens of microseconds to a few tens of nanoseconds. Radium 7. Qswitched lasers were used as powerful pulsed light sources for nanosecond spectroscopy. The chromophores and organic dye molecules have an intense transition between the ground state (So) and the lowest excited singlet state (S. = (3. Appl. Porter. D . Phys. R.Ser.” This Qswitched laser increased the time resolution of ordinary flash photolysis5 a lOOOfold. J. biological chromophores should have large capture cross sections in this region.e. 3075 (1967). (London). carotenes. Lett. Collins. A. 7. Windsor. Proc. Picosecond spectroscopy offers special advantages for the study of photobiological systems such as rhodopsin and chloroplasts. Porter. Proc. t So molar extinction coefficient E is given by the equation’ T . Soc.W. Ser. Am. As picosecond laser spectroscopy was applied to the study of semiconductor and chemical species. Perrin. G. SOC. H . in visual pigments. R. and H. Setser.
Fluorescence in complicated biological macromolecules is emitted from the lowest excited singlet state S1 because of the very efficient radiationless TABLE I.INTRODUCTION 165 where v. Since many ordinary dye molecules have a bandwidth of the order of 5 x lo3cm. which is 110 nsec. List of Abbreviations AMP AR BChl BPh BS CARS CONT CELL CSRS F 11 IR L M ML MONO Nd.4. is the wave number in cm. c Soabsorbance maximum.of the S.e.. Some of them are as follows. glass Nd/ YAG OMA P PM SC SHG SPS TEA THG TPF uv VD amplifier an tireflection bacteriochlorophyll bacteriopheophytin beam splitter coherent antiStokes Raman scattering picosecond continuum generation cell coherent Stokes Raman scattering filter image intensifier infrared lens mirror mode locked monochromator neodimium/glass neodimiumiyttrium aluminum garnet optical multichannel analyzer polarizer photomultiplier scatterer secondharmonic generator single pulse selector transversely excited atmospheric pressure thirdharmonic generator twophoton fluorescence ultraviolet variable optical delay . In addition to the application of picosecond spectroscopy to the primary photochemical processes in photosynthetic and visual pigments.is calculated by the equation to be 110 nsec. This is the reason why picosecond spectroscopy is invaluable for the study of the primary process of vision and photosynthesis.and E of the order of lo5 M . In order for biological systems to utilize efficiently the light energy absorbed by the pigments without losing the energy by photoemission processes..' cm' in the visible region. the initial photobiological reaction must be completed in a much shorter time than the radiative lifetime.1. there are a number of other important applications to biological systems. z.. i. t So absorption band. Integration is over the S. it must be in the picosecond time scale.
lasers could be used for nanosecond spectroscopy. internal conversion.2.2. Because of the interaction between these side chains and the protein constituting pigments. Comparison of Nanosecond and Picosecond Spectroscopy Flash photolysis was initiated by Norrish and P ~ r t e r who . + S.1. In Chapter 4.1310'4sec from the frequency of infrared or Raman spectra. The energy transfer should be therefore in the picosecond range. PICOSECOND LASER SPECTROSCOPY S . we can determine the structural spacings between the energy donors and acceptors. In Section 4. photosynthesis.3 examples'of the application of picosecond spectroscopy to biophysical systems. ~ applied the technique to photophysical and photochemical processes of various molecules mainly in solution.2. 4. time resolution was not limited by .1 the comparison of techniques and equipment between nanosecond and picosecond spectroscopy are made. A list of abbreviations used in this article is given in Table I. In Section 4. Not only Qswitch ruby lasers but also other nanosecond lasers such as N. At this particular stage of development. and vision are shown. After the absorption of light by antenna chlorophyll. the time constant of the radiationless process should be in the picosecond range.166 4. Nanosecond and Picosecond Spectroscopy 4.3 various methods of picosecond spectroscopy are described. is generally in the nanosecond range. The time resolution of flash photolysis was limited by the pulse width (typically a few tens of microseconds) of the excitation flash. lasers and Qswitch Nd/YAG lasers have been utilized for excitation light pulses. or for pulse width measurements. Since the radiative life of S.2 the principles of generation and the classification of picosecond light pulses are described. Therefore this process can also be studied by picosecond spectroscopy.2. In the appendix we discuss several nonlinear optical phenomena useful for generating pulsed light for excitation and interrogation. the motion might slow down to the picosecond time scale. the light energy is transferred to a reaction center without losing the energy by fluorescence emission in the transfer process.2. The application of Qswitch lasers improved the time resolution of photolysis experiments by a factor of a thousand. Since absolute rates of transfer are obtained by picosecond spectroscopy. Some of the optical elements and detectors used in picosecond spectroscopy are also described briefly in the Appendix. The motion of side chains of biological macromolecules or polymers are known to occur in 10. In Section 4. Soon after the success of Qswitch oscillation of ruby lasers.
There are two approaches to nanosecond or picosecond spectroscopy. in “Ultrashort Light Pulses” (S. In the pulse technique.4. The classifications of nanosecond and picosecond absorption and emission spectroscopy are summarized in Table 11. and that of the timeresolved emission or absorption spectrum. a perturbation of nanosecond or picosecond duration is given to a sample. L. one can determine the time course of the resulting process by observing the intensity of the monitoring light or the emission. p. 1977. Berlin and New York. The modulation method can be used only for emission processes exhibiting a single exponential decay. a pulse technique and modulation technique.). boxcar integrators. lockin amplifiers.1. ed. such as wideband (up to 1 GHz) oscilloscopes. H. Shapiro. D. several completely new detection methods were invented. and transient digitizers have become commercially available in the past 20 years and have been extensively used for nanosecond to millisecond laser photolysis and spectroscopy.2.2. 1977. the sample is periodically perturbed by excitation light with wellknown phase characteristics. p.). these devices are not suited for picosecond spectroscopy because they do not have time resolution on the picosecond time scale. J . 18. Auston. However. SpringerVerlag. L.1. yon der Linde. If an emissive species with a finite exponential decay lifetime z is excited by a modulated light source with frequency w. ed. Excitation Pulsed Light Source for Picosecond and Nanosecond Spectroscopy.‘ the ’ D. light emitted from the species is also modulated with the same frequency and a phase shift 8 given by tan 8 = o z . the lifetime of the species can be obtained. Picosecond or nanosecond spectroscopic methods can be classified as either emission or absorption methods. In what follows we discuss only the pulse technique.’”. SpringerVerlag. Berlin and New York.2. in “Ultrashort Light Pulses” (S. Shapiro. NANOSECOND AND PICOSECOND SPECTROSCOPY 167 detectors but mainly by the excitation laser pulse width. singlephoton counters.By measuring the phase shift 8. 4. L. p. 1977. In either case.). 4. Excitation light sources only are used for emission spectroscopy. 123. In order to resolve times in the picosecond region. Berlin and New York. .1 . Picosecond excitation light for the pulse method can be obtained from various kinds of modelocked lasers’: induced Raman scattering of modelocked lasers from various kinds of liquids. In the modulation technique. By using perturbing pulses with a shorter pulse width than the time constants of the phenomena to be observed. highspeed photomultipliers. there are two types of measurement: the observation of the time dependence of emission intensity or absorbance. Classification of Picosecond and Nanosecond Spectroscopy. 204.2. SpringerVerlag. Bradley. D. while excitation and monitoring light sources are used for absorption spectroscopy. Many sophisticated electronic devices. Shapiro. in “Ultrashort Light Pulses” (S. ed.
ed. Inc.rrrs2. synchronously pumped dye lasers Picosecond continuum: nanosecond laser: nanosecond flash lamp Subpicosecond continuum Su bnanosecond (1 0. 4.I nsec) Excitation light sources Detector systems Photomultiplier. N. variable delay. Kerr gate. sampling scope Qswitch ruby and Xe flash lamp: Nd.. flash lamps Modelocked gas (HeNe. dye lasers pumped by the above lasers. synchronously pumped dye lasers Passively modelocked dye lasers. TEA N. and photomultiplier Variable delay and photoinultiplier . streak camera Picosecond ( 1000. N. Phys. very widehand oscilloscope. 65 (1964). Kunz.1 nsec) Sampling oscilloscope. ((1966). Kerr gate.Am.’ Synchrotron radiation can also provide several ten or several hundred picosecond pulses. H. ’. G . “Synchrotron Radiation Techniques and Applications. l 4 V. 191 (1968). .’’ Singleshot solid lasers. Model 31A and 32A. fast photodiode. Nd/glass. Nd/YAG). modelocked solidstate lasers (ruby. Soc. variable delay and photomultiplier. such as nitrogen.!. Young. lasers and dye lasers pumped by the lasers Modelocked solidstate lasers (ruby.users 1. Lu.’ Nanosecond flash lamps such as hydrogen.YAG lasers. such as Qswitch ruby14 and Nd/glass’ lasers.I6 hydro ’ C. Xe flash lamp Ar ion) lasers. repetitively pulsed lasers.’ parametric oscillation. excimer lasers.I pscc) Echelon and O M A . Evtuhov and J . Neeland. 1979. . G . and D. deuterium. PICOSECOND LASER SPECTROSCOPY Classification of Emission and Absorption Spectroscopy in Nanosecond to Subpicosecond Time Region Monitoring light sources for absorption Time domains Nanosecond ( 1000. lasers.1 psec) second and higher harmonics of the picosecond pulse. 9. and nitrogen lamps with a pulse width of a few nanoseconds are commercially available as excitation light sources. K. and photomultiplier Subpicosecond (1 0. TRW. variable delay..” SpringerVerlag. Snitzer and C. I ” H. B14li. l 3 “Handbook of TRW Fluorimetry” Manual of T R W Nanosecond Fluorimetry Equipment. wideband oscilloscope: boxcar integrator. Ndiglass. air breakdown and H. l 5 E. streak camera.168 TAHLE II. Berlin and New York. Nd!YAG). Heard.
The chemical composition of ruby for use in laser oscillation is AI.. NANOSECOND A N D PICOSECOND SPECTROSCOPY 169 TABLE 111. laser oscillation takes place between a lower level and an upper one where the latter is populated by relaxation from excited levels.. ed. 1979.6 0.030. laser H. with 0.4.71 61. Today 31. 1 (1976). l9 ’’ H. Lasrrs4. J . laser oscillation takes place between an upper and a l7 P. A. 1068 (1965). Bazhulin.3 347. 1260 (1960). and G. Ch. Danielmeyer.2. JETP (Engl. Dye lasers pumped by excimer lasers as well as excimer lasers2’ themselves are also useful light sources for nanosecond experiments.. K . G . Phys.20. N.1 156. In a fourlevel system. 1973. ’O . Wavelength of laser oscillation. ed.530 193 248 308 3s I single shot single shot single shot single shot 10 1064 532 10 1020 1020 1020 1020 50200 1040 1050 315 12 57 L 1020 1020 20 20 30 2030 2 0. laser Solidstate lasers Ruby laser 2nd harmonic Ndlglass laser 2nd harmonic NdiYAG laser 2nd harmonic Excimer lasers ArF laser KrF laser XeCl laser XeF laser I. peak power. Schafer. J. Trans/.” SpringerVerlag. Phys. and energy are typical of commercially available lasers or homemade lasers.310) x 103 (10SO) x 106 5 1 13 0. In a threelevel laser system. Nd/YAG is yttrium aluminum garnet doped with Nd3+.2 I060 1001 < 10 0.11 0. “Excimer Lasers. Pulsed Lasers for Nanosecond Spectroscopy Typicalb repetition rate (Hz) Typicalb peak power (MW) Typical singlepulse energy’ ( J ) Pulse width (nsec) Laser Gas lasers N. Rhodes. Petrash. (nmy 337.04 ’ Repetition rate.O.3 694.)20. F.05 % (by weight) C r 2 0 3 where only the chromium ions participate in the laser oscillation phenomenon. Ewing. its laser oscillation is interpreted in terms of a fourlevel laser. Berlin and New York. and dye lasers” pumped by flash lamps or nanosecond lasers have been powerful light sources for nanosecond spectroscopy and laser photolysis. Soc.05 0.3 310 15 2030 2030 15 15 .” SpringerVerlag. P.01 200300 4060 (0. Ruby laser oscillation is interpreted in terms of a threelevel laser. Knyazev.11 0. genI7 and Nd/YAG lasers”. 32 (1978). I. G . “Dye Lasers..1 0.02 0. Berlin and New York. 22.
As the light beam passes through the active medium. ruby.1. and pulse width of these nanosecond lasers are listed in Table 111. The gain of the active medium of a threelevel system is more sensitive to temperature than that of a fourlevel system.2. the former is populated by exciting Nd3+ from the ground level to levels located above the upper level. The laser pulse increases in intensity when it passes through the active medium.2. and a laser gain medium. l b and is given as follows. The timedomain explanation is illustrated in Fig.2. Mode locking in frequency (a) and time domains (b). the beam then passes through the dye. an active medium. a passively modelocked laser is obtained by inserting a saturable dye in a cavity composed of mirrors. Generation of Picosecond Pulse. In the cavity.2. 4. . shortening the pulse width each time it passes through the dye solution.CAL RESONATOR RESPONSE CURVE FREQUENCY MODELOCKED LASER OUTPUT  LkJduL TIME (b) FIG. 1 . the light intensity in the wavelength region where the gain is greater than the loss is increased while it is decreased outside this spectral region. Experimentally. Nd/YAG. where the noise and leading edge of the pulse are absorbed more than the peak and trailing edge because of nonlinear absorption in the dye solution. The oscillation wavelength. Kr ion. peak power.170 4. Pulse trains from a real GAIN CURVE (MATERIAL RESPONSE) OPT. or dye solution). Ar ion. Picosecond Light Pulses 4. the beam passes back and forth many times. The operating principle of the modelocked laser can be explained in terms of either the time domain or the frequency domain (Fig. 1). PICOSECOND LASER SPECTROSCOPY lower level. with the final duration of the pulse theoretically limited by the gain spectrum width of the laser medium used (Nd/glass. pulse energy.
2. They can be classified as follows: (i) singleshot lasers and (ii) repetitively pulsed lasers. namely. which undergo irreversible photochemical reactions.2. 103. the output of the laser varies in the distribution of the phases and amplitude of the longitudinal frequency components. The Nd/YAG laser and passively or synchronously modelocked dye lasers pumped by an Ar or Kr ion laser are repetitively pulsed lasers. When the repetitively pulsed lasers are used for photoreactive samples. The longitudinal modes are separated in frequency by c/21. The geometrical configuration of the laser resonator and gain medium is responsible for resonator loss. eds. If a set of longitudinal modes is maintained in a fixed phase and fixed amplitude relationship.). P. With a lasing medium of an adequate bandwidth. in “Picosecond Phenomena” (C. where 1 is the optical path length between the highly reflective back mirror and the output mirror which make up the laser cavity and c is the speed of light. p.2. a picosecond pulse train with a constant time separation of c/21 can be produced. 4. Without the mode locker. SpringerVerlag. Ippen. Repetitively pulsed lasers are more appropriate for obtaining more precise data since it is easy to average data at high repetition rates. P. Classification of Picosecond Light Pulses. NANOSECOND AND PICOSECOND SPECTROSCOPY 171 resonator do not achieve the theoretical pulse width limit. Shapiro. and S. as well as dye lasers synchronously pumped by the fundamental or second harmonic of a ruby laser or the second or third harmonic of a Nd/glass laser.4. This is due to spreading of the pulse width caused by laser material and cavity dispersion. or in biological tissues must be moved by a motor attached to the sample so that the irradiated part of the sample goes out of the probing region. This E. L.2. Each laser material (gain medium) has its own gain curve. the ruby laser. in glass matrices of organic solvents at low temperatures. the Nd/giass laser. There are various kinds of singleshot modelocked lasers. the samples must be circulated using a pump and a flow cell. 1978. Shank. lppen and C. V. in polymer matrices. the output will be a welldefined function of time forming the modelocked laser pulse train. Each transverse mode in a laser cavity has a set of longitudinal modes in the frequency region where the laser gain overcomes resonator loss. The mechanism for modelocking a laser as visualized in the frequency domain is illustrated in Fig. la. . Photoreactive samples in the crystalline phases. Berlin and New York. V. E. Recently Shank et aL21 succeeded in producing subpicosecond pulses of several gigawatts at a repetition rate of 10 Hz by means of an Arlaserpumped rhodamine 6G laser amplified by a threestage dye amplifier pumped by the second harmonic of an amplified Qswitched Nd/YAG laser. Several typical picosecond light sources which can be used for research in biology are listed in Table IV. Shank. the ff ashlamppumped dye laser.
E. Phys.001 5 x IO~ 610630 10Hz 1000 0.2. V.5 Wavelength of laser oscillation.1 MHz 100 0. Lett. Ippen and C. The reason why so many different schemes have evolved is that there are various picosecond lasers. 103. 24. in "Picosecond Phenomena" ( C . Berlin and New York. detection .. 4. Appl. Shank.3. P. V. 373 (1974). In the following section. SpringerVerlag. L. E. (nm)" 694. V. C. Ippen.5 0. we give some details of several different experimental schemes summarized in Table V. Pulsed Lasers for Picosecond Spectroscopy Typicalb energy per pulse (mJ) Laser Ruby laser (amplified single pulse) 2nd harmonic Ndiglass laser (amplified single pulse) 2nd harmonic Nd/ YAG laser (amplified single pulse) 2nd harmonic Arlaserpumped passively modelocked rhodamine 6G laser' Amplified Arlaserpumped passively modelocked rhodamine 6G laserd I.3 347.). Various Methods for Picosecond Absorption and Emission Spectroscopy Biologists may be interested both in the timeresolved absorption or emission spectrum at a fixed delay after excitation and in the time dependence of the absorbance or emission intensity at a fixed wavelength. p. 1978. The former is necessary for the assignment of the transient species and the latter for the determination of the rates of the corresponding processes. Shank and E.2 Typicalb repetition rate Typicalb peak power (MW) Pulse width (psec) single shot single shot single shot single shot 10005000 100500 500010. PICOSECOND LASER SPECTROSCOPY TABLE IV. and energy are typical of commercially available lasers or homemade lasers.5 1060 530 1064 532 610630 I10Hz 110 HZ 0. highpower repetitively pulsed laser is of great use for obtaining a subpicosecond continuum which can be used as a monitoring and reference light in absorption spectroscopy. and S.000 5001000 1000 20100 210 50 5 30 3 20 1520 58 46 3050 2040 0. eds. P. P. Ippen. The continuum is generated in nonlinear optical processes such as parametric fourwave mixing and/or selfphase modulation. peak power.172 4. Shank.. Repetition rate. Shapiro.
This is an example of a repetitively pulsed light source. The monitoring light signal is averaged over many laser shots. The generated continuum is focused by L3 for use as a probe pulse and detected by an ordinary photomultiplier (PM) attached to a monochromator (MONO). 4. and M1.2. The second harmonic generated by SHG is for an excitation light source and is reflected by BS. Absorption Spectroscopy for Time Dependence Measurement Using a Repetitively Pulsed Laser. The second harmonic (315 nm) of the laser output is used as an excitation light source. It is focused by L3 on a sample cell. and M3 and focused by L1 into a continuum generation cell. NANOSECOND AND PICOSECOND SPECTROSCOPY 173 Various Methods for Picosecond Absorption and Emission Spectroscopy Time dependence of absorbance or emission intensity (T) or timeresolved spectrum (TRS) T T TRS T T TRS T. TABLE V.3. A cavity dumper attached to the dye laser is operated at 10 Hz in synchronization with the Nd/YAG laser for amplification. The fundamental pulse after passing BS is reflected by M2. an Arlaserpumped dye (rhodamine 6G) laser is amplified by a threestage amplifier (AMP) pumped by the second harmonic of an amplified Qswitched Nd/YAG laser used as a light source. In the block diagram shown in Fig. absorption or emission (A. Readers interested in a particular application can skip to the relevant section. 2a.1. and it is difficult to eliminate the effects of longterm fluctuation . the delay time dependence of the absorbance change is determined. while it takes a very long time to obtain a single absorbancechange curve if a singleshot laser is used instead of a repetitively pulsed laser. VD. VD with a stepping motor. By changing the variable delay time of 315nm used for the excitation of a sample. E) Singleshot (S) or multishot (M) experiment Time resolution method Variable delay Echelon and OMA Variable delay Kerr shutter Streak camera Kerr shutter Echelon and OMA in twodimensional mode Streak camera and OMA in twodimensional mode Figure number 2a 2b 4 5a 5b 6 2b T.4. TRS S 5b systems. averaging is easily performed by means of a repetitive pulse. The principle of this method is as follows: Monitoring light intensity is probed before and after excitation. TRS Method number. and methods to resolve on a picosecond time scale.2.
3. and R. Rentzepis. Chem. M1. of the laser intensity by averaging with this method. M2. P. illustrated in Fig. . The experimental system can be modified to be a dualbeam spectroscope in order to minimize the effects of intensity fluctuations (see p. 1 (1971). Block diagrams of (a) repetitivepulse and (b) singieshot picosecond spectroscopy equipment to measure the time dependence of absorbance of intcrmediates. M. a modelocked Nd/glass laser is used as a singleshot light source. 4. 2b. The single pulse is amplified by AMP after being extracted from a pulse train by SPS. The second harmonic generated by SHGl is for the excitation of a sample and is reflected by BS1. Phys. and (b) ML Nd/glass laser. Jones. The third or fourth harmonic can also be used as the excitation light source.PICOSECOND CONTINUUM AMP UI SPS YL LASER FIG.2. and focused on the sample cell by L1. Topp.2. P. Picosecond lasers used are (a) Arlaserpumped M L dye (rhodamine 6G) laser with a cavity dumper and a Nd/YAGiaserpumped dye amplifier. Lett. VD. uses an echelon to convert time resolution to spatial resolution. This is the big disadvantage of using a singleshot laser. This method. 630 nm 315 nm PICOSECOND LASER SPECTROSCOPY PICOSECOND C O N T I N U U M 14 SAMPLE CELL VD DUMPER STEPPING MOTOR A 5 3 0 nrn A.174 (a) 4 4 4. The second harmonic generated by SHG2 is reflected '* M.22 In the block diagram. R. 176). Echelon Method for the Measurement of Time Dependence of Absorbance Using a SingleShot Laser.2. 9.
then the interpulse separation is given by Ar = 2d cos O/c for the reflective echelon and At = d[(n2 . Both the monitoring and reference beams are detected by a vidicon or TV camera attached to the monochromator. (a) Transmissive and (b) reflective echelons used to get a pulse train of a probe or a reference beam to resolve in a picosecond region. A single pulse is converted into a pulse train passing through or being reflected by echelons.2.22 to obtain time resolution of emission intensity in picosecond region. These echelons are used to convert a monitoring pulse to a pulse train with appropriate interpulse separation in time and space. The situation is similar when a reflective echelon is used. When the step thickness of an echelon is d. It was also utilized to get time resolution of absorbance change on a picosecond time scale using singleshot lasers. M8. in the case of an echelon with steps of 3 mm ( a ) TRANSMISSIVE ECHELON (b) REFLECTIVE ECHELON FIG. The light that travels through the different steps of the transmissive echelon experiences varying retardations.3. transmissive and reflective. the normal angle of incidence of the monitoring light passing through the echelon is 6.4. For example. If the light pulses passing through the steps of an echelon can be resolved in space. NANOSECOND AND PICOSECOND SPECTROSCOPY 175 by M3 and M4 and focused by L2 into a continuum generation cell (CONT CELL). M9. and then focused by L7 together with the monitoring beam on the entrance slit of a monochromator (MONO). as shown in Fig. Both excitation and monitoring pulses travel nearly equal optical distances. and the refractive index of the glass is n. After passing through an echelon the continuum is split into monitoring and reference beams by BS2. M5. . The echelon used in picosecond experiments is simply a stack of glass plates or blocks.sin2 0)1/2 . The reference beam is reflected by BS2. The monitoring beam passes through BS2 and then is reflected by M6 and M7 and focused on the sample cell by L6. They are subsequently focused at a small angle to each other in the sample cell by lenses L1 and L6. M10. The echelon method was invented by Topp et a1. 3. The generated continuum is focused by L3 on a scatterer (SC) and collimated by L5.cos O]/c for the transmissive echelon. There are two kinds of echelons. and M11. they can give timeresolved information about the sample.
Netzel and P. 337 (1974). ''' . Lett. each step of the stack must be in optical contact and each plate surface must be parallel to all the others. and if subscripts ex and ne correspond to the case when the excitation light is exposed to the sample and the case when the light is blocked. up to about 1 nsec. the intensity distribution over the echelon steps after passing through the sample cell is different from that before passing through the cell. This fluctuation is caused by the amplitude instability o f laser pulses which induces fluctuation of the angular distribution of the picosecond continuum intensity in the optical medium where the incident laser is focused. e.176 4.3. the absorbance changes with time on a picosecond time scale after an excitation pulse. To get a highquality transmitted and reflected image. The intensity of each pulse in the monitoring pulse train is decreased or increased by transient absorption or bleaching of the sample.3. 4. for example. To remove the artifact caused by the changes in the intensity distribution across the echelon steps. A Nd/glass laser is used as the light source. L e f f . A pulse train separated both spatially and temporally.43. Therefore. L.Resolved Absorption Spectra. Sometimes the intensity distribution over echelon steps changes from one shot to the other because of fluctuation in the spatial distribution of the continuum intensity. 4. Them. TV camera. M . a silicon vidicon. The second harmonic generated T. can be observed by a detector which has spatial resolution. Phys. formed by reflecting or passing through echelons from a monitoring pulse. With the transmissive echelon.2. care must be taken that the time separation is different from one wavelength to another since At is a function of the refractive index n of the echelon material. or photographic plate.429 (1976). The fluctuation of the spatial distribution of the monitoring light pulse may give artificial data if a singlebeam (monitoring beam) method is applied. Spectroscopy for Time. Kobayashi and S . and M 1. If. respectively. Nagakura.'~" Figure 2b shows the equipment for dualbeam picosecond absorption spectroscopy. and then focused by L1 to excite a sample cell. transmissive echelons are appropriate for experiments requiring high time resolution. T. A block diagram for the measurement of picosecond timeresolved absorption spectrum is shown in Fig. PICOSECOND LASER SPECTROSCOPY thickness. VD. If the light intensity corresponding to the ith step of the echelon is represented by I o ( i ) for the reference beam and I(i) for the monitoring beam. 29. Rentzepis. Phys. The second harmonic generated by SHGl is reflected by BS. then l ~ g [ Z " ~ ( ~ ) ~ ~ ~ ( i ) / Z ~ gives ~(z)Z the ~~(~)] absorbance change at delay time At(i) corresponding to the ith echelon.. while reflective echelons are suitable for experiments with relatively slow kinetics. the interpulse separation of the incident light pulse train is 20 psec for the reflective echelon and about 5 psec for the transmissive echelon at around 500 nm. Chrm. a dualbeam (monitoring and reference beams) method was de~ised.g.
2b. 4 can be modified to a dualbeam spectroscope similar to that of Fig. A p p l . The other light beam. Kobayashi. Either a photographic plate or a vidicon with an optical multichannel analyzer (OMA) can be used as a detector. Shichida.4. 24 M. One light beam passes through a pinhole which is exposed to excitation light and focused on the upper part of the entrace slit of the monochromator and then refocused onto the A region of the OMA. A.(I) and ZB(I) for the A and B regions. If the counts of a channel corresponding to wavelength I are 1. 4. The A region covers the top half of each channel of the photocathode of the vidicon. and the B region covers the bottom half. passing through a pinhole which is not exposed to excitation light. Ohtani. by SHG2 is focused into a continuum generation cell by L2. Nagakura. Changing the arrival time of the excitation light pulse at the sample by means of a variable delay (VD) will yield timeresolved absorption spectra at different delay times. 10. Multishot Emission Spectroscopy for the Measurement of the Time Dependence of Emission Intensity.2. the plane of the photocathode can be divided into two regions A and B. Photochem. This method utilizes a Kerr shutter. 32. Yoshizawa. A block diagram showing the experimental system for multishot emission kinetics is shown in Fig. 5a. T. first applied to the observation of picosecond phenomena by Duguay and M a t t i ~ k The . The continuum for the use of monitoring light is reflected by M2 and M3 and then focused by L4 on the sample cell and refocused by L5 on the entrance slit of a monochromator (MONO). H.4. Duguay and A.2. Photobid. Suzuki. absorbance is given by l o g [ Z ~ ( n ) Z ~ ( I ) / Z l ( I ) Z ~ as ( Ia) ] function of A. Y.4. is focused on the lower part of the entrance slit of the monochromator and refocused on the B region of the OMA. Mattick. H. Twodimensionalmode OMA is useful for dualbeam spectroscopy for measuring timeresolved absorption spectra.23bIn the twodimensional mode.3. the system shown in Fig. 2162 (1971). A modelocked Nd/glass laser is used as a light source. T. and T. 1060 nrn NANOSECOND AND PICOSECOND SPECTROSCOPY 177 4 530 nm . . F V I DlCON A PICOSECOND CONTINUUM c CONT CELL L2 BS FIG. ~ ~ Kerr shutter used in picosecond emission Z 3 b K. Block diagram of an experimental system for measuring the timeresolved absorption spectrum at a fixed delay time with a singleshot laser. Opt. T o minimize fluctuations. Yesaka. 809 (1980). S.
P. Block diagram of a system of picosecond emission spectroscopy for the measurement of the time dependence of emission intensity in (a) multishot and (b) singleshot experiments. VD. Using subpicosecond pulses from a modelocked CW dye laser. 1060 nm PICOSECOND LASER SPECTROSCOPY * 530 nm * r VD UME I L SPS LASER FIG. A modelocked/Nd glass laser is used in both (a) and (b).The measured Kerr response time of CS2 was determined to be 2. 26. The second harmonic generated by SHGl is reflected by BS. The fluorescence is detected by an ordinary photodetector. Ippen and Shank” have succeeded in operating a CSz gate at the Kerr resolution limit.92 (1975). such as a photomultiplier (PM). V. The time resolution of the system is determined by the width of the pulse exciting the sample. Emission from the sample is collected by L3 and focused by L4 on the entrance slit of a monochromator (MONO) after passing through the Kerr shutter. Shank. which does not have picosecond time resolution. . Ippen and C. 25 E. Lett.4.5. and M2 and then focused by L1 on a Kerr cell to open a shutter composed of two parallel polarizers P1 and P2 and the Kerr cell. spectroscopy is opened at several delay times after excitation of the sample. M1.0 k 0. The second harmonic generated by SHG2 is reflected by M3 and M4 and then focused by L2 on a sample cell. In the process they made a direct measurement of the Kerr lifetime.3 psec. and the relaxation time of the Kerr medium. the width of the shutter pumping pulse width. Appl. Phys.
’ ~ * ’ ~ 4. M2.” To get a standard time. However. 69.2. then S(At) = F(At). Appl. 31 18 (1979). S(At) is given by the equation S(At) = C S l d F ( t ) g ( t . Olson. G. Chem. E.3. Greve. F ( t ) is the convolution of the excitation pulse shape and a function of the intrinsic fluorescence decay.At) of the Kerr shutter. S. Kobayashi. in order to accumulate data without the effect of the jitter to improve the signaltonoise ratio. or a time 26 G. twodimensional emission information from the sample is monitored by the vidicon. Rentzepis.At). K. 3570 (1978). . 0. J . Streak Camera Method to Measure the Time Dependence of Emission Intensity Using SingleShot Laser.2. This method is much easier than the multishot method explained in Section 4. The other is that the camera has a jitter that makes it difficult to average the data.4. and P. Phys. A block diagram of the experimental system for emission kinetics measurement in a picosecond range by a singleshot laser and streak camera is given in Fig.3.5. Phys. The timedependent spectrum is observed by the combination of the streak camera. L. The sample and the entrance slit of a monochromator are located at the focal points of an ellipsoidal mirror. Deconvolution can be done by applying a Laplace transformation to S(At) and g(t . 450 (1 975). and then focused on a sample by L2. ’’T. F ( t ) must be obtained by deconvolution or convolution/simulation. M. A tiny portion of the second harmonic is reflected by BS and focused by L1 on one end of a bundle of optical fibers which lead light to a biplanar photodiode used for triggering a streak camera. Phys. an image intensifier. 27. ’’T. NANOSECOND AND PICOSECOND SPECTROSCOPY 179 The signal S(At) at delay time At is proportional to the convolution of the time dependence of the fluorescence intensity F ( t ) and gate function g(t . J . R. A modelocked Nd/glass laser is used as a picosecond light source. Rentzepis. it is necessary to have a standard time.At) dt. The emission from the sample is focused by an ellipsoidal mirror on the entrance slit of a monochromator. and M4. VD. Therefore. If the width of the gate function is narrow enough compared with the time constant of the fluorescence decay.** The main portion of the second harmonic generated by SHG is reflected by M1.2. Lerr. M. and a vidicon. Appl. Jones. E. there are two disadvantages to the method. 50. 5b. Kobayashi. where C is the constant of proportionality.4. and P. When the time constants are not sufficiently long for the effect of convolution to be neglected. P. Degenkolb. One is that a streak camera is much more expensive than the Kerr shutter necessary for multishot experiments for emission kinetics. Another gate method which can be used instead of the Kerr shutter is the optical pulse gain amplification m e t h ~ d . M3. Busch.
we can obtain both types of data at the same time.2. a Nd/glass laser is used as a picosecond light source. VD. Block diagram of a system of picosecond emission spectroscopy for the measurement of timeresolved emission spectrum at a fixed delay time by the use of a singleshot laser. TwoDimensional Detection of Absorption Spectra as a Function of Delay Time After Excitation. M1. and M2. PICOSECOND LASER SPECTROSCOPY reference. and is then focused by L1 on a Kerr cell to open a shutter composed of two crosspolarized polarizers PI and P2 and the Kerr cell. Emission from the sample is collected by L3. In the diagram. The second harmonic generated by SHG2 is reflected by M3 and M4 and then focused by L2 on a sample cell. To acquire twodimensional picosecond absorption spectroscopic data. We have discussed methods in which either the time dependence of absorbance or emission intensity is measured or the picosecond timeresolved absorption or emission spectra are measured. 6 . . The timcresolved emission spectrum of a sample at different delay times can be obtained by moving the variable delay. A block diagram of the equipment necessary to obtain timeresolved fluorescence spectra with a singleshot laser is shown in Fig. By modifying the methods.3.6.2. the equipment must be slightly modified from that for the time 1060 nm 4 530 nm VD OPTICAL TRAP 1 SPS a ML LASER FIG. passed through the shutter.180 4.28The length of the optical fiber is adjusted so that scattered light at the position of the sample arrives at the exit slit with some delay relative to the light after passing through the optical fiber (for example. 4. 6.7. and focused by L4 on the entrance slit of a monochromator (MONO). The emission spectrum can be detected either by a photographic plate or by a vidicon attached to an OMA coupled to the monochromator. a tiny fraction of the excitation pulse is split by a glass plate and guided by an optical fiber to the inside position of the exit slit of the monochromator. SingleShot Experiment to Measurement TimeResolved Emission Spectra. 100 psec). 4. In this way the light pulse through the optical fiber can be used as a prepulse which is a standard for the accumulation of fluorescence intensity obtained by each laser shot. The second harmonic generated by SHGl is reflected by BS.3.
This is also done using a photographic plate. The twodimensional image on the screen gives both spectra and time dependence.3. APPLICATIONS TO PHOTOSYNTHESIS AND VISION 181 dependence measurements of absorbance described in Section 4. as well as the spectrum. OMA 11.1 . In this mode.2.3.8.O) takes place following the equation . at several delay times after excitation.3. Instead of using a vidicon in a onedimensional mode.3.I.5. Introduction. A twodimensional streak camera is now available from Hamamatsu. can be easily operated in a twodimensional mode. 4. Photosynthesis takes place in chloroplasts. It is possible to obtain data on the time dependence of emission intensity. In the reaction center. and the intensity distribution perpendicular to the slit gives the spectrum. TwoDimensional Detection of Emission Spectra as Functions of Delay Time after Excitation.3. The perpendicular slit is not for monochromatizing but for getting the total spectrum and for limiting the height of the spectrum. Applications to Photosynthesis and Vision 4.1. The exit slit width is one of the factors that determine time resolution of the monochromatorstreak camera system.Photosynthesis 4. the intensity distribution across the direction parallel to the entrance slit gives the data for time dependence. to form carbohydrates (CH. 4. reduction of CO.2.2.2.3. it can be used in a twodimensional mode. An understanding ofthe process ofthe application of light energy to synthesize carbohydrates by plants through the photosynthetic process may someday lead to the development of biomimetic systems of solar energy conversion to chemical energy or to increased agricultural output through the use of additives or chemicals for higher photosynthetic efficiency. but data processing is more difficult and the dynamic range of the photographic plate method is much narrower than that of the method utilizing OMA.4. By rotating the monochromator by 90" and setting a slit at the position of the ordinary exit slit of the monochromator perpendicular to the entrance slit. where the energy of light absorbed by antenna pigments is transferred to a reaction center. whole emission spectra can be measured at a delay time using one laser shot. This can be done by modifying the experimental equipment for time dependence measurement of emission intensity mentioned in Section 4. The image of the entrance slit on the screen of the streak camera then moves with time in the direction perpendicular to the exit slit. The new OMA from Princeton Applied Research EG & G.3.
W. Rockley. J . Tredwell. 369. and J. and chlorophyll a (CHL) at 685 nm. Porter. H.2.. JoussotDubien. Rphycoerythrin (PRE) 29 G. Zewail.). (a) Observed and (b) calculated time dependence of the fluorescence intensity of Bphycoerythrin (BPE) at 576 nm. SpringerVerlag. 1978. 0. Biochim. the transfer of energy from antenna pigments to a reaction center. From Porter ct a/. ed.” The preceding part of the primary process of photosynthesis. G. Biophys. in “Advances in Laser Chemistry” (A. was studied with the use of the emission spectroscopic technique for red algae by Porter et dZ9 The later part of the process.e. K . Kobayashi. T. Rentzepis. The rise and decay kinetics of emission from Bphycoerythrin (BPE) at 576 nm (which is excited by the picosecond pulse). allophycocyanin (APC) at 660 nm. was studied by Windsor and Rockley3’ and Rentzepis et d 3 ’ for bacteriochlorophyll. . 6 psec) of a modelocked Nd/glass laser to excite the accessory pigments in the intact alga of porphyridium cruentum. p. Searle. J. Porter et d Z utilized 9 the secondharmonic pulse (530 nm. C .). Acfa 501. Avouris. G. PICOSECOND LASER SPECTROSCOPY 0 30 TIME (psec) 100 FIG. Amsterdam. Barber. Kaufmann. Picosecond Study of the Energy Transfer Process between Antenna Pigments by Emission Spectroscopy. Details of the study are given in the following subsections. Elsevier. Windsor and M. Degenkolb.182 4.1. 4. 126. p. 3 0 M.3. i.232 (1978). M. in “Lasers in Physical Chemistry and Biophysics” (J. primary electron transfer. 1975. 7. F. Berlin and New York. ed. and E. 3 1 P. Rphycoerythrin (RPE) at 640 nm. P. W.
R.)188. and P. Kaufmann et al. Acad.29calculated the time dependences of the fluorescence intensities. ~ ~ indicate. J.118. J. 34 M .3. Photosynthetic bacteria are often used in studies of photosynthesis because they have relatively simple structure and the reactioncenter chromophores are easily removed with detergents. Science (Washington. S.34did independent picosecond experiments over a wide spectral range. it decays in 246 a 16 psec. Leigh. K. U. R26. L. Netzel.90. therefore. and the spectrum of oxidized species P + becomes evident. APC.) 182. W. Proc. and 175 psec. The results. Netzel.32observed that the 864nm band due to bacteriochlorophyll bleaches within 10 psec of excitation with a 530nm pulse. and 50 psec for Chla. respectively.C. Science (Washington. and chlorophyll a (Chla.and PX.238 (1973). that P + T. 72. Porter et al. Leigh.1. T. M.C. S. A . Natl. APPLICATIONS TO PHOTOSYNTHESIS AND VISION 183 at 640 nm. Kaufrnann. The fluorescence from the latter three pigments all showed finite rise times. and W. to the maximum emission intensity. D. The time dependences of absorbances at 680 and 610nm are shown in Fig. All of the fluorescences except BPE fluorescence appeared to follow an exponential decay law. 7b. L. L. With the use of a picosecond absorption spectroscopic system with echelon. shown in Fig. W. 7) at 685 nm were monitored by a streak camera. The experimental results of the time dependences of the fluorescence intensities are shown in Fig. Rentzepis. and J. Sci. is very often used.3. allophycocyanin (APC) at 660 nm.34. CHL in Fig. The intermediate state PFdisappears after several hundred picoseconds. Rockley. Rentzepis. 7a. 8. RPC. J. are in good agreement with the experimental observations. M. This study is the first direct observation of energy transfer between chromophores in antenna pigments. A mutant which has no carotenoid. Netzel et al. Cogdell.1301 (1975). The mean fluorescence lifetimes of BPE. M. 4. A positive absorbance change at 680 nm is due to the immediate appearance of the intermediate PF after picosecond excitation. Picosecond Absorption Spectroscopic Study of ReactionCenter Oxidation in Bacteriochlorophyll Photosynthesis. Dutton. The experimental results obtained by these two groups are quite similar to each other. Assuming that the energy transfer between chromophores takes place by longrange interaction in the order BPE to RPC to APC to Chla. of 12 psec for RPE. 24 psec for APC. P. D.3. After this very preliminary experiment. G . and Chla were 70.33and Rockley et al. The spectral change observed immediately (within 10 psec) after excitation with a 530 nm picosecond pulse is due to an intermediate state PF. The induced spectral change measured 240 psec after excitation has all the characteristics expected of the difference spectrum between P'X. Parson. while a negative absorbance change at 610 nm is due to the appearance of P' and it appears in 220 _+ 14 p ~ e cKinetics . 32 33 .4. Windsor. P. 2251 (1975).
Dutton et al. In order to clarify the electronic structure of P'X. The absorbance at 1250nm increased immediately after the excitation of neutral reaction center with 10 psec laser 35 P. Kaufmann. PICOSECOND LASER SPECTROSCOPY AA.8. 8a. indicating that P+ is formed directly from PF. Rentzepis.275 (1975). .35 investigated the 1250nm absorption band of the reaction center of R26 mutant. whereas BChl monomer has no absorbance in the region. M.34The timeresolved absorption spectrum 20 psec after excitation is very similar to that observed for chemically reduced PXafter 20nsec pulse excitation. and P. L. 610 nm 0 200 400 600 ( p%C) 800 TIME AFTER EXCITATION I ~~ ~ ~ ~~ ~~ 0 400 TIME AFTER EXCITATION 200 600 (pSeC) FIG. From the result. (a) Rate of formation of P+ (open circles) measured from the absorbance decrease at 610 nm. J. B. From Rockley et d f 4 forms directly from PF. PF disappears in 246 & 16 psec and P + forms in about 220 & 14 psec.184 4. (b) Semilogarithmic plot of Fig. The oxidized dimer of BChl (BChlLBChl) formed by the treatment with ferricyanide has absorption maxima at 1250 nm. FEBSLetr. Chance. 60. it can be concluded that the intermediate PFhas a lifetime of about 200 psec and that the lifetime can be extended to 10 nsec if the electron transfer from PF to X is blocked by the chemical reduction of X. Dutton. and the rate of disappearance of P' (filled circles) measured from the absorbance increase at 680 nm after picosecond pulse excitation. K.
PFX = c. the physiological purposes of the photosynthetic and visual processes for light energy utilization are quite different from each other. P'X. Some of the photoreceptor molecules used in the very fast first steps of visual and photosynthetic processes are similar to each other. From these experimental results. However. 18. it was concluded35 that the oxidized cation radical of the BChl dimer ([BChlLBChlJ) is a part of the intermediate state PF which is the precursor of P'X. Therefore I is concluded to be BPh.p*X = c. and. are present in most normal plants.3. It has been suggested that the PF state could be symbolized as [BChPBChl I] where I represents another reduced component.appears several hundred picoseconds after excitation.[BChl'BChl BPhlX c. APPLICATIONS TO PHOTOSYNTHESIS AND VISION 185 pulse at 530 nm. Nature (London) 132. as Wald36. the carotenoids. 4.3. The expected difference spectrum between [BChlLBChl BPh] and [BChPBChl BPh:] resembles the observed timeresolved spectrum of R26 at 20 psec after excitation.[BChl'BChl J BPhlX < I0 psec J c.. . Vision 4. retinal. J .P+X = c.. As mentioned above. light energy absorbed by antenna chlorophyl is transferred to reaction center and utilized 36 3' G. whose structure is approximately a half of a carotene is present in the eye.. Introduction.2. In photosynthesis. Wald. 905 (19341935). Wald. G. 316 (1933). Physiol.37 first showed. Cen. picosecond spectroscopy was very usefully applied to the clarification of the mechanism of the primary process of photosynthesis.4. A class of molecules. The concluding photochemical steps of bacteriochlorophyll obtained by the above experimental results are3' c2P.1.[BChliBChl 1 100250 psec BPhlX As can be seen from the above discussion.X c2[BChlBChl BPhlX c.2.3.
p. an absorbed photon finally results in triggering nerve impulses whose energy comes from electrochemical gradients across the cell membrane which are induced by light energy absorbed. Lamola. driving the entire photosynthetic process and then finally converted to chemical energy. 74. Kito. Applebury. Green. on the other hand. Yoshizawa. The absorption of the photons by the photoreceptor. Busch. Yoshizawa and Y . Proc. Berlin and New York.).2. Applebury. J. S. Callender. R. 146. 31 19 (1977).40 The second harmonic (530 nm. 645 (1977).43 However. M. 179 (1977). hypsorhodopsin could not be observed after 530 nm excitation of bovine rhodopsin in LDAO (i. Aton. M. Part 1. Rentzepis. and R. Nature (London) 269. From the observed instantaneous rise of absorbance at 561 nm. Sci. in “Handbook of Sensory Physiology” (H.41342 Proton translocation was claimed to take place in the formation process of bathorhodopsin from the experiment of temperature dependence and deuterium substitution effect of the formation rate. Peters. L. T. seen at 4K. Sci. SpringerVerlag. Nail. Vol. Monger. VII.. Two of the intermediates observed at low temperatures are bathorhodopsin. Alfano. whether hypsorhodopsin or bathorhodopsin is the first intermediate after the absorption of the photon. Acad. A . 4 3 K. L. 40 G.186 4. A.e. 6 psec) of a modelocked Nd/glass laser was used as a picosecond light source to excite bovine rhodopsin solubilized in Ammonyx LO detergent. 4 2 B. K . G . Nature (London) 267. U .39 After the observation of hypsorhodopsin. E. Sundstrom. A. Proc. Nature (London) 182. rhodopsin. Their spectra were at first observed by freezing samples in glassforming solvents at low temperatures where the lifetimes of the intermediates are long enough to measure their absorption spectra with ordinary absorption spectroscopy equipments. PICOSECOND LASER SPECTROSCOPY for. B. and P. M. 38 39 . and P. Lifetimes of these intermediates are very short. S. and M . In visual process. Ammonyx LO). M. observed by irradiating cattle rhodopsin at 77 K. H. it was observed that hypsorhodopsin is formed from squid rhodopsin solubilized with digitonin with use of the second harmonic of the T. 4. T.2. 1972. M. L. the question arose. 69.38 and hypsorhodopsin. Nail. Applebury. Dartnall. it was concluded that bathorhodopsin is formed within 6 psec after picosecond pulse e~citation. Several intermediates are formed successively after the photophysical event.3. P. Rentzepis. Picosecond Absorption Spectroscopic Study of the Primary Process in Vision. Acad. ed.2802 (1972). A. Peters. initiates the primary process in visual excitation. A . U .~’ Furthermore. Rentzepis. 4 1 V. H. R . Picosecond spectroscopy equipment was used to study the very fast roomtemperature kinetics of appearance and disappearance of intermediate species absorbing at 561 nm. 1604 (1958).
It~was ~ . T.. T. most cattle bathorhodopsins are formed by the following process at room temperature: rhodopsin (So) h S. a first phase appears at 020 psec and a final one at 80400 psec. T. ~discrepancy ’ between the formation time constant of bathorhodopsin for cattle (< 6 psec) and that for squid (50 psec) could possibly have been due to the difference in excitation wavelength (530 and 347 nm). Shichida. Phoiobiol. Shichida. i.570. 9. Photochem. 207 (1980). H. agreeing with the observed rise time of squid bathorhodopsin and decay time of squid h y p s o r h ~ d o p s i n .440. Ohtani.found ~ ~ that the primary process of cattle rhodopsin is almost the same as that of squid rhodopsin. The data obtained for 430.e. FEBS Lett.4. 4 s Y. i. 32. it is concluded that 93?z8 % (100’ yo %) of bathorhodopsin is formed through hypsorhodopsin in octylglucoside (LDA0)solubilized samples and the rest is formed directly from the excited singlet state of rhodopsin. ~The ~ . The time dependence of absorbance at seven different wavelengths (430. The lifetime of the excited singlet state and the rise time of hypsorhodopsin were both found to be 15 5 psec. Ohtani. 44 Y. and S.The ~ ~time * ~constant ~ is <20 psec at room t e m p e r a t ~ r e . 214 (1977). T. and 630 nm are shown in Fig. 600. and 630 nm) was observed for bovine rhodopsin in LDAO. or in the protein (cattle and squid). .and octylglucosidesolubilized solutions after excitation with a picosecond pulse at 530 nm. 4 The ~ ~hypso ~ ~ intermediate was found to convert into bathorhodopsin with a time constant of 50 10 psec at room t e m p e r a t ~ r e .e. APPLICATIONS TO PHOTOSYNTHESIS AND VISION 187 modelocked ruby laser as an excitation light s o ~ r c e . 570.. and both the rise time of bathorhodopsin and decay time of hypsorhodopsin were 50 f 20 psec. 550. in the detergent (LDAO and digitonin). Nagakura. Kohayashi. FEBS Lett. hypsorhodopsin. 27.3. Yoshizawa. Nagakura. and bathorhodopsin. respectively. 106. After the picosecond spectroscopy of squid rhodopsin experiment the cattle rhodopsin experiment was repeated with the use of the second harmonic of a Nd/glass laser as an excitation light p ~ l s e . The kinetics is observed to be triphasic at these wavelengths. 80. 335 (1978). 440. Kobayashi. Kobayashi. The species corresponding to the kinetic stages are assigned to the excited singlet state of rhodopsin. 4’ T. ~ ~ ~ ~ ’ From the analysis of the depth of the dip observed in the time dependence curve of absorbance change at 570 nm. Phoiobiol. Photochem. and S . H.313 (1979). ’’’ p5ec + hypsorhodopsin * 2o psec + hathorhodopsin. Yoshizawa. 4 6 T. Kohayashi. 480.
Pergamon Press. bathorhodopsin There is no significant difference in the kinetic data and intermediates between 347nm excitation of squid rhodopsin solubilized in digitonin and 530nm excitation of bovine rhodopsin solubilized in LDAO or octylglucoside. The formation and decay times of cattle hypsorhodopsin are 15 & 5 and 50 L 20 psec. and of the protein. From this fact it is concluded that. Is ’ psec.188 4. Absorbance change at 440 nm for octylglucoside sample is shown by (c). . Ltd.~ Copyright ’ There may be a small amount of bathorhodopsin formed by the following process : rhodopsin (So) h v S . Reprinted with permission from 1980. respectively. hypsorhodopsin is the main first intermediate of the visual process in bovine and squid rhodopsin.9a.0 04 0 02 0 002 0 04 006 I 1 I I I 0 06 0 04 0 02 (C) a 0 0 02 \ 0 0 0 0 0 0 4 0 06 . while those of squid rhodopsin are <20 and 50 k 10 psec. irrespective of the excitation wavelength. Squid and bovine bathorhodopsin are both formed primarily via their respective hypsorhodopsins. Kobay~hi. Time dependence of the absorbance change at 430 nm induced by 530nm picosecond pulse excitation for (a) octylglucoside and (b) L D A O solubilized rhodopsin. 0 08 0 04 0 02 PICOSECOND LASER SPECTROSCOPY a 0 0 2 . respectively. of the nature of the detergent. 0 I I I I 400 100 200 TIME I p s e r l 300 FIG.
Pergamon (b) LDAO sample. 9c. Time dependence of the absorbance change at 630 nm for (a) octylglucoside and Copyright ’ 1980. Reprinted with permission from K ~ b a y a s h i . O r FIG. Pergamon Prmc T trl 0 10  (a) 0 on  006 a 004  0 0 0 002 0  I J  o 42  (b) 0 40 0. Time dependence of the absorbance change at 570 nm for (a) octylglucoside and (b) LDAO sample. ~ Copyright ’ 1980. Reprinted with permission from K o b a y a ~ h i .016 012 2 001 00. Ltd.08  a 0060 0 0 0 04  0 002 0  I J FIci. 9b. . ~ Press.
M. and should have no singlephoton absorption cross section and a high twophoton absorption cross section at the wavelength of the laser. Giordmaine. The laser pulse optically pumps the dyes into an excited state with energy twice the photon energy. The dye molecules then relax to the lowest excited state and emit fluorescence. Phys. L. but depends on the light intensity. l . This phenomenon is called a twophoton or a multiphoton absorption process. and it should be smaller than the twice the photon energy. On the other hand. Wecht. Twophoton absorption has been used for pulse width measurements of picosecond lasers. or fluorescein is used for the pulse width measurement of ruby lasers. rhodamine B or rhodamine 6G is used.190 4. The laser beam is split into two beams of equal intensity which are then reflected by mirrors M1 and M2. Dye molecules used for pulse width measurement by twophoton fluorescence techniques should be highly fluorescent. and then the transition from the state to the final state takes place. A. Appl. and Detectors Related to Techniques of Picosecond Spectroscopy A . an intermediate electronic excited state is actually excited by one photon. the energy separation AE between the ground state and the lowest excited singlet state should be greater than the photon energy of the laser used. 216 (1967). 4 8 J.48 Using the simple optical system shown in Fig.l . Two or more photons can be simultaneously absorbed by a medium a t sufficiently high light intensities. Lerr. Rhodamine 6G. Optical Elements. Weak fluorescence can be observed as background in the tracks of two oppositely directed beams outside the region where the two optical wave packets overlap. PICOSECOND LASER SPECTROSCOPY APPENDIX. 11. Shapiro. When the laser beam has noisy peaks with picosecond pulse widths and the total pulse width is much longer than the picosecond pulse. In order for twophoton absorption to take place efficiently in organic dye molecules. For example. Nonlinear Optical A. W. In the latter process. simultaneous twophoton absorption takes place via a virtual intermediate state which is not actually excited and works as a perturbing state to generate the twophoton transition probability. At a high laser intensity the absorption cross section is not constant. and K. I . . Nonlinear Optical Phenomena. rhodamine B. S . Multiphoton Absorption. P. Simultaneous twophoton absorption is different from a twostep absorption. for the pulse width measurement of the fundamental of Nd/glass lasers or Nd/YAG lasers. The mirrors are aligned to make the beams arrive at the same time at the center of a cell containing a highly fluorescent dye solution. Rentzepis. 10. the width of a picosecond pulse can be measured.
M. The twophoton fluorescence intensity is monitored by a camera or vidicon. Ippen and C . Berlin and New York. in “Ultrashort Light Pulses” (S.APPENDIX. L. Saxman. They are focused on the same spot on a nonlinear crystal to generate the second harmonic. Mitschele. The intensity curve plotted against the position of the variable delay gives the autocorrelation function of the laser pulse from which a pulse width can be estimated.10. C. 83.). Block diagram ofapparatus for measurement of pulse width by twophoton fluorescence. Two mirrors are adjusted such that these two beams are coincident at the center of TPF cell. In many cases. 122 (1970). Second. One beam passes through a fixed delay and the other through a variable delay. Appl. 1064 nm for a Nd/YAG laser. Nonlinear processes such as second.2. while if the pulse has no substructure and it is a single picosecond pulse. the ratio should be 3: 1. The delay time is changed shot by shot when a singleshot laser is used. 1977. V. P. ed.50 A. Rentzepis. Higher ratios can be obtained by applying the threephoton fluorescence method. it is necessary to convert the wavelength to the visible or nearUV regions to excite biological macromolecules. Phys. SpringerVerlag. 17. Lett. The laser beam is split into two beams of equal intensity. and FourthHarmonic Generation.1. Shank. AND DETECTORS 191 CAMERA or VIDICON FIG. Third. . The oscillating wavelengths for various picosecond lasers are approximately 1060 nm for a Nd/glass laser. 5 0 P. 49 E.49 In this case. J. and 610630nm for an Arlaserpumped rhodamine 6G laser. The experiment can be performed in the following way. Shapiro. NONLINEAR OPTICS. the peaktonoise ratio is 2:1. It is continuously changed by sliding a stage with a stepping motor for a repetitively pulsed laser. C. OPTICAL ELEMENTS. 694 nm for a ruby laser. Higherharmonic generations can be used to measure the pulse width of modelocked lasers. p. third. twophoton fluorescence patterns obtained by this method can have a sharp peak with a picosecond pulse equal to the pulse width of the noise spike. The incident laser beam is split into two beams with equal intensity. and fourthharmonic generation are effective means of generating shortwavelength radiation. and A .
Picosecond continuum light can be utilized as a monitoring light source for picosecond absorption spectroscopy. 1977. extreme spectral broadening can be observed. heavy water.06 0. When a modelocked ruby laser is used. the second harmonic of a Nd/glass laser or Nd/YAG laser is usually used. Nonlinear Crystals for SecondHarmonic Generation" ~ ~ ~ ~~~~~ Abbreviated name ADP Crystal name ammonium dihydrogen phosphate potassium dihydrogen phosphate cesium dihydrogen arsenate rubidium dihydrogen arsenate rubidium dihydrogen phosphate Wavelength (w) 1. However. Copyright The Chemical Rubber Co. cyclohexane. Usually water. The pulse shape of the continuum is slightly modified from that of the pumping laser pulse because of the difference in the degree of modulation in the spectral region of the laser pulse. " '* N . liquids and glasses or solids are usually used for selfphase modulation or fourwave mixing media. Kobayashi. or phosphoric acid are used for continuum generation. C R C Press. ed.06 0. Presslcy. often used for higherorder harmonic generation are listed. and near IR. Ohio. ' ~ The ~'~ continuum covers the near UV.1. 147 (1979).487 (1975) T. Inc. ethanol.6943 1. Cleveland.6943 0. A. Phys. carbon tetrachloride. Lett. all of the visible region. PICOSECOND LASER SPECTROSCOPY TABLE V1. p. the halfwidth of the continuum pulse is close to that of the pumping pulse. polyphosphoric acid gives a more intense continuum than other liquids.. To obtain the monitoring continuum light in the visible region. 497. 28.06 0. Commun. Mataga. Picosecond Continuum Generation. In order to generate continuum light with picosecond width. Chem.6943 1.). @ i f . and the spectral region of the continuum is broader than that obtained with normal phosphoric acid or isophosphoric acid. nonlinear crystals. CRC Press." When intense laser light is focused into a cell containing various organic or inorganic liquids or solids.3. Nakashiina and N. J. It sometimes extends several thousand wave numbers to both the Stokes and antiStokes sides of the oscillating laser wavelength. 35. An intense continuum with very wide spectral range is obtained by focusing the fundamental of ruby laser into phosphoric a ~ i d . .192 4. In Table VI.6943 Phase matching angle 42" 52" 40" KDP CDA RDA RDP 50" 90" 80" 66" "Reprinted with permission from "Handbook of Lasers with Selected Data on Optical Tcchnology" (R.
527. Copyright The Chemical Rubber Co.4. can be used to obtain an excitation light source at different wavelengths from the fundamental or higher harmonics of lasers. C R C Press. then the energy is dissipated and the light may not be as monochromatic as the fundamental. making accurate control of the geometry of the interaction difficult.APPENDIX. Cleveland.AND DETECTORS 193 A. p. OPTICAL ELEMENTS. In order to get highly intense Raman light.1. 1977. In Table VII.') 222 460 539 656 667 992 992 1004 1315 1344 1629 2202 2817 2852 2863 2967 3056 3064 365 I Liquid bromoform carbon tetrachloride bromoform carbon disulfide chloroform benzene pyridine toluene styrene nitrobenzene styrene 1. Selfphasemodulation can also occur. From an experimental point of view.. '' . which is related to the thirdorder nonlinear susceptibility. Stimulated Raman Scattering from Various Liquids" Frequency shift (cm. If the abovementioned effects occur. C R C Press.). ed. it is important to keep in mind that stimulated Raman scattering with picosecond pulses is accompanied by other nonlinear optical phenomena. the shifts of the stimulated Raman scattering from the pumping laser are given for various kinds of materials. Stimulated Raman Scattering Light Pulse. Pressley. Inc. The stimulated Raman scattering process. Ohio. TARLE VII. One of them is selffocusing which may enhance the conversion efficiency but tends to break the beam up into several filaments. NONLINEAR OPTICS. producing considerable spectral broadening. J.2dichlorobenzene methylcyclohexane cyclohexans cyclohexane dioxane styrene benzene water a Rcprinted with permission from Handbook of Lasers with Selected I h t a on Optical Tcchnology" (R.
while the latter is related to the thirdorder nonlinear suceptibility. A. great care must be paid to the delay or time spreading of the white picosecond light with broad spectrum due to dispersion in the Kerr shutter. To reduce the probability of selffocusing. A.2. This process is represented as ('4. Parametric Interaction. Saturable Dye Solution and Cell.6. The reason why CS2 is most often used for the Kerr shutter is that the relaxation time of the anisotropic refractive index is 2. a . In order to get intense Raman scattering.1) (A4 where oL.3) A. + wi.6 x lo" esu).194 4. Optical Elements and Detectors A. Kodak A9860 or A9740 dye in ethylene chloride solution is generally used as a saturable dye for Nd/glass and Nd/YAG lasers. The three main causes are anisotropic polarizability of anisotropic molecules. + w. Before using ethylene chloride as a solvent of the dye it is necessary to pass it through a silica gel column to remove impurities and to store it in the dark at low temperature to prevent degradation. the relaxation time of an anisotropic refractive index is different. Fourphoton parametric interaction can also be obtained by mixing two waves at different frequencies. When a Kerr shutter is used in hightimeresolution experiments. signal. while the nonlinear refractive index of CS2 is (12) x lo" esu.2. Optical Kerr Effect. respectively. or Nd/glass lasers. which is of the same order as that of nitrobenzene (1. rnnitrotoluene (50 psec) or chlorobenzene (68 psec)." The two processes are written as WL t 20.41.5. . The former process is related to the secondorder nonlinear susceptibility. 0 1 + 0.1. ('4.1 psec. and idler.1. or mixing process. which is much shorter than that of nitrobenzene (30 psec). 0. PICOSECOND LASER SPECTROSCOPY the other effects mentioned above must be reduced. it is necessary to use a short cell containing a highly Ramanactive medium. it is necessary to use a lens with a somewhat longer focal length than that used to obtain picosecond continuum. and electrostriction. The optical Kerr effect has several causes.and wi are the frequencies of the laser. electronic hyperpolarizability of isotropic molecules." esu) or chlorobenzene (1.6 x l o . There are two kinds of parametric emission processes that can be attained by ordinarypower ruby. . + wi.1. Nd/YAG. One is a threephoton parametric emission process and the other is a fourphoton parametric emission. Depending on the causes of the Kerr effect. +0 2 + mi.
APPENDIX. A. Therefore. NONLINEAR OPTICS. When the first two groups of detector are used. constitute the third group.2’quinodicarbocyanine iodide (DDI) or I. The lenses should be AR coated to eliminate the possibility of satellite pulse generation. These cameras can be operated synchronously with a repetitive pulse.2. The back mirror of the cavity of a modelocked solidstate laser should be highly reflective at the oscillation wavelength to increase the Q value of the resonator. A contact mirror which forms one wall of the saturable dye cell is used to avoid the satellite pulses that may be generated by reflection from the walls of the cell when a separated saturable dye cell is used.Pulse Selector. Cavity Mirrors. with spatial resolution form the second group. Recently synchroscan streak cameras have become commercially available as new group 3 detectors. They are also useful for protection against damage of the highly reflective mirrors of the cavity by multiplying the diameter of the beam. The output mirror of a cavity usually has a reflectivity of 4060%. slightly divergent from one side to the other. AND DETECTORS 195 The light transmission at 1060 nm of the solution is adjusted to 6270%.2. Both can be flat mirrors if two lenses are put inside the cavity to compose an inverted Galileian telescope. Multishot experiments must be performed when a detector of the first group is used to obtain a single kinetics curve. which is cross polarized to the entrance one. The first group includes various photomultipliers and photodiodes that have neither spatial resolution nor picosecond time resolution.l‘diethyl4. time resolution is obtained by the optical system which makes the correlation between time and space. Streak cameras.2. which have picosecond time resolution. This is to reduce both the loss of Q value by light diffraction and the thermal lensing effect of the rod. l. OPTICAL ELEMENTS. repetitivepulse experiments can be easily carried out using group 2 detectors. If it is operated in a synchronousscan mode the resolution time becomes about 10 psec because of a jitter in each scan. The highvoltage driver is adjusted to generate a halfwave voltage to rotate the polarization plane of incident laser pulse by 90” for the beam to pass through the exit polarizer.3. One of the cavity mirrors is flat and the other is usually concave. . To avoid degradation the dye solution should be circulated. to reduce the thermal lensing effect. Detectors of the second group are free from jitter.l’DiethyI2. Three groups of detectors are used for picosecond spectroscopy.4.4’quinocarbocyanine iodide (cryptocyanine) in methanol solution is used for mode locking in ruby lasers. such as vidicons and TV cameras. A singlepulse selector is usually composed of two crosspolarized GlanThomson prisms (entrance and exit polarizers) and a Pockels cell between them. A. A.2. Single. Detectors. Detectors. High voltage is applied either by an optically triggered spark gap o r highvoltage pulse driver. The camera has a time resolution of a few picoseconds when it is used in a singlescan mode.
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L. Elson 5. The mobility of some membrane components is constrained by interactions with structures either within or outside the membrane.g. to form specialized regions on the cell surface (e. and the control of locomotion and morphology. Therefore measurements of the mobility of specific cell surface components should both indicate the progress of particular physiological processes and provide information about their mechanisms. for example. This part describes fluorescence methods for measuring cell surface motions. Goldstein. The plasma membrane not only separates the cell interior from the surroundings but also participates in many active processes.Y 20 .270. These processes often require that cell surface components move relative to each other to permit interactions. or to share membrane components between progeny cells during cell division. J . and other cellular functions. In fact the situation is more complex. “coated regions”’). Anderson. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS By Joseph Schlessinger and Elliot L. l n c All right\ of reproductinn in .695 (1977). the flexibility of the hydrocarbon chains of phospholipids has been mapped by R . W . and M .5. Motions of membrane components can be classified as microscopic or macroscopic according to the distances over which they extend. For example.ti>) lorm re*ervcd ISBN o . S. between membrane enzymes and substrates. rHODS OF t X P E R I M t N 7 A L PHYSICS. The fluid lipid bilayer that forms the basic matrix of biological membranes is expected to permit rapid translation and rotation of lipid molecules and embedded proteins. Nature (London). Brown. Microscopic motions take place over distances corresponding to molecular dimensions and consist of molecular rotations and deformations.. I97 MI. differentiation. Introduction Fluorescence measurements can reveal structural and dynamic properties of animal cell plasma membranes which are crucial to their physiological function. C‘op>riglit @ 19x2 by Acadcmic Prc\s. transport of molecules along or through the membrane.I H ~ s ~ ~ ? . At least some of these interactions are likely to have functional importance. G .1. V D L . These include the reception and transmission of biochemical “signals” (from other cells and the environment) which control metabolism.
Koppel. Peters. Poste. W. Frye and M. H. and E.) 195. Natl. respectively.198 5. Indications of segmental flexibility in membrane proteins have appeared in measurements of fluore~cence. ’ 1055 (1976). l 3 K . H. J . Schlessinger. Webb. R. A. Biochem. M. P. M. Jacobson. and T. and G. which were adapted from methods previously used in bulk solutions. C. Tews. they showed that the flexibility of the chains increases from the head groups to the center of the bilayer. Peters. D. Elson. Axelrod. and W. Elson. Acad. D. Biophys. Cone. Eur. Science (Washington. 7.209 (1976). 319 (1970). D. Lardner. L. E. P. . Elson. ~ photobleaching method was first used to measure the lateral mobility of rhodopsin in amphibian retinal disk mernbranes. and J. Sci. 11 1 I (1971). In contrast to measurements of microscopic motions. D. J. A. l 4 J .C. Changeux. W. Aria 433. Macroscopic lateral motion of membrane proteins was first observed as the intermixing of histocompability antigens in mousehuman heter~karyons. E. Chapman and D. however.307 (1977). p. G . J . E.) 196. Liebmann and G. Cell Sci. Webb. E. McConnell. Rotational mobility is often taken as an indicator of the “microviscosity ” of the membrane lipid bilayer. P. W. 91. D.^ Using spin label and nuclear magnetic resonance (NMR) methods. Science (Washinglon. Kornberg and H. A . W. D. L. Petit and M. Auchet. D. L. Biochim. 1973. E.332 (1971). A68. Edidin. M. Po0 and R. Zagyansky. Kornbergand H. Wahl. D. Nuture (London) 247. l 5 D. Webb. Acad. D. Edidin. J . L. M. L. 16. Science (Washington.C.C. W. l6 D. 438 (1974). D.466 (1976).~ Magnetic resonance and fluorescence methods have also frequently been used to measure rotational motion. Biophys. K. Wu. U .) 185. Y . A. Entine. 16. Koppel. Koppel. S. V. Academic Press.) 184.215 (1976). 1183 (1974). FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS Kornberg and McConnel12*3and other^. Y. Acad. Proc. Webb. Biophys. Chapman. Kasai.C. F. 1315 (1976). Acta433. Koppel. Jacobson. this analysis in terms of a single viscosity parameter may be inadequate to describe the constrained rotations of asymmetric fluorophores in complex anisotropic membranes. Axelrod. M. Biochemistry 10. J . D. new methods were required for measurements of motions over a range of microns on cell surfaces. Sci. and W.~ A. S.2564(1971). K. J. 73. J. E. J. Axelrod. A r i a 367. Biophys. eds. Edidin. U .). Elson. in “Biological Membranes” (D. McConnell. D. Biochim. 2. Biophys. and W. D. Schlessinger. Zagyansky and M.2409 (1976). As is discussed below. Wallach.”’’ Translational motion over tens of angstroms can be measured using R.’~~ This approach has now been generalized to fluorescent labeled molecules and provides a versatile procedure for quantitative measurements of macroscopic lateral mobility. and E. Vol. Axelrod. J . D. R. Schlessinger. Macroscopic motions extend over distances large compared to molecular dimensions. W. 18. Schlessinger.457 (1974). Proc. Edidin. New York. Bahr. Nail. Biochim. J. Science( Washington.282 (1974).
this method has found few applications. Biophys. D.237 (1975). Interactions with membrane proteins and larger molecular aggregates may further impede all types of motions. A fluorescence fluctuation method. Rotational motions are indicated by measurements of steadystate fluorescence polarization and of decay of fluorescence polarization and transient dichroism. Berlin. 819 (1978). 8. Consequently. Molecular Rotations in Membranes The extent to which planepolarized light is absorbed by a molecule depends on the extent to which one of its resonant transition dipoles is parallel to the plane of polarization. however. Some specific fluorescent probes are considered as examples. Nafure (London)264. Stryer.5. which may be useful in some limited conditions. Annu. They could influence differently microscopic and macroscopic motions and could vary among different types of membrane components. Annu. Azzi. The factors that control microscopic and macroscopic mobility in membranes are not yet well understood. These interactions are likely to be more specific than viscosity effects. For example. this relationship is Is l9 2o S. A dependence of rotational diffusion rate on membrane viscosity has been demonstrated experimentally. cholesterol increases the apparent viscosity and suppresses the gelliquid crystal phase transition of the bilayer. 179 (1974).2. Q. M. A. A useful compilation of fluorescent probes can be found in Azzi. The scope of this paper is limited to a description of fluorescence methods that are useful for studying dynamic behavior on cell membranes. The most versatile and convenient method for measuring macroscopic lateral motion is based on fluorescence photobleaching.411 (1976). M.20 These studies have shown that the viscosity of the bilayer is very sensitive to lipid composition. 3.2. Biochem. The viscosity of the lipid bilayer provides a lower bound to the forces resisting the motion of molecules embedded in it. light emitted as fluorescence is parallel to the emission transition dipole. Fernandez and R.’Oa 5. Edidin. Rev. Rev. MOLECULAR ROTATIONS IN MEMBRANES 199 fluorescence energy Up to now. Similarly. The structure of a molecule fixes the relative orientation of the transition moments. Viscous resistance should vary linearly with the viscosity of the bilayer for both macroscopic and microscopic motions. an array of stationary molecules should display a defined relationship between the polarizations of absorbed and emitted light. If the molecules rotate randomly during the interval between excitation and emission. is also briefly described. 47. Rev. but a detailed description of probes is not considered in this paper. Biophys. Bioeng. . L.
The (monochromatic) excitation light beam propagates in the y direction with vertical ( z axis) polarization. which are defined as so that P = 3r/(2 + r).2) The steadystate level of fluorescence depolarization may be measured in a timeindependent experiment. (5. 1.. EXCITATION DETECTION FIG. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS attenuated to a degree which depends on the extent of rotation.) t o the direction of polarization of the excitation light. The fluorescence polarization is usually characterized by the polarization parameter P or by the fluorescence anisotropy r. ) to the polarization of the excitation light. which is fixed in a laboratory frame of reference. Therefore. . This arrangement is shown in Fig.200 5. be related to the plane of polarization of the incident excitation light. Fluorescence is detected perpendicular to the excitation (x axis) through an analyzer which is oriented either parallel ( I . The light beam is propagated in the y direction or and the emission intensity is detected through an analyzer which is oriented parallel (IIl) perpendicula (1. 1.2. Using more complex apparatus one can observe the timedependent decay of polarization or anisotropy. typically on the scale of nanoseconds. The transition dipole is described in polar coordinates. if the mean fluorescence lifetime is comparable to the time required for molecular rotation. the rotation rate can be deduced from the extent of depolarization of fluorescence excited by planepolarized light. Geometrical arrangement for measurement of fluorescence polarization. The interpretation of fluorescencepolarization measurements in molecular terms requiresthat transition moments. fixed relative to the threedimensional coordinates of the molecule.)or perpendicular ( I .
/q. Perrin.1. For nonspherical particles C(Y) may vary with r. which depend on the size and shape of the molecule. J . T is the absolute temperature.23 in a different form: ro/r = 1 + 3 ~ . Weber” showed that ro/r = 1 + c(t)Tz. the relaxation time for rotation about one axis. A molecule of arbitrary shape can be described in terms of three rotational frictional coefficients. (5. as where k is Boltzmann’s constant.3) Here r. Then. 7 . say p l . G.” p . For a sphere of radius R . the latter quantity measures the time required for the average projection of a molecular axis on its initial orientation to decay by e’. 33 (1934).2.415 (1953). Phys. Protein Chem. J . More precisely.2. in a solvent of high viscosity).2. where the overbars indicate average values.f 2 = f 3 = 8nqR3 where q is the viscosity of the solvent. the fluorescence lifetime zo and the time required for molecular rotation.5. / p . Phys. 7 . then _ _ _ _ _ cos 4 = cos 4o e . 5. SteadyState Fluorescence Polarization The steadystate anisotropy of the fluorescence of spherical particles is determined by the relative magnitudes of two characteristic times. 1 (1936). MOLECULAR ROTATIONS IN MEMBRANES 20 1 5. In general. Perrin. More generally. Hence if d. F..o is the angle between the axis and some reference orientation at time t o and d. Radium Ser. (5. specified by p. the rotational relaxation times are related to the rotational frictional coefficients. 23 F. is the value at time t. and q is the viscosity of the solvent..g. Hence for a sphere2’ p = 4nqR3/k?: The measured anisotropy may be related to the rotational relaxation time with an equation originally derived by Perrin22. 8. 22 .4) where c(r) is a function of molecular shape and the location of the transition dipoles in the molecular framework. is the limiting fluorescence anisotropy in the absence of molecular rotation (e. A slightly more complex relationship must be used for nonspherical particles which require more than a single relaxation time to describe their rotational behavior. for example. Weber. Adu. Radium Ser.f 2 andf3.2. is related to the frictional coefficients for rotation about the other axes. 7 . the rotational relaxation time.
1655 (1975). Thompson. hydrophobic fluorescent molecule is dissolved in the lipid bilayer of a vesicle or cell membrane. B. C. however. Robbins. Shinitzky and Y . J . 29 F.2. Weber. 72.4) after determining the absolute temperature T and the fluorescence lifetime z. (5. Biochemisiry 12. W. A.^' In this experiment the rotational relaxation time of the cellbound fluoresceinCon A was correlated with the “degree of rotational mobility” of the Con A receptor sites. 81. B. phase transitions and degree of artificial3’*3 1 and order in micelles. Rudy and C. and Y. 24 2s ’’ . Mol. Barenholtz. 26 U. Gitler. W. liposomes. and E. Sachs. M. 169 (1929). Nishida. M. A. Shinitzky. Biol. Biochemistry 15. Shinitzky. 2 7 K. and T. and the steadystate polarization of asuspension of the cells or vesicles is measured. Biochemistry 15. Nail. and L. Fuchs.5hexatriene). Weber. R . J. Barenholtz. Dianoux. B. J . 32 M. Biophys. 521 (1973). Chem. and cell membrane^. A.245 (1973). M. Acad. Lentz. Litman. Inbar. Eur. S. 28 M. 250.5) This equation has been extensively applied to the measurements of the microviscosity of membranes. Mol. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS For spherical particles. and T. Proc. 5.2. R. Y. P. The fluorescent probes most commonly used are perylene or DPH (1. Cogan. E. Phys. Barenholtz. R. Biochim. 231 (1972). 38 M. Inbar. and L. c(r) = k / K a constant involving the Boltzmann constant k and the effective volume of the sphere I/: Then ro/r = 1 + kTzo/qI/: (5. Acta 288. 3351 (1975). 4529 (1976). Barenholtz. and T.The fluorescence anisotropy of the embedded probe measured in the lipid bilayer system is then compared to the fluorescence anisotropy of the same probe when it is dissolved in a reference oil of known v i s ~ o s i t y . Ann. J. Blout. a small. Inbar. Toyoshima and T. Shinitzky. Sci. 249. Parola. 117 (1974). Thompson. Biochemistry 12. Gitler.38 M . and S. E. Jacobson and D . U . Biochemistry 15. Shinitzky and M. Lipids 12. Sachs. Wobschall. Usually z is measured at a single temperature and the relation between fluorescence intensity and fluorescence lifetime then used to evaluate z at different temperature^^^ : z1h2 = IIP2. J.247 (1973). Biol. Chem. Y .^'^' In a few cases the fluorescence polarization method has also been applied to measure the rotational relaxation time of fluoresceinconjugated Concanavalin A (Con A. Chem. 2652 (1974). 35 P.2. 166 (1975). C. (Leipzig) [5] 12. ~ The ~~ membrane ~ microviscosity can be calculated by applying Eq.2766 (1976). 603 (1974).521 (1973).6) This simple approach has been used to study the “microviscosity” of 3236 membranes. Biol. 3 7 M. Inbar and M. Immunol. 3 3 M. Typically. 34. Lentz. (5. Perrin. Shinitzky. Stubbs. Shinitzky. Osawa.4521 (1976). Phys.202 5. 3 4 S. 85. 36 G. Biol. FEBS Lett. G .6diphenyl1. 30 B.3. a mannose binding protein) both in aqueous solution and bound to cell membrane^. J.
Dependent Fluorescence Polarization When a fluorescent molecule can be described as a spherical rotor in a threedimensional liquid.2.(t) 39 J . P.2. Frequently.. Easter.2. detect possible asymmetric restrictions to rotation in the system. the situation is more complex. Moreover. . Sample ~~ fluorescence is excited by pulses of polarized light from a thyratrongated air flash lamp. and make possible a more realistic analysis of the effects of bilayer "viscosity" on the motions of embedded particles. The observed fluorophore may display several fluorescent processes with different lifetimes zi. Consequently c ( r ) of Eq. i (5.4) is constant and the equation is linear in r.8) This leads to a nonlinear Perrin plot.2.2. Nanosecond Time. The measuring system most commonly used is a simple modification of a singlephotoncounting f l ~ o r o m e t e r .571 (1976).2. 16.2. H . however. R. (5. For a system of this type the average fluorescence lifetime is defined as and the average rotational correlation time is defined as follows: (5. J.7) and the rotational correlation time: r(t) = C Pie"Pi. and L. Then the behavior of the fluorescence anisotropy is correspondingly complicated. the nanosecond timedependent fluorescence depolarization method can in principle resolve the different components of zi and pi.10) While the steadystate depolarization measurement can give information about the average rotational relaxation time ( p ) using the average fluorescence lifetime (z). then both the decay of the total emission and the decay of the emission anisotropy of the molecule can be described as monoexponential functions.5. Multiexponential functions must be used to represent both the total emission: (5. Biophys.39 The decay of total emission and emission anisotropy are measured by collecting I . DeToma. the fluorescent particle may undergo anisotropic rotation due either to an asymmetric shape or to restrictions imposed by the anisotropic fluid matrix on certain rotations. Brand. MOLECULAR ROTATIONS IN MEMBRANES 203 5.
~ The ~ .~' Phase shift methods are also in use. S. 4 5 R. 2319 (1977). Chem. 4 6 R. Eiophys. J. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS and ZL(t) (see Fig. a fast phase occurring in about one nanosecond and a much slower phase.4081 (1977). Eiol. 4 2 R. Chem.44 5. 7500 (1977). Chen. E.2. Ikegami.204 5. the dichroism decayed. 66. in egg lecithin and in dipalmitoyl phosphatidyl choline v e s i ~ l e s . G. the timedependent anisotropy v(t) can be calc~lated. 2163 (1977). After appropriate deconvolution from the time profile of the excitation pulse.~ ~ comparison of these data to the results of measurements of steadystate fluorescence polarization demonstrated that the former revealed a much more complicated picture of the behavior of DPH in oil or lipid vesicles than the latter did. and L. and A. E.45 Measurement of rotational diffusion of a membrane protein was first accomplished by Cone. 252. Nuture (London). they proposed a model which allowed them to estimate the effective viscosity of the bilayer. K. Dale. ~ A '. FEES Lert. Brand.3. L.41 Analysis of microviscosity determinations based on steadystate polarization data in terms of the component lifetimes and rotational relaxation times detected in timedependent measurements has been carried out infreq~ently. Chen. ~ the ~ anisotropy decay curves into two phases.289 (1977).~~ fluorescence polarization of DPH was measured in paraffin oil. Cone. Decay of Transient Dichroism Steadystate and timedependent fluorescence polarization measurements have suggested that the rotational relaxation time of membrane proteins is on the order of a microsecond or longer. 4 3 S. Phys.46 He exposed rod outer segments to 5nsec pulses of polarized light. 20. Chem. Ikegami. From the L. and A. This exposure selectively bleached rhodopsin molecules whose absorption transition dipoles were parallel to the axis of polarization. J. and L. 236. who made use of the natural photochemistry of rhodopsin in retinal disk membranes. thereby inducing a dichroism in the rhodopsin. Dale. S. Biochemistry 16. A .timedependent ~~. Roth. New B i d . Bid. Weber. 4" 4' . Excitation and observation must be repeated many times and averaged to reach adequate precision. J. Kinosita. Cherry. R. Kawato. 55. Kinosita.A general theoretical discussion of the analysis of restricted rotation by fluorescence anisotropy decay has also appeared. Their values were about an order of magnitude smaller than those estimated from steadystate anisotropy data without considering the restrictions on rotation revealed in the relaxation experiments. Based on the temperature dependence of the anisotropy relaxation data. 252. 1). A. Kawato. J. 35 (1972). Brand. 44 K. Kawato et ~ 1 separated . A. As the rhodopsin molecules randomly reoriented because of rotational diffusion. J. I (1975).
This method was used to study the rotational diffusion of band3 proteins in human erythrocyte membranes. M. 245. the conceptual limitations. The fluorophore rotates in an anisotropic environment in which the rotations can be restricted to varying extents depending on the 4 7 K. Since tripletstate lifetimes are in this range. Schneider. R. . From this experiment the rotational relaxation time of bacteriorhodopsin was calculated to be greater than 1 msec.2. Chcrry. and D.4. Cherry et u1. the conceptual and the experimental. (t) are the timedependent absorbancechanges for light polarized parallel or perpendicular to the plane of polarization of the excitation light. NeM B i d . Measurement of the transient dichroism of triplettriplet absorption of labeled proteins yielded the rate of rotational motion. 389 (1976). MOLECULAR ROTATIONS IN MEMBRANES 205 rate of decay a value of 20 psec was deduced for the rotational relaxation time of rhodopsin in the disk membrane.^' In this system the induced dichroism did not decay during the period of observation. and G. Nature (London) 263. GonzalezRodriguez. It is clear that even a bilayer composed of a single lipid species is not an isotropic fluid. The initial dichroism was produced by polarized laser light pulses of 12 psec duration. a major constituent of the erythrocyte membrane. The anisotropy factor measured in this experiment was defined as (5. A very different result was obtained when a similar experiment was performed on bacteriorhodopsin in purple membrane fragment^. Parish. J.2. 5.2. Busslinger. It is possible to divide these assumptions into two categories. These experiments yielded a rotational relaxation time of 0.5. Cherry. It is assumed that the fluorophore rotates in the bilayer (which is a twodimensional fluid) as it does in isotropic fluids. A. J. J. Chapman. RaziNaqvi. Special Features and Limitations The interpretation of steadystate fluorescence polarization data of membranes in terms of microviscosity is based on a few crucial assumptions.4a have developed a general method based on transient dichroism for studying the rotational relaxation of membrane proteins. this time was not affected by removal of the erythrocyte spectrin network. they used as a label eosin isothiocyanate. First. In a few cases the limitations embodied in the assumptions have been ignored and data therefore misinterpreted. Biirkli. G. Moreover.5 msec for the band3 protein. Ntrfurr (London). 249 (1973).11) A l l( t )and A . R. which is readily excited to a triplet level.45. 4 8 R. Their approach was to label membrane proteins with probe molecules that have an excitedstate lifetime in the millisecond range.
which are sensitive to the viscosity and anisotropy of the fluid. S. van der Saag. From our experience we conclude that most fluorescent hydrophobic molecules tend to enter very rapidly into the cell interior. The fluorescent probe may bind to different species. It is very common to use a single average lifetime (t) in the Perrin equation [Eq. Partial quenching or chemical reaction during the fluorescence lifetime would yield a complicated multiexponential decay. (2) Mixed population of cells. de Laat. 4458 (1977). It is often assumed that the fluorescent hydrophobic probe is inserted in the lipid matrix of the plasma membrane. Proc. The location of the probe in the cell is often unknown and is difficult to ascertain.49 49 S. This includes carbocyanine and merocyanine dyes and also fluorescent derivatives of lipids. thereby restricting the motion of the probe molecule. The situation is even more complicated in a cell membrane which is composed of different types of lipids. P. A . Shinitzky. This can lead to serious problems of interpretation. W. At least one million cells are necessary for a single fluorescence polarization experiment.2. 74. and M. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS location of the fluorophore in the bilayer. In an isotropic medium without quenching processes the decay kinetics of a fluorophore can often be described by a simple exponential decay. Use of synchronized cell cultures can reduce this problem. Primary cultures are usually composed of heterogenous populations of cells at different stages of their cell cycles or differentiation. . NBDphosphatidylethanolamine and rhodaminephosphatidylethanolamine. Experimental assumptions that can lead to misinterpretations can be summarized as follows: (1) Localization of fluorescent probes.2. proteins. A second problem originates in the typical complexity of the mechanism of fluorescence decay.206 5. glycolipids. This assumption affects not only the rotational relaxation times calculated from the Perrin equation but also the estimated values of fluorescence lifetimes at different temperatures calculated from Eq. T.4)] instead of the individual components of the fluorescence decay processes. Narl.6). (5. and glycoproteins. Therefore the measured microviscosity does not necessarily reflect the fluidity of the plasma membrane but in fact is calculated from a complex average ( p ) of all the rotational relaxation times pi of probes located at different sites weighted by their corresponding quantum efficiencies. and therefore the average rotational relaxation time ( p ) which is obtained from the steadystate polarization data is determined by rotational behavior occurring in heterogeneous environments. Acad. The measurements of fluorescence polarization arc usually performed on suspensions of cells which have been labeled with a fluorescent probe. U . It is possible that the hydrophobic fluorescent probe that is embedded in the lipid matrix could also bind to some hydrophobic domains of membrane proteins. Sci. (5.
'^ The fact that the rotational relaxation time for fluoresceinCon A in water is only two o r three times shorter than the rotational relaxation time for fluoresceinCon A on the cell membrane is also consistent with the presumption of segmental flexibility. Therefore the significance of measurements of apparent . When fluoresceinCon A is bound to the cells the polarization data yield relaxation times of 701 60 nsec. MOLECULAR ROTATIONS IN MEMBRANES 207 (3) Segmental flexibility in measurements of receptor rotational relaxation times.20. ~ The ’ . These values are at least 100fold shorter than the values obtained for protein rotations on cell membrane by other methods.5. which is in the nanosecond range. Fluorescence polarization studies on artificial bilayers have precisely detected the gelliquid crystal phase transition in phosphatidylcholine dispersions. The major advantage of the steadystate fluorescence polarization method is its simplicity. Neither steadystate nor nanosecond timedependent fluorescence depolarization measurements are adequate for studying the rotation of proteins in the cell membrane. is too short compared to the relaxation time of the proteins (microseconds or more). But this time domain is adequate for lipid probes which have rotational relaxation times in the nanosecond range. multilamellar liposomes and small. singlelamellar vesicles. The fluorescence lifetime.transition are determined for both large.24 2 8 The interpretation of polarization data on intact cells is complicated by uncertaintites about the location of probes and about segmental motion of labeled proteins. Its major use is as a diagnostic tool for studying qualitative changes that are related to the fluidity and degree of order of membrane system.4548 Most values are on the order of microseconds or longer. It is very likely that the values deduced from steadystate polarization data for the rotation of fluoresceinCon A do not reflect the rotations of the Con A receptors but rather some kind of local rotation of the bound dye or a segment of the bound lectin. cells of both types were labeled with fluoresceinCon A and the mean rotational relaxation times of the cell surface labeled lectins measured by steadystate fluorescence polarizat i ~ n .” Measurements of fluorescence polarization have also provided reasonable values of the phase transition temperature and degree of order in micelles and erythrocyte membranes.309 The results are qualitatively similar to those obtained by other methods. In order to compare the rotational relaxation times of Con A binding sites on normal and transformed cells.^"^' They found that the rotational relaxation time of fluoresceinCon A in solution is 58 nsec. ~ authors ~ interpret the differences in the polarization of the fluoresceinCon A on the cell surface as an indication that the Con A receptors can rotate at different rate^. Is is noteworthy that at the high doses of Con A used in these experiments the cell surface receptors are “frozen” and cannot move later all^. The parameters of the phase.2.
D and V can be calculated from the measured time behavior of the fluorescence. such as flow along the cell surface. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS 38 rotational motion of Con A receptors on normal and transformed and the effects of malignant t r a n s f ~ r m a t i o n 38 ~~ and ~ c h o l e ~ t e r o lon ~~ membrane fluidity cannot now be definitely known. Sci. Shinitzky. Proc.48 5. Null. The concentration of fluorescent molecules is minimally perturbed by measurement. uniform in speed and direction. A . sensitivity. The method of decay of transient dichroism seems to be a promising general approach for studying the latter. Then the recovery of the fluorescence in the region due to redistribution of the bleached and '' M.2128 (1974). rapid measurement. Therefore their number is estimated from measurement of the fluorescence emitted from the observation region.1. Acad. S. 71. with velocity V. The rate at which the number of fluorescent molecules changes is determined by the size of the observation region and the magnitudes of D and V. with diffusion coefficient D. In one. The varying time course of the fluctuating fluorescence is analyzed statistically to yield D and V. the number of fluorophores and consequently the fluorescence undergo transient spontaneous fluctuations about their mean values. Nevertheless.3. . called fluorescence correlation spectroscopy (FCS). Once the size is known. In these experiments the specified molecules are distinguishable and detectable by their fluorescence. The second method. Macroscopic Membrane Motions 5. Inbar and M . U . We consider both diffusion. and simple. Therefore the mean number of fluorophores in the observation region is constant. Two methods based on these ideas have been developed. Generally. the concentration of fluorescent molecules on the membrane remains in overall equilibrium throughout the experiment.3. In principle other molecular properties could be used. This irreversibly photobleaches a portion of the fluorophores in the observation region. Basic Concepts Lateral motion may result either from random Brownian diffusion or from a systematic driven process. fluorescence polarization methods are suitable for studying the rotational relaxation of lipids but not of proteins in membranes. fluorescence photobleaching recovery (FPR). generates an initial nonequilibrium gradient in the concentration of the fluorescent molecules by exposing them to an intense pulse of light.208 5. but fluorescence has the advantages of specificity. and simple flow. The central experimental task in determining D or V is to measure the number of molecules of a specified kind in a defined open region of the membrane as a function of time.
it is convenient to use the same microscope optical system and laser excitation source for both phases.) Total Energy 0 2 4 6 t/T 8 1012 14 FIG.A/B. (a) Fluorophore concentration profile induced by a brief pulse of an intense Gaussian laser beam for different bleaching energies. (b) Schematic recovery curves for diffusion and flow.3. The photobleaching pulse must be short compared to this recovery time. Principle of the FPR method. 5. 2. The observation region is defined as the area illuminated by the excitation light and is typically a small fraction of the cell surface (< 1%).. During the recovery phase the laser excitation is attenuated by several orders of magnitude relative to the bleaching pulse to minimize further bleaching. 2. Subsequent bleach on the same spot would show increased fractional recovery.'~''(See Fig. the monitoring of the fluorescence recovery due to redistribution of bleached and unbleached fluorophores. MACROSCOPIC MEMBRANE MOTIONS 209 unbleached fluorophores is analyzed to yield the mobility parameters.Fluorescence Photobleaching Recovery An FPR experiment consists of two major phases: first.3. We shall consider FPR in some detail since it has proved to be more convenient for measurement on cell surfaces. Although other experimental arrangements have been tried.The diffusion curve shows an immobile fraction 1 . second.2. The recovery time is determined by the size of the illuminated area and the rate of motion (Dor V). . the irreversible photolysis of a portion of the fluorescently labeled molecules in a small region of the cell surface and. Fluorescence photobleaching experiments have been performed by Peters et al.5." Edidin and Zagyansky.'1s'2 and Jacobson et a l l 3 We call our approach fluorescence photobleaching re~overy. We shall then give a briefer description of FCS and compare the two approaches.
through a field diaphragm (FD) and lens L3.3. and a scatter aperture (A) and lens (Ll) to control the size of the beam as it enters the vertical illuminator. and the objective lens L2 of the microscope onto the specimen membrane. The appropriate laser line to excite the fluorophore is selected by a prism in the laser cavity.  FIG. . The beam from a krypton or argon ion laser is focused through a spatial filter. (usually water immersion 40 x NM 0.65). or light can be supplied by the illuminator (IL). The mirrors (M) and the field diaphragm (FD) can be moved to present an image to viewing eyepieces or a camera (C). 3. Dotted lines indicate removable components (see text for notation). The fluorescence radiation is collected by the same objective lens (L2). In order to observe and photograph the cell. a spatial filter (SF) to ensure a reasonably regular Gaussian intensity profile. The field diaphragm limits the volume “seen” by the photomultiplier. a beam splitter that supplies a small portion of the radiation to a photodiode intensity monitor (MON). and onto a sensitive photomultiplier tube (PMT) that is connected to photon counting electronics for measurement of the timedependent fluorescence intensity.l6 The illuminated area on the fluorescencelabeled membrane is usually a spot of 1p radius. Microscope geometry for FPR experiments. the lens (Ll) may be removed to spread the laser illumination. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS The apparatus that we have developed for the photobleaching experiments is shown in Fig.210 5. In a typical experiment the fluorescently labeled cells adhering to the dishes in which they were cultured are covered with complete growth medium or buffered saline solution. passes through the dichroic mirror (DM) of the vertical illuminator. The beam is passed through a filter (PG) to remove plasma glow. vertical illuminator.
the fluorescence concentration profile at the beginning of the recovery phase (t = 0) is given by C(r.~ ” ’ ~ ’ .3. .0) = CoeaTo’(r). Special efforts and/or multiple labeling may improve this sensitivity. Then the concentration C(r.3. The theory for FPR15 assumes that the photobleaching of the fluorophore to a nonfluorescent species is a simple irreversible firstorder reaction with rate constant crZ(r).3) (5.’ The sensitivity for observations on membrane proteins with diffusion coefficients 10. The entire apparatus is mounted on a table with pneumatic supports that eliminate essentially all building vibrations. (5. For slow diffusion.3. Z(r) is given by Z(r) = (2p0/72~ ~ ) e . a solenoidoperated neutral density filter (FF. also allow alignment of the field diaphragm with the center of the visual field and visual identification of the area selected for quantitative observation. For a bleaching pulse which lasts a time interval To short compared to the recovery time. (5. MACROSCOPIC MEMBRANE MOTIONS 211 Eyepieces El and E. The electronic system includes photon counting electronics. bleaching times as short as 4 msec are preferred with brighter bleaching intensities. The “amount” of bleaching induced in time To is expressed by a parameter K : K = aTOZ(O).2) where Co is the initial uniform fluorophore concentration.3. The duration of the bleaching pulse ranges from 5 msec to 3 sec.aZ(r)C(r. The bleachingfilter solenoid pulse is synchronized with the chopper wheel.l o cm2/sechas been limited by autofluorescence of the cells to 2200 fluorophores per square micron. t)/dt = .t). multichannel digital recording.3.4) For a Gaussian intensity profile. For the fasterdiffusing phospholipids. To supply the photobleaching light pulse for FPR experiments. OD 4 or 3) is momentarily removed from the laser beam. The fluorescence light intensity is measured by sensitive coldoperated photomultiplier to reduce dark current. etc. recorders. where W is the halfwidth at e’ height and Po is the total laser power. and simultaneously the highvoltagebias potential is removed from the photomultiplier dynode chain to avoid damaging it. (5. t ) of unbleached fluorophores at position r and time t in the absence of transport can be calculated from  dC(r.1) where I ( r ) is the bleaching intensity. it is sometimes useful to reduce the average monitor beam intensity below that set by the filters FF and NDF by limiting the exposure to periodic pulses generated with a chopper wheel (CW).5.
3. Fluorescence Correlation Spectroscopy The experimental apparatus and theoretical analysis of FCS and FPR measurements are similar in many respects. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS Axelrod et al. More complicated expressions are required for arbitrary K values. Values of D and A / B are determined by curvefitting procedures described elsewhere. For pure flow.3. 2) of fluorophores.8) where yD and yF are constants which depend on K . and simultaneous diffusion and flow are presented in the paper by Axelrod et al.' If the fluorophore is attached to a variety of molecules with a range of diffusion coefficients. The major differences between the two methods arise from the smallness of the typical spontaneous Auctua . Simultaneous diffusion and flow would yield recovery curves of intermediate properties. (5.3. It is often simplest to determine a transport rate from T ~ .. The values of yD and yF versus K .3. ' 5.3. as well as expressions for F ( t ) for diffusion.3. F ( t ) has a sigmoidal shape with zero slope at t = 0 and then a relatively rapid approach to the final value (see Fig.212 5. YD = T1/2/ZD.5) r0 = W2/4D (5. Only the existence of some independent information on the distribution or a special case such as clear bimodal recovery due to two widely separated values of D allow the deconvolution of the recovery curve to be carried out quantitatively. flow.6) and F ( t ) is the fluorescence intensity at time t." For pure diffusion. Theory was also developed for uniform flow at velocity V. If some of the fluorophores are immobile the recovery of fluorescence intensity is incomplete and the fraction recovered at long times gives the mobile fraction A / B (see Fig.' showed that for K 4 1 for simple diffusion the fluorescence recovery curves can be represented in simple form: F ( t ) = F(0)(1 where + t/qJ'. (5. the ~ .2)YD. or for simultaneous diffusion and flow.time required for half of the observed fluorescence recovery to occur: difusion = (w2/4zl.7) POW v0 = (w1Tl/2)?F7 YF zl/2/TF? (5. F ( t ) has a finite slope at t = 0 but approaches its final value very slowly.the recovery curve reflects a weighted average D which depends on the distribution of individual diffusion coefficients. 2).
Magde. Magde. Biopolyniers 13.1 pm radius). Webb. (5. however. (2) Since the observation region is small (. ganglioside analogs. 4. 5 4 E. Rev. W. Elson and D. and W.705 (1972). L. Therefore the fluctuating fluorescence signal in an FCS experiment must be analyzed statistically to yield precise values of transport coefficients. the mobilities of components in different regions on the same cell can be measured and compared. L. Magde. hormone receptors.3. Rev. L. Annu. E. The time dependence of g ( t ) is identical to that of F(t) [Eq. the narrow depth of focus of the microscope makes possible a determination that the fluorescent marker is on or near the cell surface. Biopolymers 13. W.29 (1974).4. 1 (1974). Furthermore. A detailed discussion of the measurement and interpretation of the autocorrelation function has been pre~ented. ” 52 . D. Biophys. W. and lipid probes. Elson. This contrasts with FPR.5.~’’~ Development of g(t) requires that the fluorescence signal be observed over periods of time long compared to an individual fluctuation. the amplitude of g(t) yields directly the average number of independently diffusing fluorescent units in the illuminated area. Up to now these have included surface antigens.3. 5 3 D. Webb. lectin binding sites. Lett. L. 29. It is not necessary to infer rates of macroscopic transport from measurements of microscopic motions as in magnetic resonance or fluorescence depolarization. 5. E. ~ ~ may also be used to measure chemical kinetics in suitable systems although this has not yet been demonstrated in practice. Unlike F(t). and W.6)] in an FPR experiment (for K G 1) for diffusion or flow. Elson. E. This is appropriately done by computing the autocorrelation function g(t) of the measured fluorescence. (4) Mobility of specific fluorescently labeled components is measured. Webb. in which a single recovery allows an estimate of D or V.2. Phys. Elson and W. MACROSCOPIC MEMBRANE MOTIONS 213 tions of the number of observed fluorophores and the fact that the time course of an individual fluctuation does not deterministically define the diffusion coefficients of the labeled molecules. (3) Lateral motion over micron distances is measured directly.52 FCS was originally developed to measure coupled diffusion and chemical kinetics in chemical reaction systems in e q ~ i l i b r i u mFPR .311 (1975). Bioeng. FPR and FCS: Special Features and Limitations Both FCS and FPR have features which are favorable for measuring the mobilities of surface components of living cells: (1) The use of a fluorescence microscope enables a fairly precise localization of the observation region relative to visible cellular features.
In practice FCS is useful for measuring > D > lo* cm2/sec. Unavoidable slow photobleaching of fluorophores over these long “integration” times could also lead to a significant slow decay in cellsurface fluorophore concentration which would appear as an additional timedependent complexity in the measured autocorrelation function. it is necessary to assume (and verify) a rate law for photobleaching. For slower diffusion coefficients in the range processes it is usually necessary to use FPR. however. to develop interpretive equations for more complex mechanisms of the bleaching kinetics. Elson. in an FCS experiment data must be collected over at least several hundreds of correlation times zD to obtain a precise estimate of g(t). creates difficulties in applying FCS to living systems. it directly determines the average number of fluorescent diffusing units (differentially weighted by excitation and emission coefficients in heterogeneous mixtures52). however. In FPR.214 5. This is in contrast with FPR. As previously indicated. FCS avoids observable perturbations of the measured system. In terms of diffusion coefficients this extends from to cm2/sec. FLUORESCENCE METHODSFOR STUDYING MEMBRANE DYNAMICS ( 5 ) A wide range of mobilities is accessible. while FCS on the same system would consume more than two hours.Second. diffusion or flow). During this prolonged period of data collection locomotion of the cell or local motion of the membrane could overwhelm the small fluorescence changes due to spontaneous fluctuations of fluorophore numbers and prevent the development of a precise correlation function. The requirement for a statistical analysis of spontaneous fluctuations. which  55 E.) (6) The methods are quantitatively precise and in principle capable of distinguishing among various mechanisms of transport (e.. the currently available and simplest interpretive theory assumes irreversible firstorder photobleaching. in preparation. Indeed. however. . (7) Transport rates are measured in individual cells (under physiological conditions). in which a diffusion coefficient can be determined from a single recovery curve. L. Unlike a photobleaching experiment. An FPR measurement of the diffusion coefficient of a typical membrane protein ( T ~ 20 sec) would last approximately three minutes. First. however. This capability could be useful in studying aggregation e q ~ i l i b r i a . At least in principle the FCS method has three significant advantages over FPR. ~~. on living cells. which requires the destruction of a substantial portion of the fluorophores in a small region of the cell surface.~~ FCS requires no assumptions about the photochemistry of the observed fluorophores.55 Third. It is straightforward.g. (As indicated below. FCS is less useful than FPR in the lower part of this range. thereby enabling a correlation of physiological events and morphology with dynamic properties.
the concentration of fluorophores lo00 molecules/pm2. MACROSCOPIC MEMBRANE MOTIONS 215 typically show enough membrane activity to distort FCS measurement even of relatively fast processes. .58 Direct detection of photochemical effects in the bleached region is very difficult because the number of photolabile molecules is so small (5002000). An experimental demonstration of the small extent of heating during an FPR measurement has been carried out by They used FPR to measure the temperature dependence of the Wu et lateral diffusion coefficients D of two fluorescent lipid analogs in phospholipid multilayers of varying composition. These results have been confirmed in similar experiments by Fahey and Webb. Jacobson. the duration of the bleaching pulse 1 sec. E . Biophys. 3046 (1978).56 Although these increments are probably negligible.5. A greater than 100fold change in D was detected in dimyristol phosphatidylcholine multilayers at 23"C. 4 .03"C and during the fluorescence recovery phase 1050C. the temperature at which the gelliquid crystal transition has been shown to occur by many other methods. krypton laser lines 568. Therefore information on this matter is mainly indirect but in its cumulative weight provides a persuasive argument against significant adverse photochemical effects. the maximum temperature rise is small. This coincidence of the transition temperature determined by FPR and by other methods demonstrates that the temperature perturbation in an FPR measurement must be quite small. 520. 3936 (1977). Axelrod. and D. J . Wu. K.. Fahey and W. Webb. 129 (1977). Biochemistry 17. The laser power density during the bleaching pulse is approximately W/pm2.8. The damage could arise either from heating or from chemical effects on membrane lipids or proteins due either to direct photochemistry or to free radicals generated by the bleaching pulse. the temperature rise could become significant if the fluorophore concentration or beam intensity were increased by several orders of magnitude or if the cell were to contain other pigments which absorb the excitation light.     (1) To minimize cell damage.2. P.'. during a typical bleaching pulse 0. we choose fluorophores excitable at wavelengths at which most cell components do not absorb. FPR is usually the preferred method over the entire range of mobilities. Papahadjopoulos.g. 18. W. Therefore it is essential to evaluate the possibility that the bleaching pulse in an FPR experiment damages the cell or the plasma membrane. e. as erythrocytes or plant cells do. F. and 482 nm.This heat is rapidly dissipated owing to the high thermal conductance of water so that a thermal steady state is reached within sec. 5h 57 D. Biochemistry 16. and the extinction coefficient of the fluorophore at the wavelength of the laser line lo5 M cm. These parameters yield a heating rate of 105"C/sec.3. Consequently.
W. Therefore there were no perceptible effects which depended on excitation wavelength or intensity or on the identity of the fluorophore. The fluorophores and resultant free radicals are in the aqueous phase.4. Exp.4.60FCS experiments do not require an exposure at high laser intensity as do FPR experiments. 116.8 nm. E. Webb. Elson. Barak. Wolf. C . E. hormone. W. (3) Increasing the laser intensity tenfold [into the range 103102 W/(pm)’]. and W. Webb. not embedded in the membrane. Measurement of the mobility of the lipid probe diI (3. W. . D. 6 o D. 179 (1978). Jacobson et al. D. Elson. Y. 305 (1977). L:S. 6 1 K. Henkart. ) 195. They are therefore exposed to rapid quenching processes. Hou. and J. Blumenthal. or lectin.3’dioctadecylindocarbocyanine iodide) in different cell types confirms the 5 9 P. but rather is bound to a large protein ligand. Fahey. toxin. changing excitation wavelength from 568.1. such as an antibody. L. They have used scanning electron microscopy to examine the irradiated area after bleaching with different light intensities. and P. Biochemistry 16. Wojcieszyn. Jacobson. Science (Washington. Koppel. 3476 (1977). and changing the fluorescence probe from rhodamine to fluorescein did not alter the measured diffusion coefficients or the fraction of mobile fluorophores. E. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS (2) In most cases the fluorophore is not directly attached to a cell structure. (4) Similar values were obtained using both FPR and FCS to measure the diffusion coefficient of the lipid probe diI in myoblast membrane^'^^'^ and in lipid bilayer~. F.61 5. Up to now they have detected no photochemical damage in the bleached area. a standard test of cell viability. Cell Res. R. D . J. They have also tested the effects of freeradical quenchers and light intensity on the incorporation of trypan blue.~’ Similar values were also obtained by the two methods for the diffusion coefficients of fluorescent lipopolysaccharides in artificial bilayers. as would be expected if substantial photochemical processes were occurring. E. Schlessinger.216 5 . Applications 5. L. Translational and Rotational Diffusion of Molecules in Membranes and Membrane Viscosity Published data summarized in Tables I and I1 clearly indicate the diversity in both the translational and rotational mobility of different cell membrane components. have recently carried out experiments which seek to assess in detail the extent and kind of local photochemical damage that occurs in an FPR measurement. Wolf.2 to 520.
) 185. Axelrod. Science (Washington. Acad. Elson and J. Proc. Johnson and M. K. Webb. W. Edidin. Reidler. Biochim. Elson. Rev. U . Peters. Elson. 73. Cone. S. IJ. Schlessinger. Biophys. D. Nutl. D. E. Tews. J. W. Lateral Diffusion of Cell Membrane Components Diffusion coefficient (cmz/sec) lo@ 109 Membrane component Lipid probe (diI) Analog of ganglioside GM. Schlessinger. Y. S. Axelrod. Sci. Webb. 0. A .60 0. A. S.30. 215 (1976). A . M. D. Biochim. Yamada. Sci. D. Lipid probe (diI) on unfertilized mouse eggs Lipid probe (diI) on fertilized mouse eggs Unselected surface antigens on unfertilized eggs Unselected surface antigens on fertilized eggs Rhodopsin in amphibian disks Surface antigens on mousehuman heterokaryons Unselected surface "proteins" (labeled chemically) Unselected surface antigens (labeled by antGP388) Labeled proteins of erythrocyte membrane (mainly band 3 ) Con A binding sites (SCon A on Con A at low dose) Con A binding sites (high dose) Mobile fraction 1 1 References 5  108 0.C.50.8 0 . 2409 (1976). Poste. 1183 (1974). A. U. I..448 (1978). W. Zagyansky and M. Schlessinger. L. L. D. Biophys. C. M. J.TABLE I. 691. Bahr. Sci. 179 (1974). L.) 184. Elson. and E. Nature (London) 247. 1217(1976). A. E. ' P. ' R. M. Entine. M. Yahara. W. V . A . Acud. 1526 (1980). T. in "The Neurosciences: Fourth Study Program'' (F. S. and T. < 10'0a 108. 319 (1970). Petit and M. L. Wu. and I. Po0 and R. Jacobson. D. W.8 0. A. and E.1090 0.209 (1976).Zagyansky. Wiegandt. Cell Sci. Schlessinger. Bioen.4 x lo" 10"<3 x 0. Webb. Acta 433. K. Edidin.q. 307 (1977). and H. Koppel.).438 (1974). P. Nature (London)272. Worden. Nutl. Proc. Proc. Edidin. S. Proc. Science (Washington. K.240.) 196.5 (continued) Shows two distinct components. Annu. Acad. 73. Natl. Jacobson. J . Natl. Biochem. Acta 367. E. Schmitt and F. Biophys. Sci. Pastan. Liebmann and G. A . in preparation. W.457 (1974). J. Acud. M. Koppel. Y. Massachusetts. and W. D. 7. Biophys. de Laat. V. E. and G.C. eds. Science (Wushington. ' ' .30.25 109. Edelman. Eldridge. Edidin. M. <lo"" 5 x 2 x 10~0109 2 x 2 x 10'O 4 3 x I 0. J. See K. Elson. and G. J. Science (Washington. E. Cambridge. G. E. 77.7 00.3. 1978. U . MIT Press.) 195. 74. 282 (1974). Lardner. S. L. ' S. J. . 1110 (1977). L. Schlessinger. Peters. D.14 108. H. Elson. Frye and M. Schlessinger.C. Edidin. and J. D.466 (1976). Edidin. Yamada. G.C. Rutishauser. 0. L. Acta 433. van der Saag.
Eur. Schlessinger. L. Schlessinger. Nature (London).5 msec ~. ' J. Auchet. 389 (1976). 245 (1973). I. M . Ravdin. Bio[.8 0 0. Shinitzky. Cherry. A. Barak. New Biol. Schneider. 5353 (1978). Elson. Sci. Cuatrecasas. E. K. L. scc) 3 x lo" <lo'* 3 x 1010 < 3 x 10lz Mobile fraction 0. Biochem. A . E. P. J. 249 (1973). S. Nature (London) 263. and A. 332 (1971). 73. 9. 34.8 0. K. Acad. FEBS Leti. " J. ( I M. Sachs.287 (1970). Yamdda.2909 (1977). Hammes. Mol. M . Willingham. 550(1976). Radda and D. TABLE 11. Podleski. d e 70160 nsec  f. 75. M.S. and I . e New Biol. Proc. f M. R. Aria 288. J . 81. 74. FEBS Lett. 236. S. Inbar. W . 245. W. ' R. J .Wdhl. P. 18. Gitler.4 Nanosecond time> 700 nsec dependent fluorescence polarization  h * G. Cone. M. Biochem. Proc. N ~ t w (London). Sachs. Smith. Cherry. W. E. Nature(London)264. 23 I (1972). and T. Rotational Diffusion of Cell Membrane Components Membrane component Perylene in erythrocyte ghosts Retinol in erythrocyte ghosts Rhodopsin in amphibian disks Bacteriorhodopsin in purple membrane Eosin labeled band3 protein of erythrocyte ghosts FluoresceinCon A on normal and transformed cells ANS or DNS bound to electroplax membrane fragments Method Fluorescence polarization Fluorescence polarization Decay of transient dichroism Decay of transient dichroism Decay of transient dichroism Fluorescence polarization Rotational relaxation time Viscosity (poise) References  I . Biophys. Acad. J . Pastan. Axelford. Natl. W. Parish. R. W. and L. M. G.Rudy and C. Pastan. A. 9. RaziNaqvi. Sci. G. Nail.3 110 b 20 psec 2 < 20 msec 0. A. L. R.TABLE I (continued) Membranc component Acetylcholine receptor (diffuse) Acetylcholine receptor (patch) IgEFc receptor Fibronectin Insulin receptor E G F receptor Diffusion coefficient (cm'. and D. I 4 x 1010 3 x l o . J.S. U . and J. Inbar. D. 35 (1972). P.s I. . C. and E. and G. J. J . Biirkli.8 0 0. Chapman. Webb. Schlessinger.8 References f. Proc. Metzger. S. 247 (1973). W. G. Schlessinger. K . M. and L. 4594 (1976). Elson.50. U. U. ' R. Arad. Shechter. Kasai. Elson. Shinitzky.' " " ' D. Changeux. L. Nail. Webb. A . Koppel. Y. Webb. GonzalezRodriguez. Sci. Busslinger. P. J.
A . Capaldi. R. it is necessary to develop fluorescently labeled lipids which retain their capacity to bind to membrane proteins. In order to study this phenomenon using fluorescence methods. Acad.62is similar to that of diI. Nevertheless. A . C. 70.APPLICATIONS 219 I I 0 80 160 240 TIME (sec) 320 FIG. Interaction of real membrane lipids and of diI with hydrophobic membrane proteins could also limit their rate of motion. 62 h3 . J . and G. Eldridge. E. Schlessinger. Sci. in preparation. Evidence for the immobilization of membrane lipids by hydrophobic proteins has been obtained in studies using spinlabeled fatty acids. Reidler. Griffith. S . 4a). L. The behavior of the fluorescent analog of the ganglioside GM. Jost. FPR curves for (a) the lipid probe diI and (b) proteins on cell membrane labeled with TNBS and rhodamine antiTNP antibodes. 480 (1973). H. C. Note complete and fast recovery for diI and incomplete recovery with slower diffusion of the labeled proteins.63The formation of a lipid boundary layer could influence the mobility of both the lipid and the protein. existence of a fluid membrane matrix in which membrane molecules are free to move over wide areas of the cell membrane (see Fig. It is not certain that only fluid dynamic factors limit the lateral mobility even of so simple a molecule as diI. J . 0. it may be wrong to assume that the major factor limiting the mobility is membrane viscosity. P. and H.5. U . A. Natl. The similarity of diI diffusion in different kinds of cells suggests that the viscosity does not depend strongly on cell type. Elson. Vanderkooi. Proc.4. The consistency of direct measurements of macroscopic lateral diffusion of the lipid probe with estimates derived from microscopic motions of lipids suggests that the same forces limit both.4. Its diffusion coefficient is only a factor of 2 smaller. Wiegandt.
t A quantittative comparison of mobility in disk and in plasma membranes would require that account be taken of the unusually high content of unsaturated lipid in the f ~ r r n e r . Actu 211. 72.t The additional interactions responsible for limiting the lateral mobility of membrane proteins could originate from many sources including (among others): interactions with cytoskeletal structures. Delbruck.220 5. If we assume that retinal disks present an ideal fluid membrane in which the diffusion rate of the rhodopsin molecules is mainly limited by the viscosity of the lipid matrix. and   64 65 P. ~ ’The . G. Saffman and M. Biochim. The calculated viscosity would be 20 poise. G. . It is impossible at this time to give a quantitative interpretation of the difference in diffusion coefficients of diI and the mobile labeled proteins. An analysis of this possibility could be carried out using the theory of Saffman and D e l b r ~ c kLet . A . S. One possible interpretation is hydrodynamic: the proteins are larger and therefore move more slowly because they experience greater viscous drag. S . The calculation suggests that the lipid viscosity does not control the lateral mobility of the hbeled surface proteins.l o cm2/sec. Acud. ~ ~us assume that membrane proteins have reasonable size (radius of 50 A) and that the diffusion coefficient is 2 x 10. Fleischer. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS The mobility of membrane proteins is more complex. then the mobility of membrane proteins in other cell types seems to be limited by interactions in addition to viscous drag. It is approximately ten times faster than the diffusion of insulin or epidermal growth factor receptor complexes on 3T3 cells. we feel that the presence of these unsaturated lipids will not affect the qualitative validity of this discussion. C. 3 I I 1 (1975). It is noteworthy that in retinal disk membranes rhodopsin is the major protein constituent and cannot have interactions with cytoskeletal elements. The membrane proteinsoccur in both mobile and immobile classes (see Fig. Null. N. 4b). Nevertheless. and D. 10 (1970). a viscosity of 12 poise is calculated from the diffusion coefficientsof diI and the fluorescent ganglioside analog in the cell membrane. McConnell. Proc. which is approximately tenfold higher than the viscosity estimated for the lipid matrix by many different methods. U. The mobile membrane proteins fairly consistently diffuse about 50100fold slower than the membrane lipids. The existence of immobile protein molecules suggests an interaction between them and some other molecules or supermolecular structures in the membrane or in the cytoplasm. Sci. The diffusion coefficient of rhodopsin is 5 x cm2/sec8. Nielsen. Rhodopsin in amphibian disk membranes seems to be the only system in which the lateral diffusion of a membrane protein is determined by the viscosity of the lipid matrix. data necessary to d o this are not yet available.which is approximately 20 times faster than nonselected proteins and antigens on cell membranes and IgE receptors on rat mast cells. Biophys. nonspecific collisions of membrane proteins with each other. Using the same approximation.
APPLICATIONS 22 1 interactions of membrane proteins with the extracellular matrix composed of collagen and f i b r o n e ~ t i n . Schlessinger. Hynes and J . Rev. Stoeckenius. 74. 233. Yamada and I . Bid. Blasie and C. Biophys. 39. 152 (1971).2909 (1977). M. Mol. Biochim.~ ' The rotational diffusion of cell membrane components is in many respects similar to their translational motion. 0. Pastan. A . Unlike visual rhodopsin. 70 J. Barak. The viscosity of the lipid matrix of erythrocyte ghosts is in the range of 110 The viscosity calculated from the rotational relaxation time of band3 proteins corresponds to 25100 poise. Yamada. Pastan. Proc. S. Blaurock and W. G. Acad. Its rotational relaxation time equals 20 psec. J .47This difference is consistent with xray diffraction data which indicate that disk membrane is a planar fluid array of rhodopsin m~lecules. The fast relaxation times measured for fluoresceinCon A on cell membranes by steadystate fluorescence polarization37338 and for ANS and DNS  '' R. A . Hynes. U.48 This result seems to indicate that the viscosity of the erythrocyte membrane is not the major limit for the rotational diffusion rate of band3 proteins. '' A. Biophys. S. It seems that the retinal disk presents an ideal fluid membrane in which both the rates of rotation and translational of rhodopsin are limited by the viscosity of the lipid matrix. Webb.417 (1969). 87 (1977). Worthington. M. Natl. G . K.4. 113 (1974). ~ ~ . 0. E. which corresponds to a viscosity of 2 poise.222 (1976). In addition. K. R. L.3492 (1974). M. Nature (London). Annu. and E. 1. 7 3 R.Elson. 71. Hammes. bacteriorhodopsin does not rotate rapidly in the purple membrane.72*73 The rotational relaxation time of band3 protein in erythrocyte ghosts is considerablylonger than the values obtained for small hydrophobic molecules by steadystate fluorescence polarization. Acad.~' while bacteriorhodopsin is packed in a crystallike arrangement in the purple membrane.5. K. Acta 456. Henderson. W. it was found that depletion of spectrin did not affect the rotational relaxation of band3 proteins in the erythrocyte ghosts. Trends Biochem. 6. New B i d . Bioeng. W. Sci. Lipid probes rotate at a rate which seems to be controlled by the viscosity of the lipid matrix (in the range of 110 poise). Again rhodopsin in amphibian disks is an exceptional protein. A similar value was obtained when the viscosity of the amphibian disk membrane was calculated from the lateral diffusion coefficient. M. R. Sci.48It is not clear what interactions impede the motion of this protein. 1. 68 69 66 K.73 (1976). Cell 3. . Weston. Sci. L. Yamada and J. Natl. A. U. The fact that band3 proteins can possess rotational motion without being able to move laterally over macroscopic distances" demonstrates an important distinction between local diffusion (around the axis of the molecule) and macroscopic lateral diffusion. Bye. 7 1 J. Proc.S.
251. Cuatrecasas. fluorescent derivatives of those two hormones were prepared. U. Sci. Shechter. The existence of a mobile hormone receptor is consistent with the fact that different hormones that bind to different receptors can apparently activate the same adenylate cyclase molecule. Kahn. Possible Significance of Mobility and Immobility of Membrane Proteins The scope of this paper does not include detailed analysis of the mobility of different membrane components. P. Jacobs. Proc. A . Perkins. Cuatrecasas.79The fluorescent analogs retained substantial binding a ~ t i v i t y ~ ' . A . K. 7 6 P. 15. Chem. 5.' ~ motion of the hormone receptor in the plasma membrane would enable the hormone to interact with various effector molecules. R. 74 75 . S . Chang. ~ Lateral ~ .70. 9 Y. Willingham. Annu. ' Accid. 2135 (1978). Furthermore.and ~ ' biological potency. Proc. P.261 (1976). 5353 (1978). and 1. Cyclic Nucleotide Res. The conclusion of this study was that the receptors for insulin and EGF are mobile on the cell membrane with similar diffusion coefficients D (35) x lo" cm2/sec at 2 3 T S 0Increasing temperature to 37°C yields pronounced  J . 169 (1974). Natl.J. M. J .222 5. Natl. Acad. and others located at fixed sites. Bianco. ~ ~ . FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS bound to electroplax proteins by nanosecond fluorescence polarization' do not seem to reflect the rotational relaxation of the entire protein molecule but rather some kind of independent. Orly and M. U .~ ~ Several reports have already proposed that the fluidity of the plasma membrane plays a role in the mechanism of hormone a ~ t i o n . Nevertheless. Shechter. some of which might be mobile. Natl. I (1973). 3. to illustrate the biological significance of membrane mobility we consider two systems in which the mobility or immobility of membrane components seem to be consistent with their physiological function. 7 7 C. A . Sci.2.4410 (1976). 1877 (1976). The two systems are the receptors for the hormones insulin or epidermal growth factor (EGF) and the major cellsurface protein of fibroblasts f i b r ~ n e c t i n . 7 9 3T3 fibroblasts were labeled with the fluorescent derivatives of insulin or EGF and the FPR method was employed to study their mobility. DeMeyts. restricted rotation of the probe molecule or the flexibility of a segment of the protein. 43. 7 8 J. Adu. W. R. P. Ref). B i d . Schlessinger. Padrenergic receptors from turkey erythrocytes can activate the adenylate cyclase of other cell types after the two are fused by Sendai In order to measure the mobility of the receptors for insulin and EGF by FPR.Biochem. Cuatrecasas. S. 75.S. 73. J . and P. Proc. Y . S. CI. J . Schramrn.4. Roth. Acad. A. Schlessinger. and J. Sci. Ce/lBio/. Pastan.
70 It was found that both endogenous fibronectin labeled with rhodamine antifibronectin antibodies and fluoresceinlabeled exogenous fibronectin bound to the cell in a fibrillar pattern and were immobile on the experimental time scale of the FPR method (D < 3 x cm2/sec). A . Con A binds to fibronectin and is largely immobile in areas rich in fibronectin. Willingham. Natl. This pattern is consistent with the apparent involvement of fibronectin in cellcell and cellsubstrate adhesion. in the mechanisms of hormonal activation of certain cellular functions. The amount of this protein decreases substantially after transformation. 73.S . Therefore the fibrils d o not form a barrier across the lipid phase of the plasma membrane. U . Proc. Azide. Yamada. 75. Therefore oxidative phosphorylation and some cytoskeletal elements do not seem to be responsible for the properties of the bound fibronectin. Acad. These examples should provide some idea of the kinds of information that can be obtained about the dynamic properties of cell surface components from fluorescence measurements. Shechter.B2These observations prompted a study of how fibronectin binds to cell surfaces using fluorescence microscopy and photobleach methods.4. Y. adhesiveness. or a combination of both. internalization. Acad. These static properties of fibronectin are consistent with its apparent structural function as an adhesive or anchoring component.80*81 showed that at 37°C the fluorescent hormone receptor complexes rapidly aggregate into patches on the cell surface and soon thereafter appeared in endocytic vesicles within the cell. S. S. and cytochalasin B did not alter the immobility and the cell surface distribution of the fibronectin molecules. A . The presence of immobile fibronectin fibrils did not impede the diffusion of a lipid probe. Sci. 2659 (1978). Yamada. andI. perhaps. C.1217(1976). Natl. U. Pastan. The capability for detailed analysis of the mechanisms of physiologically important processes will further improve as we gain greater understanding of the physical forces and structures that limit . It seems likely that the behavior seen in this study is involved in the regulation of receptor levels on the cell surface and also. In contrast. M. Addition of exogenous fibronectin to transformed cells partially restores the morphology.^^*^^ It forms fibrils concentrated at the edges of cells and seems to connect cells with each other and with the substrate on which they are growing. Proc. ** K . S . Sci. . APPLICATIONS 223 receptor immobilization.80~81 The large glycoprotein known as fibronectin appears in an immobile structure on cell surface^. vinblastine. M. Pastan. Schlessinger. and I . a fluorescent ganglioside analog.5. or various surface antigens. Therefore the immobilization of the hormone receptor complexes is presumably the consequence of receptor aggregation. and parallel alignment typical of normal fibroblasts. I . In another study Schlessinger et aZ.
M. Biophys. Vaz. Ann. Technical Developments Measurements of polarized emission from triplet states (anisotropy of phosphorescence or delayed fluorescence) are now being used to characterize rotational diffusion of biological macromolecules in proteins. Y. Cantor and T. C. 545 (1980).84The newer techniques of FCS and FPR have rarely been applied to macromolecular solutions. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS the mobility of cellular components. when we combine measurements of microscopic and macroscopic motions to provide a more complete picture of the dynamic state of various cellular structures. R. Koppel. 8 5 J .224 5. Recent Developments 5. 3) by an arrangement of beam splitters and a s h ~ t t e r . In particular. in “Procedures in Nucleic Acid Research ” ( G . New York. J . 29. Biopolymers 18. B. Borejdo. R.5. Barisas. This arrangement eliminates problems due to displacement of the axes of the bleaching and monitoring beams from optical wedge effects in the neutral density filter. 86 T. 2.) 162. S T D. A small but important change in the optical system for FPR measurements has been the replacement of the removable neutral density filter (FF in Fig. 1971. eds. N. J. 83 . Fluorescence measurements of rotation and macroscopic translation of macromolecules in solution have proved useful in a number of applications. 366. G . Tao. 31.2.1.3. Cantoni and D. 526 (1968). 5.53 More recently. The original demonstration of the FCS method concerned coupled translation and chemical reaction of ethidium bromide with DNA. 2807 (1979). The latter is blocked by a fast shutter except during the bleaching interval. Vol. Stryer.” L.). E. 28. Sci. M.281 (1979). and as we further refine our capabilities both of making quantitative dynamic measurements and of obtaining twodimensional images of the distribution of specific cellular components in and on individual cells. 176 (1981). The bleaching and monitoring components are recombined into a single colinear beam after passing the shutter. Bartholdi. p. Davies.86This provides a sensitive and versatile complement to the transient dichroism method described in Section 5. D. Harper. Science ( Washingron. C. L. Borejdo” workers have used FCS to study interactions among muscle components.C. L. both steadystate and timeresolved measurements of fluorescence depolarization have been used for some time in studies of protein^^^*^^ and nucleic acids. Acad. Biophys. Jovin. The ~ ’ incident laser beam is split into a weak monitoring component and an intense bleaching component. and W.5.
99 R. M. Bennett and P. Supramol. it has been shown that displacement of ankyrin from spectrin enhances the lateral mobility of erythrocyte integral membrane proteins. Natl. E.g0 A potential problem in the interpretation of FPR data is the possibility of photolysisinduced crosslinking of cell surface components.5 . A . M . 85. Proc. Koppel. S . 3114 (1979). and M. P. Schindler.^^. RECENT DEVELOPMENTS 225 Several new bleaching geometries have been proposed.2540 (1980).^^ 5. Biophys. 9 8 D.L. 77. 255. 9 2 M. Lepock. 76. J . Res.225 (1981). Golan and W. Kruuv.96 More quantitative studies using FPR have confirmed and extended these ~ t u d i e s . Sheetz. Acad. Biol. E. S.5. 77. Sci. Proc. H . Cone. Cherry. 94 C. E. A. A . however. Nafl. Acad. Origin of Constraints on Membrane Protein Mobility Only recently have the structural bases of these constraints begun to emerge. Fowler and V. J. J . Wallach. 5 .^^ Fortunately. Bennett. 2537 (1980). ~ ’ Measurements ~~* of the rotational mobility of band 3 protein. 344 (1978). Wolf. R.89 Another is a pattern photobleaching method. 96 V. Dragsten. Edidin. J . Proc. Acad.^^. Elegant biochemical work has demonstrated that the band 3 protein. R. S. U . One is suitable for simple analysis of diffusion on a sphere. y 5 V. J . Nature (London) 285. 5641 (1979). U . Sci. Struct. Stenbuck.99 The apparent contradition between the observed rotational and lateral mobility can be resolved by supposing that this protein is confined to a small domain in which it is free to rotate. 76. 289 (1979). Edidin. Sheetz. McConnell. 33. Acta 559. A model for constraint of the lateral mobility ofmembrane proteins by steric interactions with a labile cytoskeletal matrix has been D. recent work has provided evidence that these effects have no detectable influence on measurements of lateral diffusion on cell surface^. This type of crosslinking has been experimentally demonstrated under excitation conditions quite different from those of FPR measurement^. F. 8. Commun. suggest that it is free to rotate at a rate limited only by the viscosity of the lipid bilayer in which it is embedded. R. 89 90 . Biophys. ~ ~ a cell fusion method. A . Biophys. the erythrocyte anion transporter. Sci. 510 (1980). Schindler. E. which has the advantage of allowing measurement of slow or anisotropic diffusion on cell surfaces. 30. Biochirn. S . Natl. Sci. Natl. Koppel. Sheetz and D. is linked to the spectrinbased erythrocyte cytoskeleton through a protein with molecular weight 215. B. A. 9 3 D. and D.99 This confinement could be due to steric interactions with thespectrinmatrix. and D. E. and H. P. Wey. A . Biochem. W. 9 1 J . Proc. U . Veatch.3314 (1979). U. 215 (1978).2043 (1980). Thompson. and P. Clark. and M . Smith. M. M. A. 9 7 M. Koppel. J . Biophys.000 named a n k ~ r i nUsing .2. R . J. Acad. E. Chem. P. The erythrocyte provides the most advanced example up to now.
Acad. S. Schlessinger. Y. S . Proc. Proc. P. This process has been seen for i n s ~ l i n . 1. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS proposed.S. U . Sci. M. Cohen.lo4* l o 5 nerve growth factor (NGF). S. and J. D. 78. F. Pastan. which may be related to the triggering of cellular responses. Gorden. A. and. This involves the binding of the hormone to initially diffusely distributed mobile membrane receptors. U . C. J. submitted (1982). Levi. 1072 (1981). W.A. Natl. Acad.L. Shechter. S. Schindler. I . .5. has been detected by measurements of phosphorescence anisotropy. U . Narl. 5025 (1978). Carpentier. 6981 (1981). S . Proc. Mol. M. Acad. Maxfield. M . Proc. 3469 (1 980). A. l n 3 F. Sci. "* R."' and a. 805 (1978). ' ' ~ epidermal growth factor (EGF). J. Schlessinger. Schlessinger. and I. l o ' W. Avivi. Shechter. Natl. Orci. C. and M. Acad. Very recent work has demonstrated that the apparent detachment of the plasma membrane from the underlying cytoskeleton allows membrane proteins to diffuse much more rapidly than in the normal membranes and at a rate appropriate for the operation of bilayer viscosity as the major constraint of lateral motion. Cell 14. A . L.226 5. the pinching off of vesicles containing the hormonereceptor complexes. J. 75.. A. and L.lo6 thyrotropin (TSH). Proc.3. personal communication. Proc. Sci. S . Y. l o b A. P. Narl.' O 3 An early phase of the aggregation of EGFreceptor complexes.Zidovetski. Elson. The origin of mobility restrictions in the plasma membrane of nucleated cells is less clear. lo' Y. Hence the integrity of both microtubules and microfilaments is required for the effect. Sheetz. Schlessinger. A . E. A . Sci.2135 (1978). Koppel."' The relative importance of nonspecific steric interactions and of specific links via ankyrin in restricting mobility of erythrocyte membrane proteins is yet to be determined. AmbesiImpiombato. which then cluster mainly over coated pits. and T. U . and M. Henis and E.'" This reduction can be reversed by treating the cell simultaneously with both colchicine and cytochalasin B. ( 1 . 77. 78. Javin. 75. Y . 5. Yarden. Y.lo2 5. Neufeld. N u t / . Acad.'" It has also recently been demonstrated that crosslinking a critical number of concanavalin A receptors on Blymphocytes causes a large reduction in the lateral mobility of surface immunoglobulin on these cells.macroglobulin. Webb.Receptor Mobility There appears to be a common mechanism for the internalization of cell surface polypeptide hormonereceptor complexes. 78. Sci. R. Acad.lo8 l o o D. Shechter. E. Schlessinger. Pastan. Willingham. Sci. Narl. Cell Endocrinol. J. U. 3576 (I98 1). finally. the aggregation and immobilization of these receptors. Willingham. Tramontano. J. and J .
The Padrenergic receptor on liver cells has been shown to be present in a patchy. Moreover. and E.5. and D. W. L. .''~The response to the agonist is. Tramontano. Elson. D. D. Fahey. S. D. Wolf.C. L. Eldridge. Reidler. F.5. A . Acad. I . TSH receptors are mobile and randomly distributed over the cell membrane. Science ( Washington."' Acknowledgments We are especially grateful to our colleagues. largely immobile state but to be released and mobilized by the agonist ( )isopr~terenol. RECENT DEVELOPMENTS 227 Two receptors which stimulate production of adenosine 3'. J. submitted (1982). W.5'cyclic monophosphate (CAMP) show quite different mobility properties. Webb). Hekman. ' l o A. J. D. E. Koppel. and J. M."' Pretreatment of the thyroid cells with 8bromoCAMP induces the clustering of TSH receptors in the absence of TSH. however. Henis. Quite different behavior is seen for the receptor for TSH on thyroid cells. P. Helmreich. U . C. in press (1 982). D. Proc. N a f l . Webb. . In contrast to Preceptors. This work was supported by NIH grants GM 21661 and CA 14454 and by NSF grant D M R 7504509 (to W. M. S. AmbesiImpiombato. Ambesi. cAMP reduces the potency of TSH to induce the production of cAMP in thyroid cells.). Axelrod. Barak. Schlessinger. '09 Y. who have worked with us on the various studies from our laboratory discussed in this paper. Sci. W. This suggests that cAMP acts both as a second messenger and as a regulator of the level of TSH on the surface of thyroid cells. too slow to be involved in the stimulation of cAMP synthesis. Avivi.
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New York. Introduction Xray diffraction analysis (XRDA) of single crystals and of fibers has been the source of our knowledge of the atomic structures of over a hundred biological macromolecules. Bell. 20 Copyright @ 1982 by Academic Press. H. Johnson. Blow. Holnies and D.’ We touch only briefly upon the closely related technique of small (. Lattman and L. ligand binding.~ T. Part 8.100 atoms)molecule crystallography. For more detailed material on this subject see Blundell and Johnson’ and Holmes and Blow.^. Inc. New York. “XRay Structure Determination: A Practical Guide. this volume. Recently. crystals of virus particles weighing several million daltons have been analyzed in nearly atomic detail. Moore. B. Prutein Crystallography. VOL. L. 111. N. Nucleic Acid Srructure. M . describe data collection technology. In addition. 1966. G. Cochran. Vol. including such important ones as hemoglobin. Stout and L. The Determination of Crystal Structures. Mario Amzel 6. is the subject of Part 8 of this v01ume. and DNA. In this part we present briefly the principles of singlecrystal and fiber XRDA. P. “The Use of XRay Diffraction in the Study of Protein and New York. H . H.^ Smallangle xray diffraction analysis. C. STRUCTURE DETERMINATION OF BIOLOGICAL MACROMOLECULES USING XRAY DIFFRACTION ANALYSIS By Eaton E.1. No clear limits to size and complexity are yet in view. Jcnsen. “The Crystalline State. ’ ’ ’ 229 MFTHOIX O F EXPERIMENTAL PHYSICS.” Macniillan. K . immunoglobulins.” Academic Prcss. which is concerned with the shapes and interactions of molecules rather than with their atomic structures. Lipson and W. London.” Wiley (Intersciei~ce). and many other macromolecular functions. 1966. Such models provide the basis for our understanding of the mechanism of enzyme catalysis. they serve to direct and organize other types of studies of the molecules involved. 1968. All Iighh if reploduction in any form reserved ISBN 0124759629 . since it is better known and covered in many textbook^. and then present some case studies that illuminate the versatility and limitations of the methods.” 3rd ed. while correspondingly complex fiber structures have also been studied. By atomic structures we mean models in which the positions of the individual atoms are known to within several tenths of an angstrom. 1976. Blundrll and L.6.
such as s or x. An XRD experiment differs in several ways from this prototype.” is the central difficulty in the practice of XRDA. a simple process. with the computer playing the role of the lens. Position vectors in direct or diffraction space will be denoted by lowercase. In a typical microscopy experiment a thin. available xray detectors measure the amplitude but not the phase of the radiation. The method also differs from conventional microscopy in the characteristics of the specimen. the structure factor. by concentrating the diffracted radiation into certain defined directions. as F. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In some ways XRDA might better be termed lensless xray microscopy. Much of this paper is devoted to the discussion of techniques for dealing with this problem. 6. The goal of the method (XRDA) is the conversion of this pattern into an image. The situation can be improved by using periodic specimens such as crystals or fibers. in principle. The process is an intricate one. an XRD specimen should perhaps consist of a single molecule held delicately in front of the beam. The most conventional approach to the theory of XRD in crystals is through Bragg’s law. To preserve the analogy. Highquality xray lenses do not exist. In the absence of a lens the distribution of radiation scattered far from the specimen is termed the Fraunhofer diffraction pattern. equally spaced planes appears to be reflected from these planes. specimen is illuminated by a collimated beam of radiation. We show below that. The use of F for 1 F (is conventional in the protein crystallographic literature.230 6. We frequently write this vector. These increase contrast both by increasing the number of molecules in the beam and.2. sometimes called “the phase problem. It states that a beam of x rays incident upon a stack of parallel. such as A. boldface letters. which is diffracted by fluctuations in scattering density within the specimen. as a . usually aperiodic. F = A + iB. as we discuss below. Diffracted radiation can be described by a twodimensional vector in the complex plane: the modulus and azimuth of the vector represent the amplitude and phase of the radiation. The loss of phase information. The contrast in such an experiment would be vanishingly small. boldface letters. However. reconstruction of the image is. Diffraction by a General Object We begin by defining some of the notation used in this paper. This pattern becomes the only observable in an XRD experiment. A lens placed behind the specimen focuses the diffracted radiation into an image. given the phase and amplitude of the scattered rddiation. Matrices will be written with uppercase. We can break F into components in two ways: F = I FI exp(ia) = F exp(ia).
is the angle between the incoming beam and the planes. (a) Geometrical representation of the diffraction condition for a stack of = cqually spaced planes.6. I . d is the spacing between planes. For constructive interference this distance must be an integral number of wavelengths. Rays “reflected” from adjacent planes have a path difference 2d sin 8. thus nE.1) Here n is any positive integer. This is illustrated in Fig. (From Blundell and Johnson. + . when nA = 2d sin 8..2. and 8. the Bragg angle. DIFFRACTION BY A GENERAL OBJECT 23 1 mirror reflects light. When the angle of incidence differs from 0 no Bragg Incident beam \\. For this object only a path difference of 2 produces constructive interference. This artist has represented the continuous density fluctuation by four shades of grey. (6. la.A 2 ’ Reflected beam e d \ El/ FIG. Bragg’s law. thus i= ( 2 / s )sin 0. = 2d sin 0.2.’) (b) Diffraction by a sinusoidal density fluctuation of wavelength I/s.
e. It is perhaps not surprising.2) is not difficult to obtain. lb. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES reflection is observed.. we have p ( x ) = po exp[2nis0 (x . and therefore F(so) = po exp(icr). the amplitude of the diffracted wave. is proportional to the amplitude of the spatial fluctuation. This density p ( x ) has certain key features in common with the stack of Bragg planes: both have constant value on planes normal to a particular axis. the amplitude. ) ] .2. The situation is diagrammed in Fig.2) The factor l/lsol is the period of exp(2nis0 ex) and is analogous to the spacing d in the conventional Braggs’s law. m (6.2.2nis0 * x). then. It is well known that any object can be represented through a Fourier integral as a combination of such spatial waves (Fourier components). sinusoidal density fluctuation p(x) = po exp(2nis0 * x). (6.4) Thus translation of the spatial fluctuation by xo produces a phase shift of 2nx0so in the diffracted radiation. Note that only one angle of incidence 0 is permitted for this function. (6. and therefore F(so) = po exp(2niso * xo) exp(ia).232 6. that diffraction from the density fluctuation occurs when a slight variant of Bragg’s law is satisfied: 1 = (2/lsol) sin 0. and the origin (i. so that m p(x) = / / [ G ( s ) exp( .3) or p ( x ) = po exp(2nis0 * xo) exp( .2. (6.2. and so CI in the above equations is generally defined to be zero.5) . It is also clear that F(so). If the origin of the spatial wave is shifted to xo.x . This approach can be extended to analyze a general object if one considers diffraction from a spatial. We can then write m o ) = Po. and both are periodic along this axis. only differences of phase can be measured.2. Experimentally. From the diffraction by an isolated spatial wave one can thus determine the period (through d). and we omit a derivation for lack of space. phase) of that wave.2nis * x) d V . Equation (6.
2. 03 (6. The vectorss are defined with respect to the object’s coordinate system.2. W. We can thus identify the diffraction pattern of an object with its Fourier transform and write m p ( x ) = JJJF(s) exp( . and s drawn in a sphere of radius 1 / . We describe now a simple geometrical construction developed by Ewald which tells which Fourier components are in the diffracting condition for a given orientation of the object. Strategies and instrumentation to achieve this goal are discussed in Section 6. DIFFRACTION BY A GENERAL OBJECT 233 The amplitude and phase of G(s) indicate the weight and position of the Fourier component. (6. By comparison with Fig. New York. The textbook by Goodman6 provides an excellent introduction to Fourier theory and diffraction. 1 it is clear that the vector triangle OAsOB is equivalent to the one formed by the incoming and reflected rays. Consider any s which terminates on the surface of the sphere. The signal recorded at any point represents the diffraction from one Fourier component. and draw from the center of the sphere to its terminus the vector OB.4. OB. 4 (Ewald’s sphere). the wave vectors of the various Fourier components of the object. rotated in the same way. For any given orientation of the specimen only certain of the Fourier components satisfy the condition in Equation (6. Goodman. Diffraction is observed along the direction of OB. To measure the full. perforce. For any orientation of the specimen diffraction along all vectors OB can be recorded by placing a film (or any other xray detector) at some distance from the object.” McGrawHill. Clearly. If the object is rotated they are. G and F are identical. and that whenever the terminus of a vector s is on the surface of Ewald’s sphere its corresponding Fourier component is in the diffracting condition.2. OA denotes the direction of the incident beam.3.2). . 1968.2nis * x) d V . denoted s. Figure 2 shows the vectors OA. threedimensional diffraction pattern in ordered specimens it is necessary to rotate the specimen systematically so that diffraction from all Fourier components ofinterest occurs and is recorded.6) The Fourier inversion theorem can be applied to Eq.6. ’J . and is drawn from the center of the sphere to a point A which is taken as the origin of all vectors s. “Introduction to Fourier Optics.2.6) to give a result which is used extensively below. apart from a constant of proportionality.
W. 19).) We have thus far dealt with diffraction from completely general specimens. Inc.” Bell. ( a ) A sphere of radius IjA is drawn so as to pass through the origin of the reciprocal lattice A. 11. Vol. Inc. periodic specimens which produce simplified diffraction patterns (see James’). XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES FIG. . The Optical Principlcs of the Diffraction of XRays. 1957. We next discuss crystals and fibers.2. (Holmes and Blow. London. If lattice point B lies on the sphere there will be a diffracted bemi in thc dircction or the line joining the center of thc sphere to this point. “The Crystalline State. and have kept the discussion general as well (for a more complete discussion of the Fourier approach to diffraction see Goodman6).234 6. two additional mathematical R. The Ewald construction. however. Before we do so. (b) A section through the same drawing i n the plane of the zerolevel section of reciprocal space (see also Fig. James.’ Copyright @ 1966. Reprinted by permission of John Wiley & Sons. John Wiley & Sons.
ed.” Gordon & Breach. (6..2.Y is the distance in angstroms from the point. the convolution theorem states that (6. . If F and f are a Fourier transform pair and so are G and g. 2 it is clear that no vector s of magnitude greater than 2/A can touch Ewald’s sphere. New York.) t devices must be introduced: the application of the convolution theorem to diffraction’ and the projection theorem. “The Molecular Replacement Method.8a) F * G = F(fg) and that FG = F ( f * g ) .2. Rossmann. (Reprinted from Rossmann and Blow38 with permission. Image of a point after Fourier reconstruction with wavelength cutoff at I/s = 1 A. 3* and represents the image of a point object after wavelength cutoff. In Fig. As an illustration we consider the effects of the nonzero wavelength of x rays on our ability to form an accurate image. Formally this means that the diffraction pattern of the specimen is multiplied by a function with value one within a sphere of radius 2/1 and zero outside this sphere.6.2. (6. DIFFRACTION BY A GENERAL OBJECT 235 FIG 3.8b) where 9 represents the Fourier transform and the asterisk represents convolution.u ) du. G. thus all diffraction from such components must remain unobserved.2. . which is defined by f(x> * g(x) = Jf(u)e(x . This transform is shown in Fig.9) This theorem has wide application. 1972. * M. The observed image is therefore the convolution of the true image with the transform of the solid sphere.
z)exp[2ni(sx + ty + uz)] dx dy dz. y)exp[2ni(sx + ty)] dx dy.2. y. If a(x. instead of any value between 0" and 360". Setting u equal to zero and rearranging. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In protein crystallography wavelength cutoff is not usually the factor limiting the amount of diffraction data available. We now discuss the projection theorem. we rewrite Eq.10) Thus a twodimensional Fourier transform of the zaxis projection of the electron density function is the u = 0 section through the threedimensional diffraction pattern. t . Application of the Fourier inversion theorem to Eq. 0) = ss exp[2ni(sx + ty)] S p(x. Many crystals have projections which are centrosymmetrici. real numbers. They are thus signed. which states that the image synthesized using only a central section from the full diffraction pattern is the projection of the full. y) is the Fourier transform of the central section F(s. . Phases for such reflections are particularly easy to determine. 0).4. u ) = / / / p ( x . t. sometimes it is expedient for data collection to be limited by the investigator. since it is frequently very simple to record diffraction from such central sections. threedimensional image down the normal t o the central section.. (6. It is easy to show that such projections have structure factors of phase either 0" or 180". z ) dx dy dz. y.2. where smax is the radius in reciprocal space beyond which no data are obis the wavelength of the highestfrequency component served. and has a closely corresponding form for crystals. This result is independent of the projection axis chosen.e. t .10) demonstrates that that projection o(x. (6. which is the form most commonly used. Resolution is given by l/smax.2. y) represents the integral over z (that is. ~ ( xy) . (Section 6.236 6. where the precession camera. (6. =o ( x. the projection down z). On other occasions the intensity of the diffraction pattern falls effectively to zero at values of s or simply swhich are smaller than 2/A. To derive this. Thus 1/smax in the synthesized electron density function. then a(x. especially for crystals. This theorem is of great practical importance. we have F(s. This gives rise to the crystallographic definition of resolution in an image. so that centrosymmetric projections are of great interest experimentally.3) automatically provides photographs of particular planes in the diffraction pattern. y).7) using the components of x and s: F(s.
It is easy to show that h is itself a lattice (called the reciprocal lattice) with unit cell vectors a* = b x cjV.3. it is the set of points at the termini of the vectors H = ma + nb + p c . and some discussion of its characteristics is essential for understanding xray photographs and data collection in general. the Fourier transform of p. the Fourier transform of H.3.1) where V is the volume of the unit cell and x denotes the cross (vector) product. The reciprocal lattice is thus intimately related to the geometry of diffraction. where m. Diffraction by Crystals A lattice is a regular grid of points in three dimensions. Formally. and the Fourier component giving rise to the reflection. This net. It is clear that the convolution operation here amounts to placing a copy of pMat each lattice point in the crystal. Lattices are divided into classes depending upon special relations among the unit cell vectors. The reciprocal lattice is an important tool in dealing with the geometry of Bragg diffraction. As we stated above. and p are any integers and a. which is zero except at points of the reciprocal lattice. with a set of 6 functions at the lattice points. (6. The crystal can be regarded as the convolution of a single unit cell’s worth of molecules. Also. the unit cell vectors. In a crystal each unit cell of the lattice is occupied by a number (usually small) of molecules which are in most cases related to one another by simple operations such as rational rotations. the corresponding reflection. Each lattice point is in the same environment as all others and can be imagined to lie at a vertex of a parallelepiped called the unit cell. and c are any noncoplanar vectors. n. It is clear from Eqs. Crystallography 6. it is a unification that a given h will serve to label a reciprocal lattice point. These relations are the source of the name reciprocal lattice. (6. Inspection of Fig. 7 (which is discussed more fully below) reveals that the Bragg reflections lie neatly arranged at the points of a twodimensional net. the diffraction pattern of a crystal is given by FM(h). b.3. c* = a x b/V. b.. and h. and c. reflections.8b) one can show that the Fourier transform or diffraction pattern of a crystal is the product of F.6.3. which is defined by the vectors a.3. in fact. pM.   . CRYSTALLOGRAPHY 237 6. but we shall only consider specific examples as they arise.. (6.1) that a a* = b b* = c c* = 1.2. b* = c x ajV. is a section from the threedimensional lattice defined by h.1. and translations. Using Eq.
+ y$ + z‘i.2nih x)..3.c* + terms in a . where the triplet hkl is often called the Miller indices. (6.2b).2). (6.3. etc.3.3. in the second h.5) . (6.3. one finds p(x) = 1 h F(h) exp( . the position vector x can be written as x = x’d. This rather abstract derivation has shown us that only certain welldefined Fourier components exist in a crystal. and so diffraction can only be observed when one of these satisfies Eq. Thus the hkl component repeats h times along the a axis.  (6.1) all terms containing a * b*. In the first case h’. where a caret denotes a vector of unit length.2. and I’ are measured in reciprocal angstroms.4) which is the usual inner product form.3. Using Miller indices.b*.3. we have h x 9 = hxa * a* + kyb * b* + Izc .3. k.238 6. = From the crossproduct relations in Eq. Eq. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES The vector h can be written as h or = h’d* ha* + k’6* + l’i. and fractional coordinates.3b) or x = xa + yb + zc. etc. Of course. k’.3. (6. The latter notation is universal in crystallography.3.3a) (6. and 1 are in units of the reciprocal lattice translations and are always integers.3b). If one rewrites (6. and 1 times along c. must vanish. the inner product h * x is independent of the choice of formalism.2a) (6. k times along the b axis. (6. + ky + / z . (6. and Fourieranalyzed it accordingly. Recalling that aa*= bb* = cc* we have h * x = hx 1. Similarly. It is important to realize that the same Fourier components would have emerged if we had treated the crystal conventionally as an object periodic in three dimensions.* (6.2b) h = + kb* + lc*.5) to reflect the periodic nature of the crystal. Eq.
Nowf(s) represents diffraction by an atom situated at the origin. and lo). . It is therefore convenient to have a formula.its diffraction pattern is phase shifted by the factor exp(2nih * xo). New York. and that for any crystal the set of such operations forms a group when the product of two operators is defined as the result of their successive application.3.6. but deal with particular examples as they arise.” Wiley. 9. Buerger.x) d V . The sum of the diffraction by all atoms is therefore F(h) = I‘ cf.7) where p j is the electron density function of the jth atom. Crystals are made of atoms. and x j is the coordinate of its center.) This precludes the existence of mirror planes or other inverting operations in their crystals. One can also apply the Fourier inversion theorem to (6. and are usually denotedf(s). (6.” Kynoch Press. which expresses the diffraction pattern in terms of atomic positions and types. 9. IV).. Again. we pass over this entire topic by noting that symmetry operations which superimpose the whole crystal on itself do exist. we do not attempt a general discussion.6) relates the diffraction pattern to the continuous electron density in the crystal. Vol. are nonsuperimposable upon their mirror image. Vol. have been tabulated (Ref.3. Diffraction patterns for each atom type. I. Since atoms in this model are spherically symmetric. one can write that (6. Birmingham. CRYSTALLOGRAPHY 239 This is the equation used to calculate electron density function images. Formally. lo M.3. If the atom is shifted to point xo.e. often called atomic scattering factors. These groups are termed space groups.6) giving the structure factors as a function of the electron density. Equation (6. called the structure factor equation. “XRay Crystallography. and we interpret their electron density functions in terms of atoms.(h) exp(2nih j * xj).3. J .3. unit cell (6. 1952. Although symmetry is one of the most important fields of study in crystallography. The diffraction pattern of one unit cell is the sum of the diffraction patterns of all the atoms in the cell. It is worth noting that almost all biological molecules are handed (i.f(s) is also. (refs. h and x are expressed in integer and fractional coordinates by convention.5): F(h) = /JJ p ( x ) exp(2nih . England. 1942.8a) International Tables for XRay Crystallography.3. Again. and the symmetry operators are the composition of the translational symmetry of the lattice with operations that relate the molecules within one unit cell.
8b) This equation enables us to calculate the diffraction pattern from a preliminary atomic model.x ) .3. As we shall see. (Some texts say. . 46. The more general form for this factor. This synthesis and its significance were first discussed by A. The Patterson Function Electron density maps can be calculated from the structure factors F(h) using the Fourier synthesis described by Eq.8a) is based on diffraction by atoms at rest.3. However. L. We show below that the Patterson function is the autocorrelation of the electron density function and represents a complete. To take into account the effects of atomic thermal motion (James. where the value of B j .3. the structures solved typically had a limited number of atoms. the location of heavy atoms that have been bound to the protein molecules. These locations are essential data for the procedure that provides phases for the protein diffraction pattern.3.8a) is (6.5) when the phase of F(h) is known. Patterson" in 1934. Equation (6. it can provide clues leading to a structure determination. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES where xj is the position of atom j . Normally the phase of F(h) is not known. The final form of the structure factor equation (6.2. The contribution to the structure factors is different from resting and from vibrating atoms. a synthesis using the observed F2(h)which have a phase of zeroas coefficients can always be calculated. 6.Patterson. can be related to the rootmeansquare excursion of the jth atom. loosely. alternative description of the observations. and it is commonly named after him. (6.' Chapter 5) each f j ( s ) must be multiplied by the factor exp( Bjs2/4).3. but in fact it is the convolution of p(x) with p( . 372 (1934). In protein crystallography we use the Patterson function for a problem of similar complexity. Properly interpreted. L. and forms the basis of the process for refining such models in order to minimize differences between observed and calculated structure factor magnitudes. Rev. Phys.240 6. is rarely used in protein work.) Until about 1960 the Patterson function was the dominant tool for solving the phase problem in crystallography. which allows for anisotropic atomic motion. The quantity B is usually called the temperature or DebyeWaller factor. usually determined experimentally. It is well known that the atoms in a crystal are undergoing thermal motion. In this nomenclature one deconvolutes the Patterson function to recover the electron density. that the Patterson function is the selfconvolution of the electron density function. '' A.
CRYSTALLOGRAPHY 24 1 Use of the Patterson function depends upon interpreting it in terms of the autocorrelation of a function made of atoms or. where F*(h) is the complex conjugate of F(h).3. which depends heavily upon symmetry.9)] and the fact that p(x) is real to obtain This expression indicates that the Patterson function is the autocorrelation of the electron density of the crystal. h h (6.3. p(x) can be expressed as = 1 Pj(x j  xj).3.3. The expression for the electron density based on atomic electron densities can be used to further analyze Eq. and upon the concept of vectors running between atoms. As shown before [Eq.8b) and (6. ideally. (6.2.7)].3. and we cover it as briefly as possible.9) The function P(u) is centrosymmetric [P(u) = P( u)]. where xj is the coordinate of the jth atom and N is the total number of atoms in the cell.11) we obtain which can be rearranged to yield . The interpretation of this synthesis requires some further derivations. we obtain P(u) = 1 C [F(h)F*(h)] cos(2nh h * u). There is an extensive literature discussing this interpretation. (6.1 I).6.10) With expression (6. the topic can be tedious.3.3. (6. Substituting in (6.3.5) for the electron density we can use the convolution theorem [Eqs.9). In crystallographic nomenclature the Patterson function P(u) is defined as 1 P(u) = 7C F2(h)~ 0 ~ ( 2 *nu). To be frank. (6. Substituting F(h)F*(h) = F2(h) in expression (6.2. points.
Define the integral in the above expression as (6.” Wiley. the atomic electron density profiles of the kth andjth atoms are Gaussians of variances 07 and a : . XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES The change of variables x’ = x .3. J.3.k = u j ” 6. (6.. For a crystal structure containing a few atoms in the unit cell the Patterson function can be “solved (deconvoluted). this synthesis has a large peak at the origin corresponding to the interatomic vectors ujj that contains the sum of the selfconvolutions of the electron density profiles of individual atoms.xj simplifies the above to N N p(u> = j=1 k = l 1 JJJpj(x’)pk(x’ + u . Substituting (6.13) where ujk = xj .242 6. xj . . This can be done by dividing the F 2 ( h )by the square of the Fourier transform of the average atomic electron density profile. If.3. the function can be partially deconvoluted in structures which contain a few heavy atoms that dominate the diffraction pattern. A very detailed analysis of the Patterson function and of different deconvolution methods can be found in BuergerI2 (and references therein).ujk) d v . the height of the peaks is approximately proportional to the product of the atomic numbers of the relevant atoms. The peaks Pjk are the convolution of the individual atomic profiles. Sophisticated trial and error methods are used to obtain the coordinates of the atoms in the unit cell from the set of interatomic vectors.xk.ujk. translated to the Patterson origin. Pjk is the convolution of the electron density profiles of the ith andjth atoms. the peak centered at ujk will also be a Gaussian of variance fl.13) we obtain N N (6.14) in (6.xk.N peaks for the vectors ujk with k # j. In addition. In addition. for example.15) This expression describes the most useful feature of the Patterson function : its maxima occur at points ujk which correspond to the interatomic vectors of the structure. If all atoms in the structure have similar radial electron density profiles. The peaks in the Patterson function can be “sharpened” if instead of the observed F2(h) modified values are used which correspond to the same structure but composed of point atoms. “Vector Space and Its Application in Crystalstructure Investigation. 1959.14) where u’ = u .3. There are N 2 . + ” M. Buerger.3. New York.
3.4. The vectors between site A and the minor site C (AC and AC') are also present.lox The main use of the Patterson function in protein crystallography is for the determination of the positions of heavy atoms (Section 6. Projcction along thc b axis of the diffcrence Patterson function of a gold heavyatom derivative (AuCIi) or glycera hemoglobin crystals.6. (6. A single precession photograph or a short diffractomer run (Section 6. The interpretation ofthis Patterson function involves two major (A and B) and one minor (C) heavyatom sites. projections of the Patterson function are used [Eq. (Courtesy of Dr. The zero contour is dotted.4. for example. and the negative contours are omitted.4). 4.3. in many cases. Eduardo Padlan. These functions can be calculated using data from one section of the reciprocal lattice. The contour lines are drawn at equal but arbitrary intervals.10)].3) can be used for this purpose. For this purpose.4. The analysis is systematic . (6. Heavyatom derivatives are usually screened and the heavy atoms located using these projections (Section 6. The crystal in this projection contains twofold axes of symmetry perpendicular to the plane of the drawing.) . This assignment can be used to obtain the coordinates of the heavyatom sites.4). W ___c CRYSTALLOGRAPHY 243 C FIG.2.16) Data for such a synthesis can be collected very easily. For the major heavyatom sites the map shows the peaks corresponding to the interatomic vectors between a heavyatom site and its symmetry mate (AA' and BB') and between the two heavyatom sites ( A B and AB').
An example is shown in Fig. J.14 Since then a great variety of proteins have been crystallized. C. Biol. For the first time there was a tantalizing possibility that the molecular structure of the proteins within the crystal might be determined..4. Summer. If so. 5. In one version. ed. Their photographs were obtained by enclosing the crystals in glass capillaries. ’ and ~ the enzyme (protein with catalytic activity) urease was crystallized by Sumner in 1926. All of these procedures fail when extrapolated to protein crystals. 6. l4 l5 Ib .435 (1926). The synthesis of the electron density function [Eq. Most challenging of all was the difficulty in measuring the phase of all the protein structure factors. 1970. E. In the case of small (nowadays about 100 atoms) molecules this information is recovered by a number of procedures based on the Patterson function (Section 6. Crawfoot.. The symmetryequivalent points are calculated. It was not until the middle of the K. 69. J. D.5)] requires the amplitude and phase of each structure factor. The crystals often have a very beautiful external morphology. They realized that protein crystals contain a large percentage of mother liquor filling the space between molecules. The intensity of the individual Bragg reflections is proportional to the squared modulus of the structure factors. the grid point is flagged as a possible site. Reichert. Anat. Prot ei n Crysta Ilography 6. The problems to be overcome were formidable.3.3. Nature (London) 133. and the putative interatomic vectors generated. for example. Introduction The oxygen transport protein hemoglobin was crystallized in the latter part of the nineteenth c e n t ~ r y . Copenhagen. Med. Bernal and D. making careful use of the symmetry of the crystal. (6. In 1934 Bernal and C r o ~ f o o t showed ’~ that these crystals produce a rich and complex xray diffraction pattern.2) or upon various algebraic relations between the structure factors. R. The computer then checks to see if these vectors correspond to peaks in the Patterson function.1. J .4. Chem. which contain thousands of atoms per unit cell. B. F. The latter are collectively called direct methods (a selection ofpapers on this topic appears in AhmedI6). 198 (1849). and that crystals become disordered as they dry out.” Munksgaard. with welldeveloped faces and edges. “Crystallographic Computing. Muellers Arch. Wiss. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES trial and error. Figure 4 shows an example of how the positions of a heavyatom derivative can be located using a projection of the Patterson function. a computer search assumes in turn that each grid point in the unit cell is a possible heavyatom location. p.794 (1934).244 6. but all phase information is lost. Ahmed. Physiol.
4. Many other difficulties also obtruded. To provide accurate models of protein molecules these images have to be interpreted. Difficulties associated with the interpretation of electron density maps have.4. usually in light of a known sequence of amino acids within the protein (Section 6. The images synthesized from such patterns are deficient in detail. These are also discussed below (Section 6. 287 (1954). M . the diffraction patterns from even the best protein crystals fade out at much lower resolution than the patterns from small molecules (Chapter 6. Photograph o f a crystal of the F1 sector of mitochondria1 ATPase. which has proved astonishingly successful in the intervening years.4. Green. Proc. been overcome by a combination of manual and computerbased model fitting and refinement techniques. V. The bar represents I mm in the scale of the micrograph.0 A.6. . W.4. Thus protein crystals give rise to hundreds or thousands as many Bragg reflections as smallmolecule crystals. The number of Bragg reflections within a reciprocal space sphere of fixed radius is proportional to the volume of the unit cell. 1950s that the laboratory of Max Perutz” introduced the method of multiple isomorphous replacement (Section 6. London. F.5). and M. l7 D. A 225.4). Soc. Ser.5).2)typically in the range 1. Automated procedures to measure these reflections awaited development (Section 6. PROTEIN CRYSTALLOGRAPHY 245 FIG.53.3).5 . and individual atoms are not visible as they are in the smallmolecule case. R . Ingram. Further. Perutz.4. to a large extent.
One begins with a concentrated protein solution often in the range 1 % by weight of protein. 23. Crystallize protein. 9. Protein crystals suitable for xray diffraction studies are usually larger than 0. . 3. Once obtained protein crystals must be kept in the presence of mother liquor. 2. In most cases such precipitates are amorphous or microcrystalline. Determine position of heavy atoms. i. combine model and data. and the literature is filled with examples of apparently random procedures that allow the crystallization of previously resistant molecules. " ) . Make a new synthesis using calculated phases dC hut observed amplitudes F. 5. and 9 illustrates Fourier refinement. Usually about 50% of the crystal volume is occupied by solvent. however. The repetition of steps 7. are almost universally met. such as salt or organic solvent. Certain conditions. The final criterion of adequacy is their ability to diffract to the desired resolution. Matthews.4.'' and therefore the crystals are destroyed A.J. Mol. 8. Measure structure factor amplitudes for native and heavyatom derivatives. Crystallization The crystallization of proteins retains large elements of black magic. one of several methods of improving the initial model.491 (1968).e. and rate of precipitant addition. 6. Steps in a Protein Crystal Structure Determination ~~ 1. Fit a molecular model to this synthesis. Variables explored are precipitant type and concentration. Using above information calculate phases for structure factors. Anal. temperature. but occasionally single crystals result..'* who appears to have a green thumb in this regard.. Make heavyatom derivatives. W. Methods Biochem. In what follows we discuss each of these steps in more detail. The cycle is abandoned when improvement ceases. 4." 8. which causes the protein to come out of solution. 6.33. In addition. certain ions such as C1. pH. and 4cfrom the model.or C a 2 +could be required in low concentration for crystals to form. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES TABLE I. We then deal with examples of protein structure determination. Calculate an electron density synthesis of the crystal. Calculak structure factors F . Table I is a flowchart of the major steps in the determination of a protein crystal structure. To this is added either gradually or suddenly a precipitant.246 6. 7. McPherson. Biol.2. The crystallization of proteins has been reviewed by McPherson. A variety of microtechniques have been developed to allow the systematic investigation of many conditions using a minimum of material.2 mm in all dimensions. 249 (1976) B.
eds.3 keV. when deciding how to collect the data. Brown. Stanford Synchrotron Radiation Laboratory. line (8. the sensitivity to damage by radiation.4. Connecticut. The measurement of the intensities that provide the desired structure factor amplitudes is often a long and tedious process. ~ second consists of the measurement of the diffracted intensity of all observable reflections. Collection of XRay Diffraction Data Two types of information can be obtained from xray dimraction experiments using single crystals.20 but its use is obviously restricted by the access to such sources. 1952. For diffraction experiments the crystals are sealed in glass or quartz capillaries in the presence of mother liquor. Experiments for the determination of the crystal lattice and its symmetry are simple to perform and easy to interpret. The second method involves the use of crystal monochromators. No. London. The condition in 2o H. The symmetry of the crystal can be determined by careful visual inspection of the recorded regions of the reciprocal lattice.” In this case the xray beam is reflected by means of an intense Bragg reflection of the monochromator crystal. Radiation from storage rings of highenergy accelerators is also used. (6. One should take into account the cell dimensions of the crystal. 1.1)] one can obtain the unit cell parameters from measurements of the difffraction angle 8. “XRay Crystallographic Technology. a filter of Ni. Stanford. A.54& is used to reduce the amount of contaminating radiation.3.” SSRL Rep. When Cu xray tubes are employed the K. The method of choice depends on the particular problem under consideration. 1978..4. X rays for protein crystallography are usually obtained from copper anodes. . Some of the advantages of the individual methods are discussed in more detail this section. They consist essentially in recording (usually photographically) several selected regions of the reciprocal lattice. The first type involves the determination of the unit cell parameters and the symmetry of the c r y ~ t a lThe .” Hilger & Watts.’l In the first. 78/04. etc. 6..2. “Workshop on XRay Instrumentation for Synchrotron Radiation Research. PROTEIN CRYSTALLOGRAPHY 247 when allowed to dry. the size of the crystals.54 A) is selected by means of one of three techniques. which has a sharp absorption increase for wavelengths just shorter than 1. The number of reflections which must be measured for the determination of the structure of a protein to high resolution is very large (from approximately five thousand to several millions). Under these conditions they are in general very stable and can be used for data collection. Winick and G .6. Using Bragg’s law [Eq. how well it diffracts. Therefore the strategy of data collection is of great concern to protein crystallographers. Guinier.
A layer line screen of the appropriate radius (Fig.842 (1970). 84 (1968).^^ We discuss briefly below the most common implementations of these methods. thus providing a high degree of monochromatization. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Equation. W..22 In the other systems collimation is used to provide an xray beam of the appropriate cross section (usually from 100 microns to a few millimeters).” Wiley. The choice of a particular configuration for data collection involves a variety of considerations. 3.The film is always maintained parallel to the plane being recorded to assure that the image is undistorted. Press. 7. LiF. 2 5 U . The mirrors can be slightly bent to focus the xray beam for higher luminosity. Since its introduction by Martin Buerger in the 1940s.2. H. Two general methods exist to record diffracted intensities. All reflections have the same size and shape and fall in positions on the film that can be simply and accurately predicted. Nockolds and R. 1977. This is accomplished by orienting the crystal so that the chosen reciprocal lattice plane is perpendicular to the xray beam and parallel to the film (Fig. U.” Cambridge Univ. Wonocott.26 Precession photographs are particularly easy to deal with. Amsterdam. The third method consists of using grazingincidence reflection from metallized glass flats (mirrors).3.” NorthHolland Publ.1 The Precession Camera.22 For a given grazing angle there is a strong dependence ofthe reflected intensity on wavelength. “The Precession Method in XRay Crystallography. Instrum. Intensity data can also be obtained from precession photographs. Appl.248 6. W. (6. Willis. J . 6). 6) is located at a predetermined distance from the crystal and moved so as to isolate reflections from a single plane of reciprocal space. 26 C. namely photographi~’~ and . J .. 2 4 M. Buerger. and graphite are the most widely used for this purpose. 1. 1964. Sci. “Single Crystal Diffractometry. 1966. A precession angle ji is introduced and the normal to the plane precessed around the xray beam with semiangle i i . E. J . eds. S . M. “The Rotation Method in Crystallography.1) will permit only a single wavelength to be reflected. London and New York. Kretsinger. the precession camera has become a favorite among protein crystallographer^. Reflection angles can be chosen such that the shorter wavelengths (the most common contamination) can be eliminated.^^ All preliminary photographs and initial determinations of cell dimensions and symmetry are done with this camera. T. An example of a precession photograph is shown in Fig.4. Crystals of quartz. Harrison. Ariidt and A. The precession camera permits the undistorted recording of a section of the reciprocal lattice of a crystal on a photograph. Arndt and B. Crystallogr. J. 22 23 . Films can be digitized using automatic microdensitometers and the data further processed to give structure factor amplitudes. These photographs are ideally suited for the measurements of unit cell parameters. These will emerge as we discuss particular devices. New York. ~ counter ~ technique^. 6.
(6. the normal to the reciprocal plane (and to the film). Not all the intensity diffracted at a given time is recorded. other problems intrinsic to this method of data collection make it less convenient than those using other geometries. This allows all reflections of the reciprocal plane within a circle of radius AD to pass through the diffraction condition. Screenless precession photographs have been used successfully in protein structure determinations. 7). and therefore extended exposure times are needed to collect a complete data set. The plane is oriented perpendicular to the beam.6. Screened precession photographs are ideal for recording one section of the reciprocal lattice to use for the calculation of projection Pattersons [Eq.4. The presence of the screen does not permit all diffracted beams to reach the film (this is the reason for its inclusion to start with). Ewald’s sphere construction for the precession camera: (a) the incident xray beam (arrow) intersects a rcciprocal plane at a point A . (b) A precession angle fi is introduced such that C E . Screenless precession photographs can be used to avoid this problem. . However. 6. the normal is precessed around the xray beam. PROTEIN CRYSTALLOGRAPHY 249 FIG. Arta Crystallo. To record the photograph. the number of longexposure photographs needed to tion data 1/smax collect the data is unreasonably large. The reason for this disadvantage can be traced in part to the use of layer line screens. A film is placed at point Band is oriented parallel to the reciprocal plane. Freer.6 . B B27.4. A layer line screen is introduced to isolate a cone of semiangle p. for crystals with large cell dimensions (> 50A) and for highresoluGZ 2 A.” However. The simplest geometry for recording screenless photographs consists of rotating the crystal in the xray beam while recording the diffracted beams on a stationary film.Srri.. An undistorted image of the reciprocal plane is recorded in the film within a circle of radius (see Fig. forms an angle p with the xray beam. Small precession angles have to be used to avoid spot overlapping. This is the general ’’ N.H.2380 (1971).3.16)].2 The Oscillation Camera. Xuongand S.qr.3.
each photograph is obtained while the crystal rotates over a very small angle (from a few minutes to a few degrees). Note also the very large number of reflection present. to avoid overlap of reflections. The photograph was taken using a precession angle of 6".) principle of the oscillation camera.250 6. For long exposures. corresponding to a resolution cutoff of approximately 7. Precession photograph of a uranyl derivative of crystalline southern bean mosaic virus. Symmetry is casily recognized in the photograph. Speical precautions are taken to avoid backlash and overexposure of the reflections that are in the diffraction condition when the movement is reversed. Also. (Courtesy of Michael Rossmann and John Johnson. to facilitate the identification of the reflections.23 Usually. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES FIG.5 A. Cell dimensions simply related to the distance between spots. . the crystal is oriented with a unit cell axis parallel to the rotation axis and perpendicular to the xray beam. the crystals are oscillated back and forth through this angle.7.
Oscillation photograph of crystal porcine pancreatic aamylase.3. However.6. 6. (Courtesy of Paula Fitzgerald.5".) A complete data set is obtained by recording smallangle oscillation photographs for all the necessary different orientations of the crystal around the rotation axis. PROTEIN CRYSTALLOGRAPHY 25 1 FIG.8. 2) from the intersection of Ewald's sphere with reciprocal lattice planes. (For a complete discussion see Arndt and W o n ~ c o t t ~ It~ is ) . The curved rows of spots arise (see Fig. computercontrolled fourcircle diffractometers have become the . The FourCircle Diffractometer.3. such as the one shown in Fig.4. 8.The edge of the photograph shows reflections at 2A resolution.4. Since their introduction. The problems involved in obtaining a complete data set using the oscillation camera and in measuring the films are outside the scope of this chapter and will not be discussed. The oscillation angle is I . sufficient to note that there are several good computer programs available for processing oscillation photographs. To measure diffracted intensities with conventional proportional or scintillation counters it is most convenient to be able to orient the crystal and the counter in any arbitrary orientation with respect to the xray beam. several simplified geometries have been used successfully for this purpose.
8b)l and can serve as a reference to measure the phase of the protein structure factors. 4 ) which allows the crystal to be set at any desired orientation. Dickerson. 6. The crystal is mounted at the center of rotation of an Eulerian cradle (with angles w. 14. Introduction of heavy atoms is usually accomplished by soaking preformed N.1) neither the Patterson function nor direct methods can be used to obtain the structure of a protein crystal from the observed structure factor amplitudes F(h). and W . Vernon. J. R.4. the present cost of these instruments does not put them in the category of equipment that can be acquired by a single laboratory. positionsensitive detectors has resolved this difficulty. The major drawback of diffractometers is that they measure one reflection at a time and miss all the others that are formed simultaneously. 289 (1978). Acta c'rJ3. Freer.28 In any case. S. Srcr. The counter can then be set at an angle 20 from the xray beam to record the intensity of the diffracted beam. Strandberg.H.stallo. A A34. Phase Determination Using lsomorphous Replacement As stated before (Section 6.25In this geometry the counter can be rotated in the equatorial plane that contains the xray beam. This problem is especially serious for crystals that are very sensitive to radiation. x. C. Arta Crj'. '' . C . equipment incorporating these detectors is still in the developmental stage. and because their high electron density makes them stand out above the protein.yta//ogr.qr.4. I188 (1961). Nielsen. The development of large. The method most commonly used for solving the phase problem for crystalline proteins is called multiple isomorphous replacement or the heavyatom method.4.29 In this method protein crystals are modified by the introduction of atoms of high atomic number..28 Unfortunately. With this arrangement any reflection can be made to form in the equatorial plane. the heavy atoms can be located using the Patterson function or other methods from smallmolecule crystallography. the phase of the scattering from them can be calculated [Eq. and H.3. Fourcircle diffractometers are very convenient to use and are usually the method of choice for proteins with cell dimensions smaller than approximately 100 A. Because there are few of these. At least one such machine has been functional for several years. 2 9 R. The process whereby these modified crystals can lead to the determination of phase is quite simple. Xuong.252 6. bidimensional. Using the diffraction patterns from the native and modified crystals one determines the positions of heavy atoms bound to the protein. (6. Kendrew. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES method of choice. Hamlin. Once the positions of the heavy atoms are known. Computer control of the circles permits the collection of a large number of reflections with very limited operator intervention. T. E. E.
4. Substituting (6.exp(2nih j= 1 r= 1 N XJ.2) here NH is the number of heavy atoms in the unit cell and x. C. L. For a heavyatom derivative to be useful it must be isomorphous to the native crystal.2) yields F P m = FP(h) + fH(h). Kopka. This means. Borders. PROTEIN CRYSTALLOGRAPHY 253 crystals in solutions containing. Most heavyatom derivatives are found by trial and error. exp(2nih xr). L. Margoliash. (6.x j ) + 1 f.. J . J .4.VtI * N (6. Once a suitable heavyatom derivative has been prepared and its intensity data collected. Heavy atoms normally bind to the surface of the molecule. However. Heavyatom substitution and isomorphism can be monitored by comparing intensities and lattice constants and derivative crystals in precession photographs. which is exposed to solvent.3) which represents the diffraction that would arise from the heavy atoms alone. It is convenient to define the heavyatom structure factor fH(h) = C S. The structure determination of cytochrome C30 and rubredoxin (Section 6.1)and (6.1) where xj are the coordinates of the N protein atoms. that the heavy atoms have replaced an equivalent volume of solvent in the crystal.4.4. For the most part they make interactions with amino acid side chains in ways that cannot be predicted before the structure is known. ffg. Mu/.xj). Jr. and E. it can be used to determine the phases of the native crystals. .4.(h) and FpH(h) are Fdh) = j= 1 C fj exp(2nih . Dickerson.3) into (6. Au. Pt.. or Sm). 77 (1967). r (6. specific modification of the protein using heavyatomcontaining groups has also been used. are their fractional coordinates. E.4.6. certain reactions. varying concentrations of salts of highatomicnumber elements (e. ideally.nih . Varnum. E. M . Weinzierl. J .9) illustrate many of the facets of this method which we now describe.4. .g.4. However. The structure factors of the native and the heavyatom derivative F. 29. while causing no other changes.4) To use the heavyatom derivative for the determination of the phases of the native crystals it is necessary to find the position of the heavy atoms in 3" R . FpH(h)= C1 . Bid. :exp(2. such as the binding of Hg to SH groups are generally useful.4. (6. in addition to mother liquor.
4. (6. J. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES the cell.6) (6. see also Dickerson et aL3’ and Sygusch3’). R €324. sin + b.5) For most reflections F.(h) and FPH(h). E.4). 512 (1977). since in this case fH(h) = FPH(h) + FP(h). (6. AF(h) is normally used as the estimate of the heavyatom structure factor amplitude for all reflections contributing to a centrosymmetric projections. and their temperature factors can be refined using leastsquares procedures (Section 6. (6. Weinzierl.. The structure factors that contribute to this projection are real.4. When F. Sect. This synthesis is identical to the projection of the Patterson function of a crystal containing only the heavy atoms if the problem of “crossovers” is ignored. This function contains as its major peaks the heavy atomheavy atom interatomic vectors and can in principle be solved in the usual manner (see Fig. Sygusch. their percent substitution (occupancy). A “good” heavyatom derivative should contain only a few major heavyatom sites.(h) can be calculated from the structure factor amplitudes using AF(h) E FpH(h)  Fp(h).(h) < fH(h) and F . This approximation does not introduce serious difficulties and can be fully corrected once the coordinates of the heavy atoms are known. atid R. If moduli are taken on both sides of this equation we obtain IFPHI’ = IFP f fH12. The positional parameters of the heavy atoms. andf. The preferred method for locating the heavy atoms involves the use of a Patterson function in suitable centrosymmetric projections with coefficients AF(h)’. However. SWI. J. AF is an underestimate OffH.4.7) This can be shown to yield F& = ( F cos ~ C( + u ~ ) ’ + ( F . Palmer. These parameters can be used to calculate the heavyatom structure factors f H . Dickerson.8.254 6. A A33. and therefore FPH(h) = FP(h) kfH(h).ru Crsstallo.qr. the heavyatom structure factors f H cannot be directly obtained from the experimentally accessible magnitudes F. E. .)’. Actcr C‘rysfulloy. These structure factors can be used to determine native phases through Eq. and its Patterson function can in general be unscrambled.4. In crystals without suitable centrosymmetric projections a threedimensional Patterson synthesis with coefficients AF(h)’ is usually calculated. ~1 3 ’ R. 4). have opposite sign (the socalled crossover condition). Ac..(h) 2 fH(h) and thusf.4. A . Most space groups have at least one centrosymmetric projection. 997 (1968).
4. Ac. In an actual structure determination the situation is more complicated.2nih * x).. This ambiguity can be eliminated by using several heavyatom derivatives. addition or deletion of sites.7) gives two possible values of a for each individual heavyatom derivative. and b. There will be in general two values of a that satisfy Eq.tu Crystnlkogr.7) for all heavyatom derivatives. Crick. 33 D. (6. The correct value of the phase will then correspond to the common roots of Eq.^^*^^ The “quality” of the phases can be estimated from the statistics of the minimization procedure. All magnitudes in Eq. even though Eq. errors in the atomic parameters. The estimated phases and the values of F . H . are affected by errors in the heavyatom model (i. (6. (6. Therefore. For each reflection all heavyatom derivatives should give a common value of a . PROTEIN CRYSTALLOGRAPHY 255 In this expression M is the phase of the native structure factor and uHand b.(h) exp(ia) exp( .4. F . each reflection in this synthesis is weighted according to the estimated accuracy of its phase.7). (m(h)). Blow and F.4.). and F.3. M.9) The average value of the figure of merit over all reflections. The resulting set of nonlinear equations can be solved for the protein phase and for corrected heavyatom parameters that will minimize a conveniently chosen residual. (6. The correct approach is then to recognize that this is an observational equation which depends on the phase M and on the heavyatom parameters. it is therefore convenient to incorporate all heavyatom derivatives in a single observational equation. It is roughly true that m(h) = cosrerror in ~(h)].. Usually. are the real and imaginary components of fH. exp(ia) exp( .4. provides a useful indication of the overall quality of the phase determination.2nih x). This equation can be solved to obtain the value of a.4. Several formulations have been used which differ mainly in the residual chosen and in the detailed form of the observational equation^.e. The most commonly used weight is called the “figure of merit. (6.7) are subject to errors.8) This expression is identical to Eq. Use of m(h) weighting minimizes the rootmeansquare error in the electron density when certain error models which are outside the scope of this article are assumed. are experimentally determined magnitudes and a.” m(h). etc. all the obtained pairs will not have a clear common value. (6.4.5) with F(h) = F(h)exp(ia).4. C. are then combined to calculate an electron density map using the synthesis p(x) = 7 1 F. The synthesis using m(h) weighting is often called the “best Fourier”33 and is given by p(x) = 1 v m(h)F. 12. (6. 794 (1959) .6.
The atomic model derived from this density is shown superimposed. in protein crystallography. Computer displays can also be used.4. The calculation of p ( x ) provides an estimate of the electron density in the unit cell of the crystal. Balsa wood or Styrofoam sheets of appropriate thickness are usually cut to follow some chosen contour with the shape of electron density function and FIG.5. Suck of contoured electron density sections used t o visualize the electron density function of thc immunoglobulin fragment Fab New. The transparent sections are stacked with the proper spacings and illuminated for visual inspection (Fig. . but they are in general not very helpful for the interpretation of lowresolution maps. The ease of this procedure and the detail with which the structure of the protein is determined depend mainly on the resolution of the data and on the quality of the phases used. presence of helices. subunit organization. The maps are usually contoured on lines of constant electron density and plotted on transparent sheets. can indicate the general shape of the molecule. 9). when interpreted. When this separation is unambiguous.256 6. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Interpretation of Electron Density Maps 6.9. Because the crystals are periodic. we want to know the structure of the protein molecules forming the crystal. Lowresolution maps (I/smax= 6 A). and other gross structural features. each containing the electron density corresponding to one section through the unit cell of the crystal. In many cases molecular boundaries can be located as regions of low electron density separating continuous globular regions of high density. This map represents the distribution of diffracting matter in the crystal. However. a model of the protein molecule can be built. the electron density belonging to an isolated molecule cannot always be identified immediately.
and the electron density features interpreted using this information. 3 . With higherresolution maps ( 1/smax z 2 A) the polypeptide chain of the protein can be traced. D. and D. E.) assembled to represent the molecule (Fig. To aid in the interpretation of the maps they are displayed in optical comparators like those shown in Fig. 34 35 .) 197. Love. M.. 37. 3 4The comparators allow one to fit skeletal models of the amino acids to the electron density. Knowledge of the amino acid sequence of the protein (primary structure) greatly simplifies this task. Bid. which can be superimposed over the density being interpreted. McQueen. l l . The rodlike features of the model are CI helices.4. The procedure is continued until all the density in the crystal is used and the complete amino acid sequence built. For this procedure a few (510) sections of electron density are displayed at a time and viewed through a halfsilvered mirror. J . 1378 (1977). The highresolution maps can also be plotted on transparent sheets. Science (Washington. Tsernoglou. GIyrera clibrcmchiatu at 6A resolution. Petsko. Mol. A . Hermans. (Courtesy of E. Jr. PROTEIN CRYSTALLOGRAPHY 257 FIG. 10. Balsa wood model of the hemoglobin molecule from the bloodworm. Padlan and W.C. An equivalent procedure can be followed using computer display^. and this makes it possible to adjust the model to fit the density. The illusion that the brass model is floating in the contour density is often quite powerful. E. A.^' In both cases the final product is a threedimensional atomic model of the protein molecule. 10). Individual pieces fill the regions within contour lines drawn at some constant level ofelectron dcnsity.6. One then builds a brass model while looking at the reflected image.225 (1968). Richards. J . A model of this F. G .
I I . XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES LNqht ba: . Opticalcomparator or ' Richard'sbox'whichI:dcilitatescomparisonofthemolecular model with the clec(ron density map. sdvered b P r o t e i n model (bl r Lighl box Sectjonr of eleclfm nallsdvcred de L . Solution and crystallographic experiments can be designed to test or exclude proposed modes of action. The procedure just described for the determination of the threedimensional structure of a protein molecule using singlecrystal xray diffraction is long and involved. The geometrical and chemical properties of the active site(s) and other functional regions can be analyzed. .mirror Prolein model FIG. Physical. (b) Mirror parallel to sections of map. and physiological properties of the protein can be correlated with structural characteristics.') kind contains a remarkable amount of information. Secttons 0 1 electron density map o n transoorent sheets H a ll. Important insight can be gained into the mechanism of action of the molecule. chemical. (From Blundell and Johnson. (a) Mirror tilted 45" toward sections of map.258 6. but experience has shown that the wealth of information that can be obtained from such a determination is always worth the effort.
however. products.4. Therefore in the case of hydrolytic enzymes if substrates are diffused into the crystal they will be hydrolyzed before xray data can be collected. The complex of the protein and its ligand must be crystallized and the structure determined using the usual techniques.4. most functions are dependent on interactions with other molecules. Not surprisingly. Carrier proteins (i. just as if the proteins were in solution. structural correlation of functional properties can be made using the threedimensional structure of the native protein. The usual way to overcome this problem is to use nonhydrolyzable substrate analogs. When enough biochemical and physical chemical information is available.4.6.C F. This problem can be simplified enormously if a suitably small ligand such as a substrate analog.(h) exp(ia.. can be used for these experiments. Lowtemperature techniques have also been able to “freeze” substrates so they cannot react.e. Sometimes these small molecules can diffuse into preformed protein crystals (as happens in making heavyatom derivatives) and bind isomorphously to their specific binding site.. Difference Fourier Syntheses One of the goals of the determination of a protein structure is the characterization of its functional and physiological properties. When proteinligand crystals are isomorphous to the parent crystals.10a) ppL(x)= 1 V FPL(h) exp(icrpL) exp( . and binding proteins (i.) exp( . and effectors of the catalyzed reactions.271ih * x). If we use . In principle these studies involve a completely new structure determination.e. It must be realized that in most cases enzymes also retain their activity in the crystals.2nih * x).) represent other examples of proteins for which the interaction with other molecules (which we shall call ligands) can be studied.6. the binding constants are generally lower in the crystallized protein. antibodies. most of the structure of the proteinligand complex is known (i.4.) A simple technique termed the differenceFourier synthesis can then be used to localize the ligand. etc. the protein part.e. In the cases of enzymes these molecules are the substrates. receptors. For many proteins.10b) If the substrate has a small number of electrons the phases of the proteinligand crystals will be very close to those of the protein crystals. V (6. hemoglobin). (6. The electron density functions of the protein and proteinligand crystals are given by 1 pp(x) = .. repressors. PROTEIN CRYSTALLOGRAPHY 259 6.
4. In practice. Ser. Johnson. Sarma. to locate the residues involved in the catalytic steps of the enzymatic r e a ~ t i o n . ligand difference maps are interpreted by copying the contoured density into transparent sheets which are then displayed in an optical comparator (Section 6. difference maps of ligand are easy to interpret. B 167. T.4. D.. G .5). Pro(. C .B B27. Usually. Henderson and J. L o n r h .. N .FP)exp(icc. 37 C. K . If a group of protein atoms moves upon ligand binding its original position will appear as negative density in the difference map. Soc. The protein model (already built) is kept in its correct position and the ligand built into the density while being checked for favorable interactions with the protein. A . and F.) exp( . Experiments using differenceFourier maps provide invaluable information about the biochemical and physiological properties of protein molecules. 6. . R. however. The new position of the group will appear as positive electron density. Mair.. ’ Movements of side chains of the protein that sometimes occur upon binding can also be detected in differenceFourier maps. L.qv. A . A m Crjwdlo. Moffat. The simplicity of the experiments (once the structure of the protein is solved) make difference Fouriers a powerful tool for the study of the function of proteins. (6.271ih * x). Molecular Replacement The method of differenceFourier synthesis just described is invaluable in simplifying the crystal structure determination of a proteinsmallmolecule complex whenever the crystal is isomorphous to a native protein crystal whose structure has already been determined. 378 (1967). North. C .260 6. 1414 (1971).. R .pp (difference map). Blake. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES the approximation clpL = clp we can calculate the difference ppL . in principle.c. Phillips. which is given by 1 ppL . These maps are subject to large errors due to the approximation clpL = and the need to measure small amplitude differences between F. and V . the electron density corresponding to the ligand. F.pp = (FpL. Sec.11) This map can be calculated from the structure factor amplitudes of the native and proteinligand crystals using the native phases and provides.7. The proteinligand complexes obtained in this manner can be studied to characterize the interactions between residues of the protein and ligand.. The perversity of nature R.4. ~ etc. especially if the general location of the binding site is known from sequence and chemical data. C.
97 (1974). Herriott. and some simplification must be found. E. The molecular replacement allows known molecular strucmethod.4. K. many other circumstances arise when it is of interest to determine a crystal structure when the structure of the constituent molecules is already approximately known. The study of the evolutionary relationship of molecules performing identical functions in different species is one example. Further. three to specify the position of the search molecule in the unit cell and three its orientation. and W. B. 84.7. in the usual case. that such crystals are not always isomorphous to native ones. however. Rossmann and D. The rotational search or rotation function is based upon certain simple properties of the Patterson function. The known symmetry operations of the crystal allow the other molecules in the unit cell to be generated. 24 (1962). Schmid. B i d . Mol. M. Mol. Biol. Lattman. E. and J . Mech. 0. Fehlhammer. Lattman. C . perform a sixdimensional search in which the diffraction pattern or Patterson function corresponding to every possible value of the parameter sextet is compared with corresponding experimental values from the crystal. inspired by Rossmann and tures (search molecules) to be used as building blocks to construct models of crystals of similar or related molecules. reducing a sixdimensional problem to a sequence of two threedimensional ones. G. 179(1975).8) to give accurate representations of the new molecules. 15. only six parameters. Epp. Lattman. Ward. Another is the study of the same molecule crystallized under different conditionsfrom organic solvent and from salt perhapsto check the constancy of conformation. E. R. Love. are far beyond the scope of even the fastest computers. Struct. M. Wishner. One must use the diffraction pattern or Patterson function in the test because there is no experimental electron density function with which to compare the model. but it is very efficient compared with isomorphous replacement. A particular parameter set may provide a clear best fit with experiment. I . 139 (1975). Schiffer. and other molecules have been successfully determined in this A collection of papers about molecular replacement appears in Rossmann. and W. Steigemann. 98. E. in principle.6. PROTEIN CRYSTALLOGRAPHY 26 1 ensures. Blow. thus generating the desired preliminary model for the crystal structure. Such sixdimensional searches. however. A third is the study of functional conformational changes which occur upon ligand or substrate binding. Structures of hemoglobin. This method is neither as straightforward nor as foolproof as the differenceFourier method. M. 4n H. J . protease. E.8 To construct a model of a crystal using a search molecule requires. unbiased by the original search molecule structure. These models can then be refined (Section 6. B. P. Biophy. M. E. E. Colman. P. 4 1 M. To find the correct model one could. Acta Crystallogr.J.4. immunoglobulin. 38 3y . Rossmann and realized that it was possible to separate the rotational and translational parts of the search. Schwager.
The latter bit of formalism allows one to apply Parseval’s theorem6 to (6. It is small when peaks do not coincide. isolated molecule and is therefore not periodic. The volume of integration is a sphere of radius D. In practice the success of the method varies widely. upon a selfvector family in P. (6. The correlation function most frequently used for this is R(C) = JJJPM(Cu)P(u) dV. plus whatever cross vectors happen to be of length less than D. coincide with valleys in P. where D is the maximum dimension of the molecule. is the Patterson function of a single. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In the Patterson function of a crystal one can mentally divide up the interatomic vectors into two classes: vectors running within one molecule (selfvectors). often based upon Eulerian angles. coincide with peaks in P. and valleys in P . In some cases isolated. In principle the absolute maximum of R occurs for a rotation C which superimposes the whole set of vector peaks in P . R ( C ) is large when peaks in P . The families of selfvectors are identical. (6. but appear in various orientations about the origin. The integral above is evaluated numerically for each value of C. one need only seek the orientation for which P . depending upon convenience.4. but may be written as an integral over all space. If one knows the structure of the search molecule. Indeed.13) where the F s are the structure factor amplitudes of the model and crystal. and considerable difficulty may arise in attempting to pick the correct one.12) The matrix C is a rotation matrix. highly significant peaks in R ( C ) are seen. since P . and vectors running between molecules (cross vectors). one can readily calculate its Patterson function P. it is best thought of as the set of intcratomic vectors within one test molecule. in others several peaks of nearly equal height are found. All selfvectors are contained within a sphere of radius D about the origin of the Patterson function. corresponding to the orientations of the molecules within the crystal. Both the direct and reciprocal space formulations have been used. showing that R ( C ) = C Fh(Ch)F2(h).4.and a maximum in R is sought.4. As such it is formally identical (and numerically similar) to the selfvector sets clustered about the origin of the experimental crystal Patterson function P(u). superimposes upon one of these. . thereby providing as estimate of the molecular orientation in the crystal. To find the orientation of the search molecule in the crystal. vanishes outside the sphere in any case. This sphere of radius D will contain one family of selfvectors for each molecule in the unit cell.(u).262 6. P .12).
). E.4. It is sufficient to note that by assigning a trial position to a properly oriented molecule one obtains a complete trial model of the crystal. 6.tu Crysrallogr. An extensive literature exists describing variations on T ( x ) and discussing the most efficient ways of evaluating it. Lattman and W. 1972. is the Patterson function of the model with the properly oriented search molecule at position x. Also. the success of T(x)is quite variable. Refinement of Protein Structures Protein models obtained from the interpretation electron density maps (Section 6. a hybrid of the search and correct structures. Ac.8. PROTEIN CRYSTALLOGRAPHY 263 The above formulation of the rotation function42 is a simplified version of the original function of Rossmann and Blow. in “The Molecular 42 . and it is the function of the refinement process to convert it into an accurate image. Such a synthesis is. errors in the estimation of the phases introduce errors and distortions into the electron density and into E. thus completing description of the model structure.14): (6.12) in spherical harmonics and has shown that two out of the three integrals can thereby be reduced to fast Fourier transforms.4. 1854(1970). The Patterson function of this model (which now represents one unit cell of a crystal) can be compared with the experimental Patterson function using an equation like (6. New York.. Once the orientation of the molecule is known. S w i . ‘’ R.. However. of course.5) provide coordinates for all atoms in the structure. G . E.14) J J J unit cell Here P. ed. Love.6. Crowther.4. but in favorable cases it gives a clear and unambiguous indication of the molecular position. only its position remains to be found. In an important advance C r ~ w t h e has r~~ expanded the functions of the righthand side of Eq. A. BB26.4. (6. Rossmann. these coordinates are subject to considerable error. Gordon and Breach. This is usually accomplished by the use of a ‘translation function. who put it forward for slightly different purposes.’ Again. Using a structure factor equation this model can be a source of phases which can be combined with the experimental amplitudes to produce a Fourier synthesis of the experimental crystal.4. Replacement Method” (M. thus speeding up the calculation enormously.4.’ Full details of the translation function are outside the scope of this chapter. At the resolution usually attainable in protein crystals (1/slnax z z 2 A) the maps do not have enough detail to visualize individual atoms.
Diamond. from a physical point of view the models are highly inaccurate. Ahmed. 253 (1966). (6. namely the structure factor amplitudes of the native crystals.15) with At. Copenhagen.18).16) Equation (6.3. It is important to realize that in view of Parseval’s theorem6 is in principle equal to 1 V AF2(h) J (Po unit cell .~”) K.(h) = F.).264 6. Acru (’rj. R. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES the model. Usually leastsquares procedures are used to minimize 1 W(h)AF2(h).16) is an observational relation which can be used to refine the atomic parameters. Hermans and J . In addition.730 (1974). ed. Actir C‘rjxtui/o.Sect. 21. 292.. (6. The correct stereochemistry is usually imposed upon the measured coordinates using “model building” program^. An estimation of the values of this set of observed magnitudes can be calculated from the parameters of the model using Eqs. However.Diamond. where W(h) is a properly chosen weight.(h) is called the “ R factor” and is defined as (6. McQueen.^^.The most commonly used index of agreement between observed and calculated magnitudes F. Munksgaard.s/uIIoqr. if the electron density maps are interpreted in an optical comparator the atomic coordinates do not conform to the standard geometry of the amino acids.4. p. J .(h). This clearly indicates that the minimization procedure can be performed both in direct (real) or reciprocal space. Both kinds of methods have been used for the refinement of proteins.(h) and F. Hence the models obtained in this manner represent only the best efforts of the builders. E.U J 2 nv if phases a. 44 45 .4.^^ These models are in general good enough to study most of the relevant biochemical properties of the protein. are used to calculate po. see Blundell and Johnson’ and Diarn~nd. A A30.4. 4h R.qr.(h)  F. (For a complete evaluation of some of the methods. 1975. bi “Crystallographic Computing” (F. There is in principle one set of observed magnitudes that can be used to evaluate the accuracy of the parameters.
Instead they are modified by a leastsquares fit so as to preserve the stereochemistry of the model. The electron density p o is pbes.M~ (6. 250. PROTEIN CRYSTALLOGRAPHY 265 Independent of the particular residual minimized. H. C. the resulting system of equations is nonlinear.3.N =Al. The matrix elements are derived from minimizing W(h)AF2(h) 1 47 4R S . J. and J . 49 K.4. obtained from multiple isomorphous replacement (Section 6.qr. The electron density pmode. T. Reciprocal space methods refine directly the residual W(h)AF2(h). The atomic positions are refined by shifting atoms up gradients to the summit of electron density peaks. Freer. 509 (1979). Acta Crystallo. Watenpaugh.436 (1971).P m o d e ~ X ) 1 2d v is minimized. The independent variables of the refinement are then rotations around single bonds in the structure. C. After shifts have been imposed a new set of calculated phases a.6. Diamond. This information can be used in two different ways: (i) to reduce the number of parameters. Sect. Jensen. D . 131. and L. J . and (ii) to increase the number of observations. Bid.4. Alden. Jr.8)]. Therefore all methods of solution necessarily involve iterative procedures. L. (6. A. which serves to begin a new cycle of refinement..46(1975). Also.. Carter.(h)exp(ia.4) or from a map calculated using F. since the number of observations per atomic parameter is usually quite low (from less than 1 to 2 or 3). Mol.4. Chem. W.17) where the subindices represent the number of columns and rows of the matrices. to produce coefficients for an improved electron density map. . Sieker. These a. The traditional realspace methods use either electron density or difference electron density maps. can then be combined with the observed amplitudes F . which are then introduced after each Recently Diamond developed a method49 in which the residual J~llo(x) . These procedures are usually performed without stereochemical constraints. thus reducing the number of model parameters by a factor of approximately three. Biol. The shifts calculated in each minimization cycle are not used directly. For a typical protein this condition is not easily met. can be obtained through a structure factor calculation [Eqs.M6x1.). For convenience we summarize some of the possible approaches in Table 11. the problem has to be overdetermined to provide for a stable refinement. The known stereochemistry of the amino acids is normally used to overcome this problem. Kraut. R. R . A A21. is calculated based on the atomic coordinates of the model using a spherical Gaussian to represent the individual atoms. For a problem with M observed structure factor amplitudes and N parameters the linearized observational equations can be written in matrix representation 1 AN.
z)*8. etc. bond length. 6 '(observational or approximate) Gaussian. . ' The values of n and m vary for different implementation of these methods. All structure factor calculations are done with atomic scattering factors. conformational angles: atomic radii ideal. bond angle.  Pmodeb' dV. .occupancy. planarity condition. Summary of Refinement Options Important quantities in refinement programs Residual minimized (explicitly) Weights of F (explicit or implied) Atom shapeb Parameters refined (explicitly) Stereochemistry of final model Number of partial derivatives calculated for N atoms Options used in different refinement programs J(Potv. transform of atomic scattering factor (x. but they range from 3 to 50 or more. are the values of a stereochemical parameter (i.e.rl. .. point. 1 WAFz(h) + C W@.Pidexl)'P 1 O(P. .) obtained as an average of a series of very accurate smallmolecule observations (pideal) and as calculated from the atomic coordinates of the protein being refined @ ) .Pideal)' l. This is the implicit or explicit atom shape at the minimization step. 1orz> 1WAzF(h).TABLE 11.y. optimized nN. (mlv)" pideal andp.
Sussman. so that 6~ = B'C. N A 1 .(inverse of B).19) As mentioned above.N = A&.4. S.6. 800 (1977). and S . Second. (6. R.(h) and AF(h) are calculated with the present atomic parameters.Holbrook. This is.4. Another method used to diminish the number of parameters consists of refining the positions and orientation of rigid groups. First. we obtain B6x = C. M . a very expensive task.18) The elements of B and C are and Cj = h W(h)aFc(h) AF(h). This expression can be solved for the shifts 6x in the parameters by transforming to the normal equations. Church. Defining BN.5 0 5 0 J .NAN. large blocks of 200 parameters were refined to avoid working with the full matrix. ax These normal equations can be solved for the shifts 6x by multiplying (6. the number of parameters is in general very large. however. H. They can be shown to be where F. Multiplying on the left by AT (transpose of A) we obtain A~AGX =A~A. Secl A A33.4. L. there are two problems in applying this method directly to the refinement of proteins. Section 6.N = AL. in principle. PROTEIN CRYSTALLOGRAPHY 267 with AF(h) made linear on the shifts by using the zeroth. G . used for a small protein (rubredoxin.9).4. In this case.M and Ci. Acta Crystallogr. This method has the additional advantage that the stereochemistry of the molecule is automatically preserved.and firstorder terms in a Taylor expansion. . The method was. and the normal equations will thus require the generation and inversion of a 3000 x 3000 matrix for a large number of cycles.18) by B. M . (6. M is usually not much larger than N . In a typical case there will be more than 3000 parameters. Kim.4..
62. J . Otherwise they are very small. if they are coordinates of two atoms that are bonded or belong to a rigid group).). biological relevance. Hcndrickson and J.4. S. the method of conjugate gradients5’ used for solving the system of equations takes full advantage of the sparseness of the matrix. It is therefore very difficult to choose a “typical” case.S. Indian Academy of Sciences. Rrs.9. Nut). providing further savings in computing time. 6. Hestenes and E. Wiley. Venkatsan. In addition. in “Computing in Crystallography” (R. phase determination. 1960. eds. in a typical case only 1 of the matrix elements have to be calculated and used.01.Pideal)’ k h (see Table I1 for definitions). p. leading to the establishment of a Protein Data Bank (Brookhaven National Laboratory. There are probably about 100 proteins whose structures have been determined (to some resolution) using singlecrystal xray diffraction techniques. ( P ~.). Sfand. thus using the stereochemical information as additional observations. complexity. we have settled on a 5 1 W. S. Such models have become of interest to a wide range of investigators. New York 11973) from which atomic coordinates from a rapidly expanding number of molecules can be obtained. This implies that the matrix elements of B and C will now include terms from the stereochemical equations. S. Rainaseshan.268 6. Konnert and Hendrickson found that the system of normal equations is very stable even if the matrix elements not containing stereochemical constraints are omitted. Stiefel. ’’ . XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In a recently developed method Hendrickson and Konnert5’ proposed refining the residual C W(h)AFZ(h) + C W . They differ in function. etc. A. They were solved using different methods for data collection. Diamond. and refinement./dxi)(dF. 1980. Konnert. Ralston and H. Upton. for this 1 % of the matrix elements x h W(dF.)49. A Case History: Rubredoxin To illustrate the different aspects of protein structure determination we next analyze the process as applied to a specific case. and K . H. In addition. 13. p. eds. Wilf. Ncw York. M . size. Refinement has led to greatly increased rigor and quality in the protein models obtained by XRDA./dxj) is small compared to the terms arising from stereochemical constraints when x i and xj are not coordinates of the same atom. These terms can be very large if parameters are related by a stereochemical restraint (for example. These approximations lead to very substantial savings in computing time for the generation of the sparse matrix. Beckman. Bangalore. in “Mathematical Methods for Digital Computers” (A. With this approximation. F. (C/. Bur. R.409 (1952).
Sect.’. rubredoxin. has been solved to high resolution. the authors built 54 amino acids and were able to identify about 40 of them with “reasonable certainty.” The four cysteines were assigned as amino acids 6. The rubredoxin literature uses a hexagonal unit cell (as is usually done). Watenpaugh. Using sequence data from other rubredoxins.~ but its discussion is outside the scope of this book. The crystals are flattened rhombs and can be grown to more than 1 mm per side in a few weeks.9 The unit cell of the crystal can then be represented in terms of three identical axis of length a with the same angle a between them. Sicker. The final map calculated with phases obtained from multiple isomorphous replacement included data to a resolution of 2. 12.40’. L. Most rubredoxins have an iron atom and four cysteines per molecule of approximately 6000 daltons. and 42 in agreement 5 3 K. 54 K. 39. A singlesite HgIi. and L..’ were used for phase determination.4.54 The protein was crystallized by precipitation with nearsaturated ( N H 4 ) . C. but the rhombohedral cell is more appropriate for the scope of this book. D. The crystal is then said to be rhombohedral and to belong to space group R3.77 A and a = 112. R. Herriott. L. S 0 4 at pH 4. Several electron density maps were calculated and interpreted. PROTEIN CRYSTALLOGRAPHY 269 protein that is small. R. 359 (1971). The structure was solved by the multiple isomorphous replacement method. 36. Heavy atoms were prepared by the usual soaking technique. Quant. and L. 9. Herriott. Jensen. H. Acra Crystallogr. The tracing of the polypeptide chain was unambiguous and the iron atom and the four coordinated cysteinesulfurs were located. The sequence of the rubredoxin from C . J. Examples of the electron density corresponding to several side chains are shown in Fig.derivative and a multiplesite UO.6. Rubredoxin is a nonheme. pasterianum was not known at this time. Data were collected using a fourcircle diffractometer. Heavyatom positions were refined and phases were calculated For phase determination the using a program written by Dickerson et authors used additional diffraction information (anomalous ~cattering). H.47. Cold Spring Harbor Symp. The cell parameters of these crystals are a = 38. D. Xray diffraction experiments indicated that the only symmetry of the crystal is the presence of a threefold axis at every lattice point. Jensen. easy to describe. ironsulfur protein that has been found in a number of anaerobic bacteria. and for which more than one refinement method has been used.0 A.53.5A resolution by Watenpaugh et a1. The positions of the heavy atoms were determined using AF2(h) Patterson syntheses. namely. C. . Watenpaugh. 943 (1 973). At least 25 investigator years have been invested in this project. B B29. Sieker. The structure of the rubredoxin of Clostridium pasterianum was determined to 1. Bid. J.
7 ICl FIG. at resolutions of 3.0 2.5. Electron density through aromatic side chains in rubredoxin. Copyright 1971 Cold Spring Harbor Laboratory. 2.0. and ( c ) tryptophan. (b) tyrosine.) .270 6.0. s 3 with permission. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES 3. 12.5 Ib) 30 25 2. The side chains reprcscnted are (a) phenylalanine. and 1.0 1. 2. (Reproduced from Watenpaugh et a / .7 A. Note how the rendition of the groups improves with resolution.
The ironsulphur complex involved in the redox activity of the protein is also shown.6. . thc intermediate circles sulphur. L.0 3. Quant.4. Jensen. D. PROTEIN CRYSTALLOGRAPHY 27 1 FIG 13. (6. The large circle represents iron. Cold Spring Harbor Symp.4. This gave a grand total of 424 atoms.0 and 1. A temperature factor B (Section 6.389) indicated that the quality of the atomic parameters TABLE 111.0 2. Watenpaugh. and the small ones carbon. H. only 45 % of the reflections between 2. 36. It was found that the average intensity of these reflections decreased rapidly with decreasing l/s (Table 111).3 93. For example.3.6 ('x) 5.5 A were then collected in order to improve the resolution of the model. The value obtained (0. (Reproduced from Watenpaugh P Z u I . Siekcr. Intensity data for reflections with l/s 2 1. represented by line segmentsconnecting rcarbon centers. Although a chemical sequence was still not available. 12). 23 peaks in the electron density map were interpreted as water molecules and their oxygen atoms assigned coordinates. Summary of Intensity Data for Rubredoxin"** Possible number of reflections Number of reflections exceeding 20 218 Range of d (4 Reflections exceeding 2u 91. Biol. with ~ ~ permission. 359 (1971).0 d < 2. C. Also. J.7A resolution was calculated and showed the expected improved resolution (Fig.) with the sequence data of the rubredoxin from Micrococcus aerogenese.0 1.15)] was calculated. Copyright 1971 Cold Spring Harbor Laboratory. about 95% of the nonhydrogen atoms in the structure were assigned atomic Coordinates. An electron density map including data to 1.5 A as measured by the authors had intensities larger than two standard deviations above background.7 86. Figure 13 is a stereo pair drawing of the FeS complex and the path of the polypeptide chain.0 d < 3. R.5 < < < < d d < 5. and L. Herriott. Polypeptidechain backbone of rubredoxin.0 224 816 2478 4254 165 2151 1899 '' From K.9 44.1) of 15 A2 was assigned to all atoms in the structure and an R factor [Eq.
2 A. As the refinement proceeded the authors introduced additional water molecules to explain properly located positive peaks in their difference maps. The standard deviations of the interatomic distances for C.4. The value of these factors was restricted to the range 0. The refinement was then carried out using differenceFourier maps. The course of the refinement was also analyzed by computing the R factor for reflections grouped in ranges of i. detailed analysis of the maps indicated that the temperature factor used (12 A’) was not suitable for all atoms in the structure. With this addition. Even for this extremely small protein. only the protein molecule and the tightly bound water molecules are included in the calculation. This distribution reflects several of the intrinsic problems of calculating structure factors for protein crystals.372. giving an R factor of 0. In this refinement individual temperature factors were used.3.. The temperature factor was then adjusted in two steps t o a value of 12 A’.272 6.e. At this stage a leastsquares refinement minimizing w(h)AF2(h) (Section 6. occupancy factors proportional to the peak height were introduced for all atoms. Also.10. The final R factor of 0.O. The rest of the unit cell is filled with randomly . 14 that the reflections in the intermediate ranges have the best agreement between observed and calculated structure factor amplitudes. The apprehensions derived from the imposed limitations (i. while the atoms at the outside required larger values. Problems intrinsic to this low overdeterminancy were overcome.It is clear from Fig.1. low overdeterminancy and use of diagonal blocks) werc soon dissipated. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES was reasonably high for an initial model. The assignment was made depending roughly on the distance of the atoms to the center of the molecule. leaving only a few atoms in the structure with uncertain atomic parameters. Atoms at the inside of the molecule seemed to require lower B values. only two observations per parameter were available. The course of the refinement through this and the subsequent steps is shown in Table IV. First. The authors found no general tendency for the parameters to diverge or oscillate. The few cases where it did occur werc corrected using differenceFourier maps between cycles. and 0 atoms were found 10 be in the range 0.8) was initiated.12 (Table IV) is remarkably low. In order to restrict the number of parameters only seven B values ranging from 9 to 25 A’ were included.06 were found to have very large errors and were eliminated from the calculation. In most of these cases the atomic parameters stabilized with subsequent refinement. They partitioned the matrix in 1012 blocks involving 200 parameters each. The authors chose the simplest possible approach. The leastsquares refinement behaved surprisingly well. probably because of the high quality of the diffraction data. the refinement involves a 2000 x 2000 normal matrix. N. Six reflections with s I 0. Furthermore.
312 0.241 0. < 0.154 0.123 0.03 weighted 0.118 0.328 0. D. S. and L.250. H.242 0.261 0. C. S.31 0.392 0.112 218 212" 212 212 212 212 212 212 212 212 190b 190 190 0.289 0.31 0. Fo map From 2 A res.224 0. Sieker.239 0.346 0.301 0.O and occupancy 23 23 23 22 22 22 23 106 127 130 130 130 1 1 R 0.389 0. Course of Refinement for Rubredoxin" R and number of reflections as function of sin O / i Overall Coordinates From 2 A res.663 0. including 256 H atoms 4th L. 36.263 0. 6 reflections with sin Qji.179 0.139 0.10 0.373 0.5 12 12 920 730 640 640 .100. Watenpaugh. 359 (1971).389 0.550 350 2107 2107 286 1 1 1 1 1 100 0.394 0.188 0.397 0.262 0. Herriott.31 0.222 0.333 0.392 0.092 0.170 0. Jensen.141 0.385 0.192 0. Copyright 1971 Cold Spring Harbor Laboratory.372 0.271 0. L.461 0.389 0.1670.153 0.080 0.25 0. I67 0.183 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 Number of reflections 5033 5027 5027 5027 5027 5027 5027 5027 5027 5027 5005 5005 5005 B 15 13.05 weighted 0. Quant.390 0.31 0.131 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 0. J.260 0.092 0. S.31 0. 28 reflections with sin @/A < 0.225 0.TABLE IV. Cold Spring Harbor Symp.126 00. B i d .389 0.504 0.145 0. including 256 H Number of H.292 0. cycle 3rd L.31 ' From K.192 0. ' . cycle F.307 0.321 0. cycle 2nd L.132* 0.288 0. R.181 0. Fo map From 1st AFmap From 2nd AFmap From 3rd AF map From 4th A F map From 4th A F map 1st L.078 765 165 765 765 765 765 765 765 765 765 765 765 765 0.209 0.239 0.192 0.306 0.148 0.312 0.159 0. S. Fo map From 2 A res.376" 0.
3 FIG. This omission introduces errors in the reflections with low s (lowfrequency components. (=is) for different stages of refinement. large s). Chapter 6.214 6.2).3. represents leastsquares refinement and AF denotes difference Fourier refinement.) distributed solvent molecules that are usually ignored.14. On the individual curves L. .. determination of the rubredoxin structure represents a highly successful attempt to obtain a very accurate model of a protein molecule. (Reprinted from Watenpaugh er a/.S.30 R 0.1 sin @ / A 0 2 0.2c 0.IC 4 t h L S 130H20 2 5 6 H atoms 1 1 1 I 1 L 0 0. The number of water molecules included is also shown. Other factors affect mainly the highresolution reflections (i.40 0. Overall. R factor as a function of sin O/l.s4 with permission. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES 0. and to the use of isotropic temperature factors (Section 6. They are related to the approximations made during the leastsquares refinement which lead to false minima.e.1).
which we now discuss. Fibers are also classified by the ways in which the threads are packed side by side to form the specimen. several chains are wound around one another in the unit. where Bessel functions play a prominent role.5. and amino acid side chains in the case of fibrous proteins. These groups are bases in the case of nucleic acids. They are bundles of threadlike or rodlike objects. In any structure determination of this type side chains can only be visualized as average masses. Biological fibers can be divided into classes depending on the nature of the units that build up the fiber. Amsterdam.55They contain periodicities along one axis. These polymers normally have rather few atoms in their monomer and generally come with one of a variety of side groups attached to each monomer. FIBERS 275 In this case it is probable that the extra effort will pay off. as in most polynucleotides and fibrous proteins. For these reasons the theory of fiber diffraction is most conveniently expressed in cylindrical coordinates..1. rather than small monomers. Perhaps the most familiar unit is a polymer chain.” NorthHolland Publ. Frequently. as the knowledge of the precise geometry of the FeS cluster and of some of the interacting amino acids could become very important when analyzing the function of the protein. and are nonperiodic in the other two dimensions. The broad definition of fibers given above conceals a number of distinct types. rodlike object. Rodlike viruses such as the tobacco mosaic virus (TMV) and the tail of the bacterial virus T4 are important examples of this type of fiber. are polymerized to form the unit. arranged in an extended or helical conformation. . The packing schemes have great variety 5 5 B. The selfassembly of these units provides interesting systems for physicalchemical study. called the fiber or z axis. and also on the way the units pack together to form the fiber. 1966. also belong to this class.6. Monomermonomer interactions are usually noncovalent.Fibers 6. “Diffraction of XRays by Chain Molecules. whole globular proteins. Vainshtein.5. which form the basis of many contractile systems. K.5. In the other major class of fibers. The double helical structure of DNA is perhaps the most familiar example of this. The fibrous proteins actin and myosin. 6. The side chains normally occur in a random sequence and do not obey the symmetry of the unit. Definition and Description Fibers form the other major class of biological specimens studied by XRD.
" L. Crick. Appl. 273 (1978). Crystaiiogr. Vand. H. Nevertheless.2. Klug ~ et ~ aLS9It does not offer any tool as simple as diffraction " S. These bundles are then packed with random azimuths (rotations about z ) into the fibrous specimen. C. s9 A. Fiber Diffraction Theory et ~ Fiber diffraction theory was first presented in seminal papers by Cochran 1 and . 5. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES and complexity. 31 (1973). F. the unit rods have random azimuths and diffract independently of one another. Am. The diffraction pattern from an oriented gel is the azimuthal average of the pattern from one rod and displays a continuous distribution of intensity along layer lines. A variety of other specimen types exist. The canonical specimen in this group is TMV. as are specimens of some simple polymers.4. Such a specimen yields a diffraction pattern composed of discrete spots confined to layer lines normal to the fiber axis.qr. Arnott. Certain preparations of DNA are of this type. in which the averaged pattern of a unit rod is observed. have been used. finally. and V. a smearing of the pattern arising from lack of parallelism among threads. fiber diffraction photographs are usually characterized by arcing. H. W. Crystailogr. in which various stages of semicrystallinity are present. great experimental art is required for preparation of fibers suitable for xray diffraction study. 9. WyckoR. C. Makowski. In crystalline fibers unit rods pack side by side to form small.5. The effects of arcing become more serious as one looks farther from the center of the photograph until. 5 8 W. In the oriented gel. or in which disorder such as dislocations or irregular alternation of the sense of rods is present. Kluy. such as extrusion and quick stretching.581 (1952). layer lines may merge into one another. on the other hand. Acta Crystailogr. The problem is especially severe for oriented gels. The same pattern would be given by an individual bundle if it were rotated through 360" about z during photography. setting limits on the resolution of the useful data that can be collected. Cochran. The information contained in these patterns is much more confused than that in the diffraction by single crystals. truly crystalline bundles.. It is remarkable that enough information can be recovered from the rotationally averaged diffraction pattern of TMV to generate an image which reveals most of the atoms in the constituent globular protein monomer. J . and we mention only a few prototypical cases. such as polyethylene.~' It should be reemphasized that all fibers give rise to azimuthally averaged diffraction patterns. 11. 6. Acta Crystallo. Crick.276 6. .56 Frequently. which we discuss in detail in Section 6. 11.5. Assoc. 199 (1958). and a wide variety of techniques. F. H . Attempts to deconvolute the smearing from the diffraction photographs have had very promising initial appli~ation.
z ) expC2nirR cos($ .5. is the Bessel function of order n. Z)in exp(in$). Z.2) Using the identity expC2nirR cos($  4)] = c Jn(2nrR)inexp[in($ 'x m  4)]. so that F(R. From Eq.3) Note that F has merely been expanded in a Fourier series in the periodic angular variable. X Then XX Thus = R cos $.5. To emphasize this the triple integral is often denoted G.5. F(R.in+) exp(2nizZ)r dr d 4 dz. We define polar coordinates by x = r cos y. FIBERS 277 from a spatial plane wave in the crystallographic case. (6.7) F(X.1) = + y Y + ZZ = rR(cos 4 cos $ + sin 4 sin $) + zZ = rR cos($ .5. (6. Z (6. We begin by writing down the diffraction pattern of a general object in cylindrical coordinates. Z) = c G.6. Y R sin $.(R. $.5. z ) exp[2ni(xX + y Y + zZ)] dV.$)] exp(2nizZ)r dr d$ dz.4) + zZ.2. The Fourier coefficients are the triple integral which multiplies exp(in$). $. where J .in$) exp(2nizZ)r dr d 4 dz (6. (6. y = = r sin 4.5) is a type of FourierBessel transform. z. we find x exp( . (6. Z).5.4) The factor i" remains by convention. The relation x exp( .(R. Z ) = l//p(r. $. z = 4. but useful and conceptually simple special cases do exist. . k: Z ) = JJ/p(x.
. periodic function..5.5. and z . . This is a slight generalization of discrete Fourier coefficients for a onedimensional.(r)Jfl(2nrR)r dr (6. are complex. 1 (6. so that it can be expanded in a twodimensional Fourier series The gn. are slight generalizations of the usual Fourier coefficients and are given by It can be shown that Gn. It is useful and insightful to make an analogous expansion of the electron density function: p is now periodic in d. d z .11) This shows directly that if one can determine the individual G n .5.(R)J.(R)i" exp(in$). (6.. (6. the Fourier coefficients gn. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Thus far F is the diffraction pattern of a perfectly general object in cylindrical coordinates.6) G.. by extension of the previous notation..7) The coefficients G n . = (6. will play a central role in the description of the rotationally averaged pattern. or.(2nrR)R d R . [which .. however.5.in$)r d r dd. then F will be identically zero except on layer lines of spacing l/cthat is.10) and that g n . . except when Z = I/cwhere 1 is any integer. If p has period c along z .(R) = 271 j).5.278 6. m = 2x JornGn. of the electron density are directly available through Eq... Thus exp( .11). The period of the fiber can be readily introduced.
$.5.6) for the diffraction pattern of a periodic object we find x Jn(2nrR)exp( .1 2) where we assume that the helix passes through the point ( r o . Such an object can be formally represented as the product of two b functions (6. F R .14) ('"f") To integrate over 4 and z a change of variable is helpful: define Then 4 = n(u + u).5.  ( 1) c = n exp(in$)Jn(2nr. Inserting this function into Eq. Helicity in the fiber introduces certain simplifications into the diffraction pattern which permit one (at least in simple cases) to deduce the geometry of the helix.6.5.5.dzd4. (6.(u  0). $. First we analyze the diffraction pattern from a wire bent in the shape of a helix of radius r o and repeat (pitch) c.5. giving F R. Substituting I? = ).O).i m ( u + v)] exp[xil(u  u)] du du. We approach diffraction by helical objects in stages. 0.15) . and make possible the generation of an image from a rotationally averaged diffraction pattern. FIBERS 279 It is almost universal that the rodlike elements in a biological fiber are themselves wound into a helix of one sort or another. (6.  ( '1 = nc x c Jn(2nr0R) exp(inrl/)i' n Io2 jo*6(u) exp[ . R)i" x Jozn J)(& .in$) exp ('Y'")r __ dr d 4 dz. (6.5) exp(in4) exp .5.13) The integral over r can be done by inspection. (6.
and a similar set of line segments FIG. This pattern is greatly simplified compared with the pattern from a general fiber. 16.l/c) = (2n/c)i'J. 15. only a single Bessel function occurs on any layer line. with permission. This is illustrated in Fig.17) I. F R . (6. although not its exact equation.n)] du dv. The factor 271c is normally omitted.R) exp(inI))i" Jo Jo6(u) exp[ and integrating over v. Because of the form of the Bessel functions J 1 this gives the diffraction pattern an x shaped appearance.280 6.16) J. move farther out from the origin as n increases in magnitude. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES simplifying. (Reproduced from Vainshteinss. and the first maxima of the J .) .5.18) F(R.5. one arrives at im(Z + n)] exp[inu(l . produce fringes outside the main x .5. Figure 16b shows that a helix can be roughly expressed as the sum of a set of parallel line segments forming one face of the helix. with the order of the Bessel function being equal to the layer line index 1. can be deduced by a very simple argument from the general shape of the helix. I). when (6. First.(Znr.(2nroR) exp(in$)i" This integral vanishes unless n = exp[inu(l . (6. Bessel functions as a function of order (n) and argument (u). 15.n)] du. I). shown in this figure. is at the origin. As shown in Fig. the first maximum of J . Subsidiary maxima of the J . ~ ( I) = nc n J. The x shaped form of the pattern.(2nro R ) exp(il$). Thus the main bars of the x are formed by the first maximum ridge of the J .
2niz/c). A simple choice for the helical case is suggested by the expansion for p(r. FIBERS 28 1 (a I (b) FIG. We choose p ( r . The two rows of points comprise the two arms of the x . We will shortly generalize this for a helix made of atoms.(2nrR)R dR.) forming the back.5. z/c) given in Eq. and which also had a very simple diffraction pattern. A uniform helical wire is not a very useful model for most systems. Diffraction by helices: (a) values ofthe Bessel function J. Substituting which represents one term in the expansion. we find that . = (6.5.10). . in Eq. with g. G 1 . one Bragg reflection.5. for G.16. This parallels the case of crystals. Note the ‘’ x ” drawn through the first maxima of the Js.. (b) the x shaped diffraction pattern is related to structural features in the helix. (6.. the helix is approximated by the sum of two stacks of sloping line segments. 4. (Reprinted from Vainshtein”. forming the arms of the x . (6. We discuss here a generalization of the helical wire whose diffraction pattern is particularly simple.19) J .8). z/c) = Jl(2nrR’)exp(id . The existence of a strong reflection therefore implies the existence of a correspondingly strong Fourier component in the object. by computing the diffraction pattern of a set of beads (atoms) spaced uniformly along a helix. with permission. Each set of lines gives rise to a diffraction pattern of a row of points normal to the stack of line segments.6.5. sinusoidal density fluctuation we could produce an object that could be used to synthesize any specimen. (6.5. In projection.. 4. ( R ) = ~~~J1(2nrR’)J. Dill’raction from these stacks occurs along directions S perpendicular to the stacks. so that crude ideas about important periodicities can be quickly obtained.. where we showed that by generalizing the usual stack of Bragg planes to a spatial.20) . that determine the intensity of the Ith layer line in the diffraction pattern of a continuous helix.
Therefore in this special case the rotationally averaged diffraction pattern from a specimen is identical to the unaveraged one. In DNA. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Using the orthogonality conditions for Bessel functions we realize that this integral vanishes unless R' = R . and is often termed the rise per residue.282 6. all the 3' oxygens another. Welberry. $. Press. F(R. Only the phase factor exp(i@)varies. A. .. Harburn. Thus (6. 17 and 18. vanish. For the case of a helical wire the pitch P was also the true repeat. and value zero elsewhere. "An Atlas of Optical Transforms. This model can be readily generated from the continuous wire model by multiplying it by a function that has value one on a stack of equally spaced planes normal to the helix axis.21) while all other G." Cornell Univ.. Note also that the intensity of the diffraction pattern.2 This model (the discrete helix) is of great general utility. New York. for example. R . Taylor. by convolving the Fourier transform of the stack of planes with the Fourier transform (diffraction pattern) of the continuous helix. This implies that the whole diffraction pattern is . and so on. l/c) = 6 ( R . This is consistent with the idea that. ~ ' To express this result formally some relation between p and P must be assumed. The diffraction pattern of this structure can be generated by use of the convolution theorem. We now proceed to the example of point masses strung on a massless helical wire. but their radii and azimuths will vary. 1975.5. These helices will all have the same pitch. all the phosphates on one strand would form one such helix. one view of a helix can be used to generate all others. The spacing p between planes becomes the z distance between atoms. because of helical symmetry. For other examples see Harburn et ~ 1 .22) which is a ring with periodically varying phase on the first layer.5. vcry strong reflections on particular layer lines can be related to the existence (in the object) of helical waves or structures of the form chosen in this example. since any helical specimen can be represented as a sum of such helices. Using this result. the diffraction pattern from any view of the object is sufficient to reconstruct an image.')in exp(i@). This is illustrated in Figs. This convolution amounts to reproducing the x diagram at each meridional point. "" G . Since the phase factor is known u priori if a helix is assumed. It is readily shown that the transform of the stack of planes is a set of points spaced l/p apart along the meridian.. is independent of azimuth in these two cases. FF*. and T. C. Ithaca. (6.
This illustrates the convolution of the x pattern with rows of points in reciprocal space.17.FIG. (b) system of planes of repeat distance C*. being the transform of a system of planes in real space. DiKraction pattern. (c) convolution of the previous two systems.) FIG. (Reproduced from V a i n ~ h t c i n ~ with ~ . . permission. 18. over the layer planes. The example drawn is for seven residues in two turns. which is a system of planes of repeat distance C* defining the position of the layer lines.. (d) distribution of the first peaks i n J. Formulatlon of thc diffraction pattern of a discontinuous helix: (a) system of points with a repeat distance C'*. from a continuous helix and from helices having closely or broadly spaced atoms.
and the phase shifts involving 4 j and z j reflect the positions of the atoms along their individual helices.in$j ( + 2xilzj C ).5.24).5. It is often convenient to separate the angular and spatial variables in this equation.26) where the atomic scattering factor4 has been introduced to give shape to our points. we can directly write the diffraction pattern as F R. only one or a few terms contribute significantly to F. Given this analysis. Thus helical structures in general have diffraction patterns much simplified compared with a general fiber.5. exist on all layer lines. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In a discrete helix.27) then (6. are forbidden by the selection rule. We can then write h i " J. at Z = l/conly certain J . and amounts to the structure factor equation for a helical object instead of a crystal.z j ) is given below.24) where rn is in principle any integer. On layer line lthat is.5) C ) (6. The helix in this example was initialized by assuming one atom at the point r = r o .284 6. z = 0.25) where the sum is over those values of y1 consistent with the selection rule (6. there need not be a whole number of atoms per turn. 4 = 0. those which fall on this layer line through the convolution process.5.(2xr0 R) exp(in$).5. for which all J . If we write'by analogy with (6.(2xrj R ) exp in$ . The full formula for a family of helices with atoms at ( r j .23) where t and u are integers. in practice only small values of rn generate an n for which J .  ( 1) = 1 i"J.5. We therefore write the true repeat c c = tP = up. This expression serves to set the relation between the interleaved x s in the diffraction pattern. n (6.5.4 j .5. is nonzero within the resolution of the photograph.28) where again the sum is taken only over those values of n that satisfy the selection rule. however. (6. The allowed values of n are then given by 1 = tn + urn. (6. . because many of the J . (6. In general. $. can appear.
) . Note that point B does not give rise to any point on the tilm. Therefore d$. The observed intensity I is I = (FF*) dl . b'. b'. (6. Reprinted by permission of John Wiley & Sons. and c) give a'.. (6.5. b. one must know the phase and amplitude of i n meridian xrays 4 . Lines drawn through the intersection of the disks with the Ewald sphere from the center 0 of the sphere represent the scattered ray directions.5.= ( 91 Gn. FIBERS 285 As mentioned before. The points of intersection of the disk C with the sphere (a.30) If one wishes to reconstruct an object directly from the diffraction pattern. and c' form a layer line. respectively. / equator .' Copyright @ 1966.6.(W. and c'.in$)( 2)") n. John Wiley & Sons.19a. Fiber diffraction geometry: the shade' qks represent the Fourier transform of a fiber..27c (1 I' G. Inc. on the film./(W2... (From Holmes and Blow.5. The points a'.29) The integral vanishes unless rn = I R./ exp(in$)i")(x G$ exp( . rather than to guess a model. . . / (a) FIG. Inc. the observed diffraction pattern is rotationally averaged.
5. Inc..286 6. diffraction from fibers is confined to a set of planes in reciprocal space. John Wiley& Sons. complex F(R. Since the data are rotationally averaged. (6. $. this process has actually been realized in the case of TMV (Section 6. From these one can calculate the full. one can use Eq. the problem of recovering the Gs from the Is is sometimes referred to as separating the Bessel functions. Note that the sphere intersects each reciprocal space . Figure 19 shows the Ewald sphere construction for a fiber. and use these to generate p from Eq.. Same as (a) but the fiber axis has been tilted so a& to bring the point B into the reflecting position.28) and then synthesize the object by Fourier transformation of F./.5. l/c) via Eq. Since each G . . Remarkably enough..* Copyright @ 1966.5. Inc. As we have seen. Reprinted by pcrmission of John Wilcy & Sons.11) to generate the g.5. Now that poini B projects onto the meridian at B’ and that thc layer line is no longerstraight. there is no variation in intensity as one moves azimuthally in any plane. Alternatively.8).1%. (From Holmesand Blow. (6. Thus all the available data are contained in radial scans running from the meridian to the limits of resolution in each of these planes. A word about how the intensity data are measured is appropriate here.is generated from only one J.4). XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES (b) FIG.. .) each G. (6..
.3.6.5. is often modeled as an ellipsoid or a cylinder. The required photographs are stillsneither the fiber nor the film moves. a single photograph provides a complete data set. only a very small number of the first models may be possible. D. in a sense. J. while constraining the lengths of bonds and the angles between them to canonical values. Later. A AM. In the case of polymers whose elements are fairly simple monomer units. Sect. This was accomplished by using rotations about single bonds as the independent variables. Problems can be simple because the fiber structure itself is simple (e. Joint use of electron microscopy is very common for these specimens. Techniques for modeling various types of disorder have also been incorporated into the analysis. so that lowresolution images upon which a model can be based are frequently available. They can also be simple because the diffraction patterns are poor and contain so little information that only a crude structure can be determined. Watson’s description in “The Double Helix”6’ of the thought processes leading to the model of DNA is a readable example of this method. Early on. when the specimen is a polymer having only a small number of atoms in the monomeric unit). “The Double Helix. 61 62 J. FIBERS 287 plane along an arc which provides almost all of the desired radial scan. P. it was necessary to reduce the number of parameters in the model as much as possible. Only a region near the meridian is missingthe blind region.4 and examine here the simpler problems that are more characteristic of the field. If data from the blind region can be ignored. To deal with the relatively small number of data. Arnott.5. and. modeling techniques have also provided images of specimens in which the repeating unit is a whole protein. The geometry and chemistry of the monomer often provide severe constraints on the model. such models were adjusted by trial and error to give improved agreement with the observed data. which in any case. Watson.5. A c f a Crystallogr. atomic models are possible. The second panel shows how tilting the fiber can help fill in the missing data. Solution of Simple Fiber Structures We defer discussion of TMV to Section 6. New York.” Atheneum.. Arnott and and others6’ made use of the structure factor formula as an observational equation for leastsquares refinement. 6.g. the “linkedatom least squares” (LALS) method. This. has been successful in providing accurate atomic models for a variety of polynucleotides and polysaccharides. In these cases no internal detail is seen in the subunit. 1968. CampbellSmith and S. On a much larger scale.3 (1978). similar to the process described earlier for crystals.. Polypeptides and polynucleotides may fall into this category.
and is also the first system in which native proteinribonucleic For this reason we present a acid interactions have been seen in short review of this project. and was only made certain "'G . Warren.65 Because the helix is so shallow. A ~ PProtein . 64 A. 216 (1977).4. or whether it only appeared to be so because of arcing. the opening angle of the x in the diffraction pattern is very small. and K. Three nucleotides are bound per protein subunit. D. long and 90 A in radius. 37 (1963). Stubbs. and it was experimentally difficult to decide whether a reflection was actually meridional. 7. so that the rise per subunit is only 23 A x & = 1. L. 20. 18. Caspar. as its structure was understood in the early 1 9 6 0 ~is . Thus the value of 49 residues in 3 turns was in dispute for some time. The RNA follows the same helical path and is positioned at the edge of the central hole. and the R N A wound in the lumen of the central. cylindrical cavity. D.~') 6.4A. S . Virus Res.225 (1960). shown ~~ in Fig.288 6. Adu. Tobacco Mosaic Virus Structure As mentioned earlier. Nuture (London)267. Caspar. some 3000 8. The pitch of the helix is 23 A. A drawing of the virus. D.5. Chew. Klugand D. L. It represents by far the most complex fiber analyzed in this way. . with a central hole of radius 20 A. Drawing of tobacco mosaic virus. Note the Dutchshoeshaped protein subunits on the outside. Identical protein subunits build a helix with 49 subunits in 3 turns. TMV is a rodlike virus. Holmes. an atomic model of TMV has been obtained from fiber diffraction studies.20. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES FIG. (From Ca~par.
An example of such a pattern is shown in Fig. The effects of interparticle interference are very small.6. E. since every third layer line has intensity near the meridian. The selection rule is I = 3n + 49m. Acfa Crjstallogr. 21. on the hh R. (Courtesy of Gerald Stubbs. . 213 (1958). C. Holmes. It is immediately clear tl. so that the pattern is essentially the rotational average of the diffraction intensity from a single rod. When I = 0. Fr.inklin and K . FIBERS 289 FIG. consisting of the loworder Bessel functions from the central x in the diffraction pattern.at the repeat is 3 turns.21. 11. All the native data nccded to reconstruct the fiber are contained In this photograph.5. The rods form an oriented gel which gives diffraction patterns of very high quality. Photograph of the dill'raction pattern of a fibcr of tobacco mosaic virus containing data to 3w resolution.) by Franklin and Home@ with the use of a heavyatom derivative.
.5.28)] or p [Eq. Barrett.. . This heavyatom derivative will give rise to a diffraction pattern built from the Gb.290 6. W .5.35) 6 7 A . J. /= G"..33) Then I' + B: + A: + B: + . which is only a small fraction of its peak value at this point. Let .5.27) that while the observed intensity is 1 = (FF*). However. The great wealth of detail in this photograph is readily apparent. the zerothorder Bessel function appears. .28) and (6. and the next is J .+ h . Qinmt.8)]. + a 1 ) 2 + (B. (6. = 1G " . . + u2). and P. C. C. . (6. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES equator. 1 = A: (6.1. Mandelkow. = ( A . . + .5.32) (6. . We recall that from Eqs. Holmes. . to a specific site in TMV. Barrington Leigh. + (B.which are simply related to the native G. G .. N . n It is sufficient to find the values of the individual G . which becomes nonzero at about 10A resolution. (6.. and write as well G =A + iB. . R . 36. since these lead directly to either F [Eq. . + b 2 ) . only the sum of G 2 is observed. (6. and mixes hardly at all with J.." where a generalization of the isomorphous replacement method was devised to deal with this problem. Leberman. von Sengbush. Cold Spring Horhor Svnip.5. y = u + ib. where m runs only over all heavy atoms. The final stages of this project were carried out in the laboratory of K. .5. E.5. which we can determine. . as in a protein crystal. Holmes in Heidelberg. . K./+ Yn.433 (197 I). ) 2 + ( A .34) (6.. and generated the original hope that the atomic structure of TMV might be revealed by it. B i d .. We pass over for the moment how this might be accomplished.. Imagine now that a heavy atom is bound. .5.
Nuture (London) 254. A A31. Sect. The coat protein subunits ’’ G.5. Bessel functions occurring on the same layer line differ in order by integer multiples of 49.34) is therefore the most accurate of the set.~’ Consider. In the actual TMV case seven heavyatom derivatives having five independent sites were used. so that the as and bs are subject to considerable error. it must be emphasized that Eqs.5. They must be solved at a large set of finely spaced sample points to provide the desired information. the difficulties in assigning accurate parameters to the heavy atoms are even greater in fiber diffraction than in protein crystallography.35) are for the I s and Gs at one point in the diffraction pattern.34) has only six terms in itthree A s and three Bs.5. This means that Eq. within a given radius of reciprocal space. the others will all be very small. Second. Thus. since they can be calculated from the known heavyatom positions. Acta Crystallogr. and proved very powerful in eliminating false solutions at certain points. The 4Aresolution structure of TMV published in 197769makes clear the fundamental soundness of the method. A minimum of six heavyatom derivatives. and U. First. two convenient circumstances make this possible. only a limited number of Bessel functions can contribute to the diffraction pattern. FIBERS 29 1 An additional equation like that for I’ arises for each heavyatom derivative. J . say 1/(4 A). 709 (1975). J.5. the additional constraint inherent in the smooth variation of the individual Gs was taken into account in a global way. G.34) and (6. for example. The determination of heavyatom positions was fraught with diffic~lty. Diamond.6. Holmes. (6. Given the radius of the virus rod. 192 . The proof of all these elaborate methods lies in their ability to produce an interpretable and plausible electron density map. E. that the azimuthal angle 4 for a single heavy atom cannot be determined from a rotationally averaged diffraction pattern. Equation (6. one can readily show that out to 4A resolution at most three Bessel functions contribute at any point along a layer line. it is characteristic of Bessel functions (Fig. Only when cross derivatives involving two different heavy atoms are examined can a A 4 between the heavy atoms be calculated. and required great insight and care. The polynucleotide chain with its electrondense phosphorus atoms is the most prominent feature in the maps. ‘’K. so that very few loworder J s can appear. Solution of these equations is not trivial.68 First. What remains is to calculate the As and Bs. C . Stubbs and R. (6. (1975). In the technique actually used the heavyatom equations are solved using (6. Stubbs. because of the selection rule for the TMV helix.5. All the as and bs are assumed to be known. Mandelkow.5. one for each term. is required to solve these equations.35) as a constraint. Further.. Further. Gallwitz. 15) that they remain zero until their argument is about equal to their order.
(Reproduced from Stubbs Pt al. Fortunately. R a n d 2 represent the radial and fiber axis directions in the structure. and the R N A model derived from this density is superimposed. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES 1 0' I 20 I I 40 I I 60 80 I WR FIG.5 8. has been FIG. radius of the virus.63with permission. The region shown contains RNA. 22. and in this area chain tracing was not possible. and these made possible the tracing of the protein chain through most of the subunit. For technical reasons the effective resolution of the map drops to about 5. Perspective drawing of sections of contoured electron density function of TMV. this portion of the molecule can be visualized in a closely related structure. An intermediate in the assembly of TMV. termed the TMV disk.) . Schematic drawing of two adjacent TMV coat protein subunits.292 6.23.) contain extensive stretches of ahelix. The cylinders represent helical segments. near the outer 808. The interaction of the RNA with the protein is also shown. (Reproduced from Stubbs P I dh3 with permission.
J. eds. Cohen. Microtubules are built from two closely related monomers. Klug. 24. Diffraction takes place where there are large fluctuations in electron density. New York. ~The ’ outer portion of the subunits are clearly seen in this structure. aand Ptubulin. For microtubules this occurs at the inner and outer surface of the hollow rod. Additional refinement is now being carried out at 3. but are much less rich than the pattern from TMV. Bricogne. Pror.. Microtubules have been studied by electron microscopy in the laboratories of K l ~ and g ~ ~ E r i ~ k s o n . Natl. Thomas. In many 7 0 J.A. 60.5. and A. ” provides an interesting example of the amount of information that can be gained from a lowresolution study. 22 and 23. Looking at Fig. each staggered axially with respect to its neighbor by about 9 A. 1976. Cell Biol. J.5. Cold Spring Harbor. Butler. Acad. For the first time considerable detail of proteinnucleic acid interaction can be made out. G . A schematic of this arrangement is shown in Fig.” Cold Spring Harbor Lab. U. which readily form an up heterodimer.4 resolution (G. N. personal communication).5. A section of the RNA binding site of TMV and a schematic drawing of the structure are shown in Figs. It also illustrates very useful interplay between electron microscopy and xray diffraction. J. 24 one can see that there are many different helices which might be drawn to connect the tubulin monomers.74.3370 (1977). E. We now consider in a crude and simple way how a microtubule diffracts x rays. and an improved version of the TMV model is expected shortly.S. 6. and other motile properties of the cell. 153 (1974). Rosenbaum. P. G. “Cell Motility. A. Bloomer. 523 (1974). The diffraction patterns they produce are also the rotational average of the onerod pattern. In the virus the R N A chain follows a complex path ranging from 35 to 50 A from the virus axis. Klug. Goldman. An xray diffraction study of brain microtubules by Mandelkow et a f . Cell Sci. P. ’* . ” R. Mandelkow. Sci.6. FIBERS 293 crystallized and analyzed in the laboratory of Aaron K l ~ g . The spacing between tubulin molecules within a protofilament is about 40 A. 7 3 L. 14. The dimers readily polymerize into microtubules with increasing temperature. Pollard. 74 H. ’These ~ groups showed that the exterior of the tubules was characterized by an array of 13 socalled protofilaments running parallel to the microtubule axis. Amos and A L. J . and C. T. J . C. and J. Erickson. Stubbs. transport of materials from one part of the cell to another.20 ( 1976). Book C : Microtubules and Related Proteins.71Like TMV they are hollow rods. and these all wrap around one segment of helix (termed the left radial) like a roll enclosing a hot dog. There are three bases associated with each protein subunit.. Nature (London) 259. Champness. Structure of Microtubules Microtubules are cellular organelles involved with cell division. A .
FIG.) 294 . (Reproduced from Mandelkow Z I r d 7 ’ with permission. Each lattice point is represented by a black disk.24. The vcrtical protofilaments ( I 3start helices) and the 3start helices are drawn in. Surface lattice of the microbule.
~ ~ permission. (a) View along tubule axis. 25. Note that the highrelief helices are differenton theouterand inncrsurfaces.FIG. (b) view from outside of tubule. (c) vicw froin insidc of tubule. Slyrofoam modcl of reconstruction of microtubule. (Reproduced from M a n d e l k o w ~ f n lwith .) 295 .
27) for the G. the major feature is the tenstart helix: the protofilament separation and subdivision are very weak.22).. . since each problem in fiber diffraction is unique. However.5. Rigorous demonstration of this would require a knowledge of the phase of the relevant reflections.” so that the slanted family of lines in Fig. so that the same families of helices can be drawn. Next. the tenstart helices on the inside and outside are not in register. (with the same order) for the outer tubule radius.5. In practice some of the helical lines connecting subunits will be in higher relief than othersor the spaces between them will be more deeply etched. which are themselves clearly divided into subunits with a strong modulation from a tenstart helix (Fig.296 6. Conclusion With these two examples we have tried to illustrate some of the scope and limitations of the fiber diffraction method. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES cases several helical lines have to be drawn if all the subunits are to be included. Eq. Because there is very little density fluctuation within the rod wall. (6. It is these particular helices that will contribute most to the diffraction pattern. (6. For lack of a direct way to phase the reflections. strong reflections on the first layer line were examined. 6.. 25) running at about 45” to the tubule axis. Also. Clearly. using the ideas that went into the derivation of Eq. additional progress on this structure can only come with the use of heavyatom derivatives. Reflections arising from the inner and outer walls of the microtubule were identified by their distance from the meridian. It was found that on the outside (Fig. The inside and the outside of the microtubule. it is not necessary that the same families be prominent both on the inner and outer surfaces. most problems are solved by resorting to specialcase reasoning. Further. or other arcane methods. respectively. have the same arrangement of subunits. of course. It is worth emphasizing that this analysis is using as a model the familiar helical wireonly here the wire is a ridge on the surface of the rod. each new problem illuminates and extends some part of helical diffraction theory. Such bimodal G. are indeed observed.5. A reflection corresponding to the spacing between the 13 protofilaments was also observed. for the inner tubule radius and a J . Inside. The investigators concluded that the inner and outer radii of the tubule were 70 and 150 A.. 24) the predominant feature was the separation of protofilaments.. at least according to this preliminary model. The analysis began by examining the equator. 24 is a threestart helix.lreduces approximately to two terms: a J .6. In some sense this is a hopeless task. Each line is said to be a “start. electron microscopy. which contains information about the projection of the tubule down its axis. Extremely clever deductions from the diffraction patterns are a .
In addition. grant GM25432 (to L. and N.Furthermore.I. Mario Amzel). Except for quite small structures. when very indirect reasoning is used. however. A few generalizations do hold.5. research career development award AI00271 and N. Lattman). Acknowledgments This work was supported by N. atomic information is difficult to obtain.I. .6.H. FIBERS 297 common feature of the literature. research grant AI14820 (to Eaton E. We find this area of XRD continually entertaining and intriguing. it is actually possible to get the wrong answer in some cases.I.H.H. lack of good specimens is a limiting factor in many cases.
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20 Copyright 0 1982 by Acddeniic Press. The intensity of the scattered light falling upon a small area varies accordingly.7. Not only do lasers provide intense. to familiarize the reader with various classes of applications. such as measurement of the diffusion coefficients of macromolecules. assaying for the swimming speeds of motile microorganisms. Our purpose is not to provide an exhaustive review of biomedical uses of laser light scattering but. VOL. LASER LIGHT SCATTERING By Ralph Nossal 7. Recently. Emphasis primarily is given to fluctuation spectroscopy. some excellent review papers have appeared in which particular biological 299 METHODS OF IXPERIMENTAL PHYSICS. Spatial variations in refractive index result in a diffraction pattern which fluctuates with changes in the structure of the scattering medium. 1). In Chapter 7. The changes which are measured then can be correlated with molecular reorganizations of the sample. rather. Chapter 7. detection of changes in blood flow. Inc All rights 01 reproduLlion in m y form rcsxvrd ISBN 0124759629 . In so doing we hope to communicate some of the spirit of the physical modeling required to interpret measurements. determination of the electrophoretic mobilities of biological cells. Introduction Light is scattered when it passes through an optically heterogeneous material.1.2 we outline the basic theory by which laser inelastic light scattering phenomena are understood. at a rate which depends upon the kinematics of the refractive index changes occurring within the sample (see Fig.3 contains a discussion of typical instrumentation and certain common experimental procedures. but we also discuss some kindred aspects of classical light scattering techniques pertaining to the intensity of the scattered signal. and measurement of the elastic moduli of soft biological materials. The remaining chapters are concerned with specific topics. but the coherence properties of their emissions allow one to relate the diffraction pattern detected at one time to that detected at a different time. essentially monochromatic sources. The availability of lasers has facilitated measurement and analysis of such fluctuating refractive index distributions.
Bioclzem.421 (1981).eds. Schurr. 133 (1970). Pecora. 10. B. Jr.” “Depolarized” components of scattered lighti. t ). The proportionality in general is described by a tensor polarizability a(r.243 (1975). C. A. 48F. we assume the scattering medium to be optically isotropic. Chu. V. Pike. however. Bloomfield. Chen. New York. B. The intensity of scattered light is a strong function of the scattering angle 8. 1977. Theory When a substance is illuminated by an electromagnetic field. and R. Biophys. Ford. Academic Press. 1981. for weak illumination. Pike. “Scattering Techniques Applied to Supramolecular and Nonequilibrium Systems. is proportional to the incident field Eo(r. Biology. 9b S. Cummins and H .e. Bioeng. ~ ’‘ ’ 7. C R C Cri/.” Academic Press. Chu.Laser Light Scattering. 371 (1977). 193 (1972). Op/. . 4. B. and Physics. “Photon Correlation Spectroscopy and Velocimetry. Z. with a field strength which is proportional to d2P/dt2. which. R. 1976. New York. those whose polarization is perpendicular to the incident polarizationarise when the offdiagonal elements of the polarizability tensor are different from zero. in “Chemical and Biological Applications of Lasers” (C. K. R..300 7. Chaptcr 5. Cummins and E. A good bibliography is given in S ~ h u r r . The latter is defined to lie between the axis of the laser beam and a line drawn ’ H . B. H. Berne and R. ihid. Lim. J.2. several books7. eds. “Photon Correlation and Light SeatingSpcctroscopy. the electrons and nuclei within the material move oppositely to each other and produce an oscillating dipole field. J. ed. Reu. N. Z. Ware. C h ~ mScr. D. R.8. eds.. Rei.” Plenum. t). ’ B.). 1974. Carlson. In this case the plane of polarization of the scattered light is parallel to that of the incident field. Vol. Prog. H. H. LASER LIGHT SCATTERING applications are described.. Swinney.. if the scattering medium is optically isotropic the offdiagonal dements of a are zero valued and the diagonal terms are equal. New York. New York. 2. 1974. Annu. Bloomfield and T. F. Nossdl. Depolarized scattering is generally very much lower in intensity than polarized scattering. Z. New York. ‘ ’ . Unless otherwise stated..” Plenum. This field is characterized by the macroscopic polarizability P(r.” Wiley (Interscience). Cummins and E.. A n oscillating dipole is itself a source of electromagnetic radiation. Moore. ‘. 2. M . New York. and we say that the scattering is “polarized. 1977. MethodsEnzymol. t ) . “Dynamic Light Scattering with Application to Chemistry. 4. and thus has not been of much concern in biological applications of quasielastic light scattering. and conference proceeding^''^ contain extensive discussions of some of the topics that follow. V. 415 (1978). L. In addition.” Plenum.
A.0. 40.k * r)]. which are induced by the incident field. In idealizations of the scattering process the incident electromagnetic wave usually is taken to be a plane wave Eo(r. for which sin cp = cos 0. 1. ZEEE53. but it is assumed that the detector is positioned sufficiently far from the sample that the resultant wave itself is spherical. B. The inset shows the scattering from an individual dipole: I. the factor sin cp is I for any scattering angle 8. Benedek. and the output is vertically polarized. Benedek and T. and G . 1164 (1967).g. and the scattered wave is taken to be spherical." Chapter 5. mode. there is an additional field component which induces dipoles oriented in the horizontal plane. the intensity of which fluctuates when thc scattering centers move. Proc.2. I n this case the angles cp and 0 are related as cp = $7 . Wiley. is the locus of directions of equal scattering intensity. Lunacek. Dubin. 1). S. T a n f ~ r d ' ~ Referring ).i(u. 1. B. 57. B. Sci. Acud. 1604 (1964). from the phototube surface to the sample through a set of collimating pinholes in the collection optics (Fig. Nu/[. 1961... Phys. Usually. e. Dipoles are induced in the vertical plane. Proc. Tanford. the laser is operated in the TEM. If an unpolarized source is used. exp[ . J. t ) E. G. reradiate electromagnetic waves which interfere with emissions from other scattering centers. New York. Greytak. THEORY 30 1 Frc. U.. t . The phototube views a small part of the resulting diffraction pattern. 1623 (1966). lo I' . The derivation which follows is similar to several which have appeared in the various essential features of which first were put forth by Lord Rayleigh (see. to Fig.7. J . l 3 C. "Physical Chemistry of Macromolecules. Dipoles. and if the detector is in the horizontal plane. we see that the detected electromagnetic  R . C k m . Schematic o f a typical light scal(ering experiment. H. (Each dipole in the sample reradiates energy as a spherical wave.) Within these limitations we now derive an expression to relate the time autocorrelation of scattered light to the spacetime correlation of density fluctuations occurring within a sample. Pecora. which is proportional to sin cp.S.
t ) is the local electric field within the sample. t ) can be written as P(r3t ) = a(r. According to classical electromagnetic theory. Kerker. P(r. (In Chapters 7. t) is the polarization at point r. we restrict ourselves here to the RayleighGansDebye theory.r l/c is the “retarded time.) A simple expression relating the scattered field to the structure of the scattering medium can be obtained when the distance R from the sample to the detector is much greater than any of the dimensions of the scattering volume (the “ farfield approximation). 377. and t’ = t . Lifshitz.” Academic Press.1) where cp(r) is the angle between the axis of the induced dipole moment at position r and a line joining that point to the point of observation. c is the speed of light. t ) is the polarizability and E(r.7 we discuss scattering from microorganisms and other large particles. C. New York. 1960. the determination of E(r. “The Scattering OF Light and Other Electromagnetic Radiation. 1957. AddisonWesley.2. and indicate modifications which are necessary in such cases. V is the volume of the effective illuminated region of the sample. Massachusetts. t ) may become quite complicated because of multiple scattering or absorption.” where n is the index of refraction of the scattering medium (c/n is the propagation speed of the emitted wave).2) where the scalar a(r. Van dc Hulst. LASER LIGHT SCATTERING field is the superposition of the fields radiated by each of the oscillating dipoles that have been excited within a sample by the incident electromagnetic field. If the sample is optically dense.14 this quantity is given as (7. 1969. ‘I’ M.’ 3. New York. P(r. . For a planewave incident field ’*“ ” I J L.2. “Electrodynamics of Continuous Media.n 1 R . and I R 1 is the distance from the detector to the origin of the sample. “Light Scattering by Small Particles.302 7.” Wiley.” p.5 and 7. t). t)E(r. In other words. Reading.1 which holds for scatterers which are small compared with the wavelength of light or whose refractive index is close to that of the surrounding solvent. Landau and E. l 5 H. M. D. (7. Because we here restrict ourselves to optically isotropic media. Here we assume the relatively simple case that the sample is composed of a dilute collection of nonabsorbing particles which are small enough that an electromagnetic wave traveling through the medium does so without significant reduction in intensity or retardation of phase.
For a suspension of particles. ordinary transmission. (r. where the wave vector of the scattered beam k' is directed along the line of observation but has the same magnitude as that of the incident beam. (7.(R.2.3) thus indicates that the scattered electromagnetic field can be used as a probe of the Fourier components of the spatial variations of the "excess polarizability " E(r.7.2.r).rl FZ k . then the integral in Eq. (Note that 8 lies in the plane defined by the incident and scattered beamssee Fig.2.2. (7. (7. (7. of course. i. (7. t 1 e i Q .2.e. We also noted that the temporal change of the polarizability is slow in comparison with the oscillation of the electromagnetic field.r E. THEORY 303 E = Eo exp[i(k .2).3) To derive the latter we substituted (won/c)lR . V%A3 where S(Q) is the Dirac 6 function. The quantity Q = k . t ) FZ . that R ):f x Eo sin cp exp( . t).oo t ) ] we find.2.) If the sample is optically homogeneous so that a(r. exp[i(k' R .i o . 1.3) becomes 1 a(r. f) as the difference .1) and (7. r d3r 2 1 M(~>S(Q>.2. the polarizability can be expressed as a(r. ao(t) might be taken as the polarizability of the solvent and C(r. where A is the wavelength of the incident radiation.k'I = 21klsin)O = (4nn/A)sin)6'. I k' I z I k I = won/c = 2nn/A. From geometricarguments onecan show that themagnitude ofthe Braggscattering vector is given as IQI = Ik . t ) = ao(t) + i(r.k') r] d3r  (7. t )expii[k * r ~ c o o t 'y  '711 d3r N R x J. so terms of O(d2ct/dt2) could be neglected.5) and Eq. t ) = a(t).coot)]  t ) exp[i(k . ( R .2.t ) I V a(r..4) where 6' is the scattering angle. In general.k' is known as the Bragg scattering vector. In this particular case scattering occurs only in the forward direction. from Eqs. t).
304 7.2.7) that the particles will be invisible if their index of refraction and that of the solvent are identical. (7.9) where N is the number of particles in the scattering volume. Early light scattering techniques involved measurement of the time average of the intensity I = I E t E .6) where Qi is the spatial extent (volume) of the ith scatterer. To derive Eqs.Rwot) R c i aieiQ. R .3) and (7. We find since A = V/L. can be related to the ensemble average of the spatial distribution of scatterers according to Eq. t ) = (y)2 f"  ___ e i ( k ' .6) and (7. respectively. LASER LICiHT SCATTERING between the solvent polarizability and that of the particles.a of an electromagnetic field is given as the square of the field amplitude9 = E*E. 7.7).2. (7. we implicitly assumed that the incident field is not appreciably disturbed as it passes through the sample and that multiple scattering can be ignored.2.. Also.(R. for sufficiently long measurement times. if the particles are much smaller than A.5) that the scattered field is given as Es(R.2.w o r ) scatterers c JQ.7) we considered that scattering caused by local variations in solvent density is insignificant and can be neglected.2. = w0/2nc.2.1. (7.r.ii(r)eiQ'r d3r. one has i where ri and Ei are.(. . In this case we find from Eqs.2. t ) = (yr  Eo sin 4) &(k'.7) It is apparent from Eq. the position and excess polarizability of the ith scatterer. If the particles are uniformly distributed throughout the scattering volume and.2. however. which. (7.2.2. (7. For identical particles we thus have <xi cj xi I / I o = (167c4 sin' ~ J / A ~ R ~ ) ( E ) ~ N . We then find E. Classical Light Scattering The intensity . 1. (7. (7. if they do not interact with one another. Therefore the scattering process is sensitive to the optical shape of the scatterers if particle dimensions are comparable to the wavelength of light. the ensemble average E i E j eiQ'crir~)) is simply the sum E'. also.
2. eiQ’crlrJ)) can be expanded in terms of virial coefficients.7).(dn/dp)p = 4nNcl.2.11) where the structure factor Y ( Q ) . (If several sizes of particles are present in the sample.2.9) we find Because p = N M / d .2.2.I7 Second. p ( r ) is the distribution of inducible dipoles within a particle. which generally is taken to be proportional to the mass distribution.10). Zimm. THEORY 305 Equation (7. the only angle dependence ” Is drp(r)eiQ” l2 (7.2.10) in principle can be independently determined. where M is the molecular weight of a scatterer and d is Avogadro’s number.6) rather than Eq.2. (7. we should start with Eq.. where p is the mass concentration of particles in grams per cubic centimeter. that the particles were assumed to be noninteracting. we have n ’  n: % 2n.12) B. Phys.Thus. 16. Here. .’3 First. J .) Two important assumptions have been invoked which. interactions can be probed since the expectation C. 1093 (1948).pM L4R2d (7. Consequently.2. (7. the “ zaveraged ” molecular weightI3 is found. H. if no is the index of refraction of the solvent. we have 1 10 4n2 sin2 rpng(dn/dp):.1 = 4na. If the density of scatterers is sufficientlyhigh. imply modifications of Eq. (7.7.defined as Y ( Q )= is sensitive to the shape of the scatterers.2. For small particles. the molecular weight of the scatterers can be ascertained by measuring the total intensity of scattered light. when particles are large.9) can be put in a somewhat more familiar form by the following manipulations. (7.9) is (xi (7. Chem. (7. for identical noninteracting randomly oriented particles. the analog of Eq.2. (7.10) Since all quantities other than M that appear in Eq. From Eq. if relaxed. We recall. we note that the index of refraction of a medium n can be related to the volume polarizability a according to n2 .2. first.
Appl. However. and. Suppose we have an instrument that can measure the joint expectation of the intensity at one instant of time and of the intensity at a later instant delayed by an increment z. Lather. Cross and P. most importantly. information on the size and structure of a particle can be determined. is given as l 3 .306 7. Two essentially equivalent schemes can be used to extract kinetic information from a timevarying signal: analysis of frequency components or formation of timedelayed “ autocorrelations” of that signal. Intensity Fluctuation Spectroscopy We have just seen that classical light scattering techniques relate the intensity of scattered light to certain molecular properties. temporally and spatially coherent radiation makes such studies practicable. that the system is in a stationary state. For didactic purposes we now consider how the motion of small particles (e. monochromatic. and. These time variations are related to local changes in refractive index of a sample. Classical light scattering from bacteria and other biological cells is discussed in papers by Wyatt * and Cross and Latimer.. By limiting the aperture of the collection optics.e. we can determine the autocorrelation function (. Method. and add the product thus obtained to the first. Other applications are discussed below. fluctuations in the intensity of the light that falls upon the detector can be discerned. A. J . D. quasielastic light scattering is concerned with extracting information from the temporal behavior of the diffraction pattern. l 7 1 + cos’ 0. Assuming that the average properties of the sample remain constant. a measure of the kinetic behavior of the scattering medium can be obtained. for large particles additional angle dependence affects the intensity through the Q dependence of the structure factor. i. 8. In contrast.. where those modifications of the basic theory which are peculiar to each case are indicated. Wyatt. when unpolarized light is used as the incident source. Opt. protein molecules of molecular weight 80.g. consequently.2.7 Microhiol. l9 7. . 11. divide by the + + + In l9 P. by measuring the angle dependence of the scattered light. LASER LIGHT SCATTERING of the intensity is through the term sin2 cp. Consequently. The recent availability of amplitudestabilized laserssources of intense.2. 1225 (1972). 183 (1973).000 daltons) can be studied with this technique. repeat the measurement at some other times t’ and t‘ z. repeat the measurements at yet later times and add those results to the former. The coherence time of ordinary light sources is too short to permit resolution of interference fluctuations arising from changes in refractive index of typical biological samples. so that we form the quantity (Y(t)Y(t 7 ) ) .Y(t)Y(t + T)} in principle as follows: Multiply the intensity at time t by that at time t z. which. finally.
Swain. A : Math. E.*. ) (A3A4) + ( A 1 A 4 ) ( A 2 A 3 ) . Pike.). R. Pike.2.2. A . Phys.14) ) The complicated expression given in Eq. eds. t)eiQ" d3r li (7. (7. THEORY 307 number of additions to the sum. A4 are Gaussian random variables. 2 2 B.3) and (7. we can write for the autocorrelation function of the detected intensity W(Z) = (j(t).2. Considerable study has been given to the manner in which finite sampling effects the reliability of deduced kinetic paramet ers. Jakeman. (7.2.4) we find that W ( t ) is proportional to the joint expectation of four quantities which contain information about the time dependence of the local polarizability of the sample.(t + z)EsC(t + T)).16) E. (7. Cardoso. the closer will this experimentally determined quantity be to a theoretical function whose properties derive from the stochastic characteristics of a representative sample.(t)E. J . 517 (1971). 1897 (1973). J . Phys. Nucl.2. 6. Because the instantaneous intensity is the square of the field amplitude.2. A . A z .2. (7. namely. 1974. . Phys. .7. Saleh and M. it ( A i A 3 ) (AzA4) then follows that (AlAZA3A4) = ( A .13) and from Eqs.14) becomes + (7. in "Photon Correlation and Light Beating Spectroscopy" (H./(t + t)) = (E. Cummins and E. p. . 75. Jakeman.15) where io(Q) = O ' Ilv a(r. R. t + T)eiQ" d 3 r . for a scattering process which obeys Gaussian statistics. F. Analysis of the joint expectation of four parameters generally is a difficult matter. E..(t)E. Eq.14) must next be related to some interesting material parameter via a physical model for the kinematics of the sample. and S. The more often one repeats the measurement. (7. A : Gen. Fortunately. New York. t + 7)eiQ" d3r)* Jv a(r. Thus. A .*. Z. E. if the statistical behavior of the scattering assembly can be characterized as a Guussiun random process. 4. x (1" a(r.2. an important simplification occurs: If A .2. Gen. Plenum.
t ) .17)by assuming that the local polarizability a@. z). As before [cf. (7. Schaefer and B. however. t ) = a. t)eiQ'd3r)* f. J. that for large scatterers. Of course. then by the law of large numbers the sum will be Gaussian distributed and the factorization indicated by Eq. additional features will be present in the scattered light ~ p e c t r u m . Berne. t ) is the "density fluctuation" and the expression within the angular brackets is the "densitydensity correlation function. However.18) is required (see Chapter 7.2. (7. Phys.475 (1972) R. we shall see. a generalization of the expression given by Eq. Voss and J. t + z) d3r (7. Rev. one situation for which it most likely holds is the case when refractive index fluctuations derive from movements of Brownian particles.2. F. 28. for which intraparticle scattering must be considered. . t)eiQ"d 3 r can be expressed as t i eiQ'ri(f)." Equation (7. such as macromolecules in solution.2.Generally. (7." Suppose.1 ( E ) 2 ( x eiQ""" 1eiQ'rJct+'r) j \ (7.a(r. Gen. Phys. t ) = [io(Q)]. + (aa/aC)aC(r.5). Lett. We note that the quantity eiQ"i(t) is the Fourier transform of the density operator. A : Math.2. when the scatterers are identical particles much smaller than the wavelength of the incident radiation. Eq. z) (the "intermediate scattering function") is defined as I l(Q. if scattering arises from a large number of statistically independent random events. (7. that the scatterers are indeed undergoing Brownian motion. t ) is given as a timeinvariant average quantity plus a term whose value depends upon the local deviation of particle density from the statistical average a(r.18') also could have been derived from Eq.15) will hold.2. the integral a(r.17) one has ) ci xi i I Z1(Q.2. . (7.17) The physical properties of a particular sample are related to acquired data through the quantity I l(Q. LASER LIGHT SCATTERING and I l(Q. now.7)]. ~ ~ * ~ ~ however. . if these conditions are not satisfied. 9. J .18) where hC(Q. The assumption of Gaussian statistics is oftentimes difficult to substantiate.308 7.2. . thus. z) = [io(Q)] (( Jv a(r. Clarke. + 24 D. from Eq. 561 (1976).2. the concentration of scatterers and the size of the scattering volume must be sufficiently large that fluctuations in the total number of particles in the scattering region are insignificant. W.
2b. = (2DQ’)ground term plus a simple exponential whose decay is given as z Thus.15) is often called homodyne scattering.19) is shown in Fig.2..19). certain kinds of particle motion cannot be detected by this technique. In such cases the ’.18’) thus becomes I . the diffusion coefficient would be determined from a representation of data such as that shown in Fig.2. (b) inverse relaxation time r. (7.. plotted versus the square of the amplitude of the Bragg scattering vector Q = 4nni.2.19) where D is the translational diffusion coefficient.13)(7.15) and (7. However.2. It has the form of a constant back. 2. Determination of the diffusion coefficient of a suspension of monodisperse noninteracting Brownian particles: (a) Exponential decay of the timevarying portion ofthe photon autocorrection function.2. (7.7. I . ( Q . for example. movements of particles with constant velocity V.4. The experimental situation depicted in Eqs. Eq. by measuring the autocorrelation at several scattering angles. ’ . sin $0.2.2. The most common application of laser inelastic light scattering heretofore has been the measurement of the diffusion coefficients of macromolecules and small particles according to Eq. Consider. The photon autocorrelation function derived from Eqs. (7. In this case components of the scattered light field mix with other components of the field at the photodetector surface to provide the fluctuating signal subsequently processed. (7.2. 2a. T) = e’Q2‘. THEORY 309 The motions of noninteracting Brownian particles can be described by Fick’s diffusionequation. (7. FIG. As shown in Chapter 7.
2. Relationship between Photon Autocorrelation and Spectral Analysis The previous section emphasized autocorrelation techniques for analyzing the fluctuating intensity of scattered light. by the WienerKhintchine theorem.(t). (7. we note that heterodyne scattering can always be used to test whether the “Gaussian approximation” given by Eq.*.21) where the righthand side of the equation also contains a negligible “homodyne” component..1. (The added field is analogous to the local reference oscillator in heterodyne radio detection and therefore sometimes is referred to as the “local oscillator field.”) If we assume that an experiment is designed so the amplitude of ELO is much greater than that of the scattered field E. Finally. (7. information about V could be obtained by performing a socalled heterodyne experiment in which a portion of the source light does not pass through the sample but is diverted to the detector.as indicated by Eq.2.13) is g(z) x ((E:oE~o)~+ > 2(E&JL0) Re(E. then the analog of Eq.’ From Eq.3. we could as well have considered spectral analysis of that signal. (7.15). I Z 1 ( Q .(t)E. . since the information contained in the frequency distribution of a timevarying signal is equivalent to that which can be extracted from autocorrelation. (7.(t + z)) + . (7..(Q.2.2.(t z)) cos(Q * VT). LASER LIGHT SCATTERING position of the ith particle at time t T is related to that at time t as ri(t = ri(t) + VT.2. Instrumentation and General Techniques 7.(t)E.20) Consequently.15) may be invoked to simplify data analysis.2. Suppose we designate this unscattered field as ELO(t) = ELOei(mot+q). However. so that the total field at the phototube surface is E(t) = ELO(t)+ E.. z) = + + T) [io(Q)]’(E)2N ei(Q’v)r. 3. However.3.  + 7. the autocorrelation function would not contain any information about the drift velocity of the scatterers.t ) as 1 rw (7..1) The equivalent nature of these functions is illustrated in Fig. .3. . Indeed.*..18) becomes I. o)is related to the autocorrelation function V(Q. (7. (7. the power spectrum of the scattered light S(Q. t)I2 = Z:Zl = ( t i ) 4 N 2 / [ i O ( Q ) ] 2 .20) we see that Re<E.310 7.2. so W(T) in this case does indeed provide information on the drift of the scatterers.so that Eq.
3.2) The frequency width of this function contains the same information as does the decay of the autocorrelation function.g. (7. (7..  (7. t ) e2DQ2z [see Eqs. and (c) particles drifting with uniform velocity. the related spectral power density is Lorentzian. I f ( Q . (7. INSTRUMENTATION AND GENERAL TECHNIQUES 31 1 ? FIG.3.3. . Processes whose autocorrelations (a) (b) (C) FIG. (b) subcutaneous blood flow or randomly swimming microorganisms. I).3.19) and (7. Correlation functions [Il(r)'] and spectra [S(w)] are related according to Eq.3.7.15)]. Thus fluctuations in the number of photon detections per unit time [ t r ( r ) ] contain the same information as docs spectral analysis ofthe photocurrent amplitude  Ci(01. The average number of detected photons is proportional to the intensity of light falling upon the photodeteclor. 4.1) are shown in Fig. Typical results for (a) diffusing macromolecules.2. Examples of the transformation effected by Eq.4. e.2. If the photon autocorrelation function is exponentially decaying.
and theoretical considerations of photoelectron emissions from a photocathode’ indicate that correlation of electron pulses with themselves gives rise to a shotnoise contribution to the photocurrent correlation function. efficient collection and processing of primary data presently require photon autocorrelation techniques. (7. (7. also. Therefore. where (i)’g2(z) is proportional to the quantity given by Eq.6) and standingwave oscillations of soft gels (Chapter 7.2. (7. Characteristics of Measured Autocorrelation Functions Although theoretical analyses based on Eq.312 7.3) 1 ‘v (1 AriZ Z(t Ar/Z + s)ds J Z(t .2. One really does not directly analyze the field intensity when an autocorrelation analysis is performed on the scattered light.9)]. manipulations are made on the photocurrent emitted by the detector (Fig.e. [gz(T) is related to the autocorrelation of photopulses n(z) as + S A T ) = <n(t)n(t + ~)>l(n(t>>2.3. 7.7) and subcutaneous blood flow (Chapter 7. Also. the expression given in Eq. an instrumental autocorrelator necessarily samples the scattered photon field over discrete intervals of time. LASER LIGHT SCATTERING consist predominantly of a small set of periodic functions are usually best analyzed in terms of spectral components [examples are the motion of charged particles in an electric field (Chapter 7.2.1 In practice however.Ar/Z + z + s’) ds’ ) + z + s’)ds’ ). Thus the photocurrent autocorrelation function C. lo)]. if the scattered signal is very weak or the time scale of the fluctuations very fast.13) pertain to continuous signals.2. i.13) is only an approximation of the measured autocorrelation function for yet another reason. Systems whose scattered fields contain a broad continuum of frequencies are oftentimes most easily related to theoretical models via timeautocorrelation functions [examples are the spatially isotropic swimming of microorganisms (Chapter 7. when spectral analysis is performed the shotnoise term provides a constant background which is subtracted from the remaining signal.. the correlated components really are integrated functions.3. %&) =(A212 1 (J Ar/Z n(t Ar/z + s) ds J Ar/Z n(t Ar/Z .(T)= e(i)h(z) (i)2gz(z). 3). Rather. (7.14).Ar/Z but minimal distortion will occur if the clock interval AT is short compared with the characteristic decay time of the true autocorrelation function.(T) is given as1 C. the signal in the first (zeroth) channel of an autocorrelator is blocked or neglected and the shotnoise term e( i)6(z) ignored. . Similarly.
Phys.2 but such considerations are beyond the scope of this introductory article. (7. the total intensity of light falling upon the photocathode will be almost constant and the ratio of correlated signal to background will be very small.3. Jakeman. 7. and sample concentration are changed. J . if the pinholes through which the sample is viewed by the phototube are very large.3. Phys. and E. . The proportionality term fi accounts for statistical errors introduced by clipping (see Section 7.e.3 below) and coherence “losses” at the photodetector surface. Oliver. (7.3. In the case of diffusion coefficient measurements of monodisperse samples.. Applications involving low count rates and short correlation times. N (1 + BCIi(Q. . L45 (1970). seem to be most successful when spectrum analyzers are employed (see Chapter 7. the greater will be the accuracy of the parameters that can be extracted from the data..3. channel width. C. 2)12). A : Gcn. the fidelity of g(Q. i. Equipment Data can be processed either with a spectrum analyzer or with a pulse autocorrelator. p w 0. even though the diffraction pattern of the scattered light fluctuates. Theoretical analyses of the statistical variability of experimental results are quite difficult and have been performed in detail for only a few casesZo 2 z . one’s intuition of this matter can usually be formed from repeated measurements on prototype samples in which instrumental settings and experimental variables such as count rate. An exhaustive analysis of the ways in which p depends on various instrumental parameters has been performed by Jakeman and associates.2.15) also is but an idealization. theoreticai and experimental analysesz5 indicate that an accuracy of approximately 1 % can be achieved for quite reasonable experimental conditions (e.. however. Velocimetry experiments. R Pike. Of course. The latter arise because the scattering volume “seen” by the photodetector is of finite size so that. z) also depends on the count rate (i.3.g.4) where p < 1. some of the information contained in that pattern is lost. where scatterers move in preferred directions. such as measurements of the diffusion coefficients of small proteins.6). The choice of instrumentation largely depends on the uses anticipated for the spectrometer. using a 50mW HeHe laser). the total number of scattering events).I.7. INSTRUMENTATION AND GENERAL TECHNIQUES 313 The factorization gz = 1 + f: as shown by Eq. scattering angle. The longer one accumulates a signal. ’’ E. measurement times less than 2 min for a sample composed of 5 mg/ml haemocyanin. The correlated signal actually appears as U(Q.e.20. are best done in a photon counting mode utilizing autocorrelation analysis. 3. Indeed.
Peacocke. In some laboratory installations the correlator output is fed directly to an electronic calculator for immediate manipulation and storage. B. P. LASER LIGHT SCATTERING Two kinds of correlators have been used for photon counting applications. Clipped autocorrelators were first used because they afforded finer time resolution and faster operating speeds than other available analyzers and because they were relatively inexpensive to construct.314 7. One. the autocorrelations formed by a clipped autocorrelator operating at zero clipping level will be virtually identical with those formed by a full autocorrelator. less than 0. Opr. Wood. J . and an option for up to 1096 channels for resolution of the correlation function. Asch and N. Chen. as well as a complete laser photon counting spectrometer. (7. Rer. a clipping level of 2 would produce a “ 1” if the number of counts in the interval is 3 or greater).g.. an autocorrelator was designed by Asch and Ford2’ which utilizes a fourbit shift register to accomplish an almost exact computation of the full ideal autocorrelation function. Yangosand S. including a builtin display. if the average count rate is low (e.j AT)is replaced by either 0 or 1. Sci. Pike. a multiplicity of interfacing options. the actual number of delayed counts n(t .. Oliver. Veldkamp. Massachusetts) markets a full fourbit realtime digital correlator having the capability of choosing the sampling time (clock width) to be as short as 100 nsec. E. Early R. Clearly. and C . E. and theory of such devices has been described by Pike and his associatesz6 and Chen et al. R . C. z9 M.g. Jakeman. 51. LangleyFord Instruments (Amherst. C. Instrum. E. Malvern Scientific Corporation (Ronkonkoma. J . 46. 17. H. Ntrtitre (London) 227. J . California) is producing a spectrometer incorporating a clipped autocorrelator which contains a microcomputer dedicated to diffusion coefficient determinations. 3197 (1978).Sci. The construction. that of a “singleclipped’’ autocorrelator. of clipped autocorrelators. the ability to simultaneously accumulate correlation functions at different sample times. Nicomp Instruments (Santa Barbara.e. 44.27 Later. operation. 242 (1970). and A. R . Jr. 506 (1973). H. 344(1980). R .29 Although an investigator previously had to build his own correlator. depending on whether a preset level is exceeded (e. it has many desirable features. it has many of the attributes. Ford. Ref. *’ S. it repeatedly performs multiplications of the actual number of counts appearing in the various time intervals. W. Instrum. R E P . Chen. Chen. Blagrove. Several firms manufacture excellent realtime spectrum analyzers.. In the other case. Foord. i. New York) markets similar signal correlators. a “full” autocorrelator.3. Lai.Sci. Appl. 2 8 R. 1356 (1975). essentially performs the operation indicated by Eq. such as short sampling times and moderate cost. Instrum. *’ . Holz and S.3).5 pulses per interval AT). H. several highquality commercial instruments now are available.. C..
e. INSTRUMENTATION AND GENERAL TECHNIQUES 315 efforts to use laser light beating spectroscopy relied upon sweptfrequency analyzers which were slow and cumbersome. and should have a polarized output. Pinholes must be used so that the portion of the detected diffraction pattern will be small enough that changes arising from material fluctuations within the sample will significantly change the amplitude of the signal (i. and has a low probability of producing afterpulses.7.. Several tubes are available which have such characteristics. 46. as is vibration isolation. The design of the sample holder or cuvette generally depends on the anticipated application.4 Good temperature control for the sample is usually advisable. Rev. Ware4). Sci. because of their generally higher power and shorter emission wavelengths [scattering intensity varies as see Eq.6) require particularly sophisticated engineering skills.. W. Electrophoresis experiments (see Chapter 7. The detector optics must be designed so that adjustments can be made to delineate the region of the sample which is being observed. Lowpower HeNe lasers are quite adequate for scattering from structures such as biological cells and tissue (Chapters 7.586 (1975) . A variety of HeNe and argon ion gas lasers have been used. The situation is now greatly improved. various pulse shapers and/or amplifiers are used to interface the photodetector and analyzer (see. New Jersey) currently offer suitable instruments. The laser must have good amplitude and beam direction stability. the detected signal must arise from only a few “coherence areas”’). Instrum. and for many applications spectrum analyzers are at least as convenient to use as are correlators (see Chapter 7. S. However. (7. ” J . emits narrow pulses. in many instances high power can be a nuisance. Argon ion sources.9). Flynn.10).2.3. Depending on the particular instrumental configuration.67. New Jersey) and Princeton Applied Research (Princeton. causing heating artifacts or providing signals which overload the detectors and analyzers unless attenuated with filters. Lowlightlevel photon counting applications require a phototube which has fast response time. 3o Inexpensive photodiodes can be used instead of phototubes for certain applications involving high signal levels and slow fluctuations (see Chapter 7. however.7). the performance of which has been reviewed in a paper by Gethner and Flynn. Nicolet Scientific Corporation (Northvale. correlators are preferable for studying rapidly fluctuating phenomena because the short delay times AT available with those instruments correspond to highfrequency ranges which are not accessible when using commercial spectrum analyzers.g. Gethner and G .10)] seem to be favored for research applications where low concentrations of small macromolecules are being probed.e.
(7.4. t)/& = DV2C(r. J . Westhead. t). ’ ~327). O)e’Q2‘. For example. (7. E. Ford.19). JsC(Q.g. W. y~ the viscosity.4. from which an expression for the densitydensity correlation function then follows as (SC*(Q. t ) = SC(Q. Once D has been determined. (7. (7.Ptrj7 12. so that Eq. the intermediate scattering function ll(Q. t ) ) = ((SC)2)e’Q2‘. (7.2.2. t). . The derivation of that expression follows directly from Eq.Co. R. we find.18’) and the diffusion equation for the time dependence of the concentration of particles C(r. X ( r . The effective particle size may be somewhat larger than that of a “bare” molecule if solvent is dragged along with the moving scatterer. information about the size and shape of a particle can be deduced. the diffusion coefficient for ellipsoidal particles is given by the Perrin equations (see e. if the scatterers are spherically symmetric the particle radius can be determined from Stokes’s law.namely. 3’ 32 R. C. 349 (1975).. Cohen and G. if we subtract a constant term Co and define 6C = C .2) would be a proper expression for analyzing an experiment even if the bare particle were not spherically symmetric.1) The coefficient D which appears in the exponent of Eq. Benedek.1) is the translational diffusion coefficient. Examples which involve measuring particle interactions are studies of the assembly and disassembly of ribos o m e ~and ~ aggregation of antibodycoated microspheres by antigen. Immunochemi.2) where k is Boltzmann’s constant. However. B. p. t ) is given by the expression (7.4. The solution is SC(Q. B.316 7. Diffusion Coefficients The most common biologically related use of laser inelastic light scattering is the determination of the translational diffusion coefficient of a collection of macromolecules or supramolecular assemblies. D = kT/6nqa. The results of such a measurement can then be used to obtain other information. t). T is the temperature. LASER LIGHT SCATTERING 7. and N . if the scatterers are grossly asymmetric a different model is necessary.. Thus.4.4. in particular. Gabler. 528 (1974). Biophys. after taking spatial Fourier transforms. T a n f ~ r d . t ) / & = DQ26C(Q. Another effect of solvent association might be to smooth out the shape of a nonspherical particle. O)SC(Q. ie. 14. such as indications of changes in molecular conformation or of modified interactions between various constituent subunits. and a the effective particle radius.32 If a sample contains identical (monodisperse) Brownian particles.
Measurements are frequently performed on particles such as proteins. D.~.. Selser. if the particles are small and. (7. let us again refer to Eq.(z) as f = M . Viruses. Koppel.2. The upper limit on particle size in part is set by one’s ability to interpret the effects of intraparticle scattering which occurs when the scatterers are large compared with the wavelength of light (particularly if the sample is not homogeneous in size). 29.^^" Complexities occur if the scatterers are not all of the same size. = rc(r)dr= M. N. One procedure which can be used to analyze data from polydisperse samples is the “method of cumulants. M.3). Bioph. ( Q . D..[ln I . the size of small peptides. ’’ ’’ .(t) = f : G ( r ) K r r d r with l. 960 (1974). Y. then the moments of G ( r ) are related to the time derivatives of Z. also.’ J P‘(u)[ii(a)]’ eD(a)Q2r da. 505 (1978).s. Rioc. and R. [A generalization of Eq. J.33 vesicles of sarcoplasmic reticulum.e. in Eq. J . Gaskin. Franklin. . (7.ys.” first described by K 0 p p e 1 . (7. W. Yeh. ~If ~ I . Schaefer. Biop/iI.4.4.4. Gcthner and F. Carlson. 24.hrwi. D.4) [ln I . Considerable attention has recently been given to studying large spatially anisotropic molecular assemblies.z) = [ i o ( Q ) ] . Biophy. and phospholipid vesicles. J .19) is I1(Q.3) is derived in the Chapter 7. CC can be taken to vary directly with the mass of a particle (frequently a good approximation). Because the intensity varies as the square of the mass. 3Sa J.7. the analog of Eq. 48 14 (1972). d dz r)2G(r)dT = d2 dz (7.t ) for a mixed collection of particles. CameriniOtero. =  I I(r . a practical lower limit on the size of the scatterers is presently 20003000 daltons.1 If. the radii of which fall within the range 15 A 5 a 5 lo3 A. ribosomes. if it can be assumed that the density of scatterers is so low that interactions between particles are rare. P/iys. ( T ) ] ~ = ~etc. D. and R. Uicm.4’P(a>[E(u)]’ du and b ( u ) is the relative density of scatterers having radius a. Koppel. such as micro tubule^^^ and actin filament^. i. However. (7. Newman and F. J . DIFFUSION COEFFICIENTS 317 Particles of many differing types can be studied. Baskin.s/r~~ 13. 57. S.3) where io(Q) z . 37 (1980). J . t) is represented as I. R.. P. To compute the intermediate scattering function I1(Q. E. J . Pusey.34 and small bacteria have been studied. J .17).2. G(T) dT = 1. ( T ) ] ~ = ~ . then a scatterer contributes to the scattering intensity in proportion to the square of its molecular weight.4. 337 (1976). E. The situation for large scatterers is complicated by the fact that intraparticle interference requires integration over the contours of the particles. (7. 16.5. C.4. D.
such as protein dimerizations or polypeptide helixcoil transitions. .318 7. The titre containing the unknown amount of antigen inhibits theagglutination reaction bycompetingfor antibody(Ah)(seevon S c h u l t h e s s c t ~ / . (7. which. The ratio M . in which case data have to be fit to a weighted set of Lorentzians.4. lrnmunoassay in the agglutination inhibition mode. parameter determinations may be facilitated. since r = DQ2. z) in the limit z 0 is proportional to an average diffusion coefficient 6. is a zaveraged quantity. Generally no more than a few (two or three) exponentials can be used to fit data if parameters are to be unambiguously determined.5) If the molecular weights of the species are known. (Similar considerations arise when frequency analysis is used. however. if the particles scatter light in proportion to their molecular weight.4. it is often more effective to fit the data to a weighted sum of exponentials.3) becomes xi (7. Specific antibody is used to "crosslink" antigencoated ( A g ) spheres. LASER LIGHT SCATTERING f Thus. the relative concentration of particles can be determined and one can deduce the equilibrium constant for simple reactions.4. An analysis of errors expected in values of D is given in Koppel's paper. that is. In this case Eq. / M : is a measure of the relative width of the distribution. Equation (7.36 If only a few particle species are present in the sample. if intensity fluctuation measurements are combined with conventional light scattering measurements. ~ * ) . particularly if the time scales for the exponential decays pertaining to sample and dust are widely separated.5) is also quite useful for analyzing data from samples which contain a small amount of residual dust or other largesized contaminant.) Ab free Ag FIG. where pi is the weight concentration of scatterers. the first derivative of Il(Q. D could be represented as B = piMiDi& p i M i . 5.
This condition is satisfied if the phase shift experienced by a plane wave transversing the scatterer is small. Excellent discussions of classical light scattering from large particles may be found in the wellknown books written by KerkerI6 and Van de Hulst. B. by which we mean correlated scattering from several points within a particle. R. .” In recent years attention has increasingly been given to similar inelastic light scattering situations.5. of the order of a few microns or less (10°K) is easily effected if the socalled RayleighGansDebye approximation’s. First. i f I 5 2(n . i. fmniunochemisfry 13.nO)AK1L < 1.7. 3 8 G. and G. We recall that the results derived in the preceding section were obtained after assuming that the scatterers are small compared with the wavelength of light. J. von Schulthess. The effects of size have been of concern ever since light scattering was proposed as a spectroscopic technique almost 100 years ago. the structure factor Y ( Q )may have a strong Q dependence so that in a heterogeneously sized sample the relative contributions to the light scattering spectrum from the various constituents change with the scattering angle. Beiiedek.32. Large Particles Analysis of light scattering experiments generally is more complicated if a sample contains particles whose dimensions are the same order of magnitude as the wavelength of light. J . A modification3* (the “inhibition” mode) utilizes antigencoated spheres and probes the degree to which an unknown antigen sample inhibits the agglutination of the spheres by a specific antibody (see Fig. voii Schulthess. and G . K..5.e. The essential assumption in this case is that an incident electric field is not significantly distorted before it interacts with a scattering center within a particle. and in certain instances can approach the sensitivity of radioimmune assays. ” G. A generalization to the case of particles whose sizes are. Changes in B are used as an indication of the agglutination of antibodycoated latex microspheres by polyvalent antigen. 7. B.e. K.37The assay can detect very small amounts of antigen..g. Cohen. LARGE PARTICLES 319 An interesting and potentially important application of this technique is a laser light scattering immunoassay for detecting antigenantibody interactions. can have two effects. Y55 (1976). fmmunoc~humisrry 13. 5). even when all scatterers are of identical size and shape. N. but only a few special cases have thus far been analyzed in detail. Benedek. Sakato. Intraparticle interference.. Second. Cohen. if the particles are not spherical the Q dependence of the relaxation times of the autocorrelation function may differ from that of spherical particles. that the presence of other scattering centers can be neglected in considering scattering from any particular center.’6 is permissible. i. R.963 (1976). e.
t ) is a weighted distribution of scattering centers. (7.5. (7. A useful transformation to a bodyfixed coordinate system can be accomplished by writing r = R(t) + p. L is the maximum dimension of the particle.(R. 6). which for optically homogeneous scatterers is simply proportional to the mass distribution within a particle.(R.6) that the scattered field may be written as E. and A is the wavelength of light. Transformation to a bodyfixed coordinate system.2) where P(p. / FIG. however. scattering from a large complex particle can be evaluated only by resorting to the solution of Maxwell's equations. t )  1 particles i(r)eiQ'r d3r. J (7.5. t )  eiQ'R(*)d 3 p ~ ( p t)e'Q'P. LASER LIGHT SCATTERING where FI . we see from Eq. In general. Thus the expression for E.no is the difference between the refractive index of the particle and that of the solvent.(R.3 20 7. t ) can be rewritten as E.1) where Q ( t ) indicates that the integration is to be performed over the volume and orientation of the particles. where R(t) is the position of the center of mass and p is a vector from R to a point within the particle (see Fig. . .2.6 .' If the RayleighGansDebye approximation holds.
(Q. and Z1(Q. t) ( eiQ’AR(t) d 3 p’ / 1 d3 p P(p’.e. O)P(p.Qa cos Qa)}’ (spheres). (7.5. even if a particle were to be so large that Mie theory (solution of Maxwell’s equations for the field within a scatterer”) is necessary. Eqs. i.5) . Note that Q generally is fixed by the geometrical relationship between the source and the detector.5. It is easy to show” that in the RayleighDebyeGans limit. This perhaps is clearer if we write Eq. (7. These last comments hold only if all particles are the same size..15)(7. LARGE PARTICLES 32 1 Let us here assume that the scatterers are identical and noninteracting. Y(Q.2. p t ) + Y(Q).5. so d3p P(p. Orientation and translation are strongly correlated when flagellated bacteria such as E . the time dependence of axial reorientation will affect the photon autocorrelation function.18)] can be written as I. Thus. Hence the particle structure in this instance would affect the intensity of the light scattered at a particular angle.5. however. so the computation of the expression given in Eq. Various model calculations in this case indicate that the Q dependence of the relaxation time of the autocorrelation function is affected by particle orientation (see Chapters 7. Then the intermediate scattering function Z1(Q. the particles have spherical symmetry. Y(Q) is given as  Y(Q) = {[3/(Q~)~](sin Qa .” given by the integrals within expression (7. (7. (7.3) ) where AR(t) is the displacement of the center of mass at time t. if a particle is nonspherical and. These organisms are cigar shaped and have internal structure. t). further.3).t ) Y(Q)(eiQ’ARc‘)). Y ( Qp . For example.5. (7. If.3) as (7. t ) [cf.o . A similar conclusion holds whatever scheme is used to compute the electromagnetic fields within the scatterer.7). but not the Q dependence of the decay.4) where pLtis the cosine of the angle between the scattering vector Q and the internal reference axis of a particle.7. In a heterogeneous assembly the Q dependence of the structure factor can affect the form of I1(Q.5.2. pol p.4) is difficult. t)eiQ’P’ eiQ‘P. coli locomote. if its translational motion is correlated with its spatial orientation.5. the dynamical structure factor depends only on the magnitude of Q and not on particle orientation. we now consider the diffusion of an assembly of large solid spheres.) is the “dynamical structure factor. t)eiQ’P implicitly depends on the spatial orientation of a particle because of the term eiQ‘Pin the integrand.
6) In the limit that a is small. 64. . artifactual scattering arising from dust contaminants in a sample can in part be eliminated. J . 503 (1977). since large particles usually move more slowly than small particles. Chen. Tartaglia.Qa cos Qa + Qai cos Q U ~ ) } ~ (shells).(Q. Opt. Thus.1) are carried out.05pm particle. Jr. the signal from larger particles will be relatively less significant as the scattering angle is increased . H . Binelrim. P h p . for a heterogeneously sized collection of scatterers. when a sample is illuminated with light from a HeNe laser (red). P e ~ o r a was ~~ the first to recognize that. R. Ardgon. in contrast. T. For example. Chem. reorientations of long. thin molecules would contribute features to the scattered light spectrum from which information about the rotational diffusion coefficients of the scatterers could be extracted. is Zl(Q. "R . T) = [io(Q)]' [ 9 ( a ) Y ( Q ~ ) [ t i ( a ) eDca)Q2r ]~ da.5pm particle decreases by a factor of almost 300 as the scattering angle is increased from 20" to 90"..7) where a is the outside radius of the shell and ai is its inside radius. Pecora and S. and B. Chen. the intensity varies by only 15 % over that range. contributions to the autocorrelation function which decay slowly will be deemphasized and the apparent decay rate will increase at large scattering angles In this way.sin Qai .5. P. This expression is important in analysis of the scattering from phospholipid ~ e s i c l e s ~ ~ . J. (7. Acta 470. E. 4 I26 (1 968). F. consequently. Pecora's expression. Pecora. Appl.a:)] (sin Qa . (7. Chrzeszczyk. Y ( Q a ) + 1 and the expression given in Eq. 187 (1977). The situation for rodlike scatterers is more complicated. Chu. K. J .. Day. vanishingly thin rods of length L.34 Chen et ~ 1 have .322 7. 48. 0.3) is Z. the relative intensity of the scattering from a 0. Holz. 230 (1972). and S.5. derived ~ ~ a similar expression for coated ellipsoids and have investigated relationships for dynamic light scattering which arise when the integrations indicated by Eq. 1 (1974). C. 16. under certain circumstances. . and P. Chum. 4 J S. (7. Sun.3) is recovered.3403 (1976). Lipids 13. for a 0. A. however. t ) = Bo(QL)eQZD' + B1(QL)e(Q'D+6err + . Thus the analog of Eq. (7. 4" 4' 42 R .8) '' D. T. Ho. which was derived for rigid. (7. Phys.5.4. Y ( Q a )decreases markedly for large values of Qa.4. Lipids 8. Kunze. Bioplys. Churn.. J (7. C h m . ~ ' and membraneous structures such as particles formed from sarcoplasmic reticulum. LASER LIGHT SCATTERlNG where a is the radius. The structure factor for spherical shells has been derived by Tinker3' and by Pecora and Aragon4': Y(Q> = {[3/Q3(a3 . R. Phys. M .5.Tinker. Phys.
S. J . D. B i d . Daune.9) and (7. information about 0 can be obtained. M.5. Murayama.The ~ ~ . the intensities associated with higherorder terms in the expansion are quite significant. the length of which is approximately 3000& and for which diffusion coefficients are found to be4’ D N 2. Fujime and M. M. for large scattering angles and particle lengths (e. I. Fujime. (7. Opt. LARGE PARTICLES 323 where B. S. However. in which case a single exponential characterizes the autocorrelation function.518 (1969) M . Appl. ( x ) are defined as (7. 4s 46 . 68.g. First. Cummins et ~ 1 used .8). C.8 x cm2 sec’ and 0 1 : 320 sec’. 2470 (1979). sizes of the order of several microns). 4 7 S. J . Additional features in the autocorrelation function may arise from molecular configurational fluctuations if the scatterers are not rigid. if correlated with particle orientation. Second. Z. 237 (1973).10) where j . Biophys. Almost all of the scattered intensity is represented by the B.5. F. Biopolymrrs 16. Schmitz. Kotlarchyk. light ~ ~ scattering techniques to measure the rotational diffusion coefficient of tobacco mosaic virus. RayleighGans theory was used to compute the form factor and results were found to be different4“ if particles are so large that Mie theory” must be used (e. and J. Cummins.5. 347 (1972). Woods. If the correlation times for translation and rotation are comparable.. ~ ~ H. 4 8 S.(x) and B . term if QL is less than 3. such that QL > 5). Carlson. S. it was assumed that particle orientation and direction of translational diffusion are uncorrelated. Caloin. Chen. Mol.g.5. Wilhelm. Internal modes of motion such a stretching and bending may thus be discernible when long. Asakura. J. several other assumptions were made to obtain expression (7. flexible molecules are e ~ a r n i n e d . Herbert. B. and G . 2091 (1977).8). 49 M . In addition to the requirement that the scatterers be rigid. Macromolecules 6. is the spherical Bessel function (of the second kind) of order 1.5.. Lin. Schurr. Eiopolymers 16. Calculations performed45 for particles so large that their rotational correlation times greatly exceed those of translational diffusion (so that the rotational terms essentially contribute only a flat background term to the correlation function) indicated that translational diffusion.7. 583 (1976). 18. and S. and S. Lee. and M. ‘I’ W. K. leads to deviations from the simple exponential decay given in the first term of Eq. T. Asano. 9. H.internal ~~ dynamics of DNA have been studied by quasielastic light ~cattering:~but molecular entanglements and hydrodynamic interactions which occur in congested solutions of such molecules are a c o m p l i ~ a t i o n . Murayama.
2.6.t ) )  eDQ2r cos VF * Qt.6. t ) [see Eqs.(Q. where E is the electric field acting upon the particles and u is the electrophoretic mobility. were an electrophoresis experiment to be performed in this configuration. which satisfies aC.15) and (7. the scatterers move preferentially in certain directions. We thus have aclat = D V ~ C . LASER LIGHT SCATTERING 7.6. the measured ) related to the square of the densityphoton autocorrelation function V ( T is density correlation function I SC*(Q. with the current J now taken as the sum of a “diffusion current” J. O ) e . the useful signal is then given as )’  Il(Q. (7. A kinetic equation to describe movement of particles in an electric field can be obtained from the continuity equation aC/& = .# E . is the fluctuation of scatterers about an equilibrium value C.6. as shown in Eq.21). O)SC(Q. however. In a homodyne experiment.2. If. V(T) 1 + fle2DQ2r.V * J. (7.4) Analogous expressions are realized for other processes in which drift is . O)SC(Q.6. In this situation the spectrometer is operated in a heterodyne mode and. (7. the scattering function Z.t ) . (7. the solution of the above equation is (jC(Q.C.18‘)]. (7. Thus.DVC and a drift term J. in which only components of scattered light are mixed on the surface of the photodetector (see Chapter 7.2.2)./at = 0.Determination of Electrophoretic MobiIit ies In the preceding chapters we have assumed that the translational movements of the scatterers were spatially isotropic. This feature forms the basis of a laser inelastic light scattering assay for electrophoretic response in which particles move along an electric field vector and thus impart a corresponding Doppler shift to the spectrum. we would obtain the result normally seen for diffusing particles. (7.vc. In order to extract information related to electrophoretic drift a fraction of the light emitted by the laser source must be diverted to the photodetector before it passes through the sample.2) where 6C = C .Re (SC*(Q. t ) will have special characteristics.324 7. = . = uEC. Q ) * .3) where vE is defined as VE = U E . t ) = SC(Q. namely.1) When the effects of boundaries are neglected.( D Q 2 + i u E .
In this case the relative height of the spectrum at a given frequency o.[ild. Ph. s . (7. Thus. 7). S .= Q * vE.3). I17 (1972). '25. nor are they all of the same size. ~ Certain difficulties not present in other applications must be resolved when engineering an electrophoretic light scattering experiment..S. resulting in convection. A. ~ w m w dMrt/iods . . Uzgiris. 2388 (1976).'^ Ware has recently reviewed work in this area emanating from his own and other investigators' l a b ~ r a t o r i e s . E. a specially designed pulse generator must be used to cycle the electric field in coordination with data accumulation by the spectrum analyzer. B. Sci. Ware. Cornrnuri.7.53 Recent applications of electrophoretic light scattering include an assay for antigenantibody reactions. 10. Smith. 5. I<. P w c Ntrtl. the autocorrelation function would be given by Eq. 53 R. R . E. A . In fact. for large particles such as biological cells. the chemotactic response of ba~teria~lsee Chapter 7. Op/. Fig.  S(O)  1/[(0  Q * v E ) ~+ ( D Q 2 ) 2 ] . One practical application of this technique has been an investigation of the electrophoretic mobilities of lymphocytes and monocytes obtained from the blood of leukemic patients52(see Fig. if diffusion can be ignored the entire distribution of electrophoretic mobilities of a sample can in principle be determined quite easily. P. Siege1 and B. a constant applied electric field can cause a sample to overheat. '' D. Massachusetts (1977).= Q vE.73. Nossal and S. It also might cause electrode polarizations. a n d K. Several other studies of cell surface charge are discussed in Smith. the particles in a sample generally do not all have identical charge on their surfaces. Smith. the scattering cell and electrodes must be carefully designed in order to obtain a determinable electric field in the scattering region. i. 4c). R. Thesis. Harvard University. Warc. were the scatterers to have identical surface charge the spectrum would appear as a sharp spike (cf. Cambridge. Weiner. DETERMINATIONOF ELECTROPHORETIC MOBILITIES 325 superimposed upon spatially isotropic motion ( e g .5) Note that as the diffusion coefficient decreases so does the spectral width. A(.7). If a collection of diffusing particles all were to have the same drift velocity.D.e. The spectrum of scattered light would be given as a Lorentzian shifted from the origin by an amount o. .is directly proportional to the intensity of light scattered by particles whose electrophoretic mobilities are consistent with o. although we implicitly assumed that the electric field is time independent.6. For example. Extraction of electrophoretic parameters from the data can be somewhat complicated if several Lorentzians overlap. Consequently. 304a (1979). B ~ ( J ~ / ~JJ. J . Cheii. A . and the sample holder 5' '' B. (7.6. However. though. U . Furthermore. H .54 and a study of the fusion of secretory vesicles with membrane receptor^.6. 85 (1976).
the implication of which is that the rootmeansquare (rms) speed or. at which times changes in specd or direction take place. very small ( 5 5") scattering angles must be employed in order to reduce spectral broadening relative to the frequency shifts arising from electrophoretic drift. These considerations are discussed at length in Smith's thesis. the entire distribution of swimming speeds of a sample can be deduced. 8). Motility Measurements When a suspension of motile microorganisms is illuminated by laser light. Finally. if measurements are made on protein solutions. In this instance a relatively simple model can be used to compute I1(Q. We first assume that the scatterers are homogeneous in size. These straightline trajectories occur over distances generally much greater than a length which is the inverse of the Bragg scattering vector Q . the diffraction pattern that results fluctuates at a rate dependent upon the swimming speeds of the scatterers.53 7. Electrophoretic light scattering spectrum of mononitclear cells obtained lrotii the blood of lcukcniic patients. in some circumstances. Certain applications require high field strengths. wave front matching to accomplish adequate signal heterodyning must be done carefully. in which case one runs the risk that particles will be leached off the surfaces of the electrodes and contribute an artificial signal. and. LASER LIGHT SCATTERING FIG. d o not interact. and are spherically symmetric insofar as their light scattering properties .7.326 7.t). (Adapted from Stnitti should be cooled to effect adequate temperature control. In many cases the tracks of motile microorganisms can be represented as essentially straight lines with intermittent stops.7. and the organisms' velocities seem to be essentially constant over similarly long distances (see Fig.
7. an analysis of the twiddle movements predicts functions which are similar and in good agreement with data. where V is constant.(Q. The first term in expression Eq. Holz and S.2. For motile bacteria. (7. Berg and D. Nature (Loiidoii) 239.c( therefore represents the resting fraction. The second term follows if we assume that movement occurs only by diffusion during the resting phase. Chen. movements during the rest period are in fact generated by the microorganism itself. 500 (1972). Certain strains of flagellated bacteria stop and "twiddle" between successive straightline portions of their tracks (see Berg and Browti'"). however. MOTILITY MEASUREMENTS 327 FIG.7.2) where LY is the fraction of the population of microorganisms locomoting at the instant that the sample is probed. These " twiddling" movementss6 result from uncoordinated oscillations of flagellar bundles and do not necessarily lead to a correlation function of the form given in the second term of Eq.18) that Z.2) results from an assumption that during locomotion the trajectories are given simply as r(t + z) = r(t) Vz. '' H.7. (7.a)eD"*'. (7. For the model illustrated in Fig. 15 (1978).7. Brown.7. J .t ) is given as (7.1) hi LY(eiQ'vr ) + (1 . .7. 1 . are concerned.57 + '' M .8. H. A. Approximate pattern oi' the trajectories of motile microorganisms. (7. C.2). 8 it follows from Eq. Biophys. 23.
R.58 If Eq. J . (7."') will superimpose.g. J .~' e. unless data for I ' ( X ) can be fitted by an analytic function. the procedure indicated by Eq. "I '' G. Marsh. so that the distribution of velocities is spatially isotropic.) 33CI.4) But. T.865 (1974). 22. [The distribution W (V )is related to the velocity distribution of the microorganisms P(V) by integrations of the latter over dire~tion. Craig and J. (7. Nossdl. . Biophys. R. (7. LASER LIGHT SCATTERING If no external forces act upon the microorganisms.2) takes the following form: (7. 171 (1972). 203 (1978). a mixed correlation function such as that given in Eq. (7.7. 1 The most striking property of Eq.7. Consequently. In contrast. 14.7.7. the term (eiQ'vt)in Eq. other procedures such as spline fitting5' or assumption of a specific analytic form for the swimming speed d i ~ t r i b u t i o n ~ ' *have ~~."' 1 1 ': P(V) sin q dq do. (7. N o s 4 and S. Fr. Biophys.4) may be impractical because of uncertainties related to truncating the transformation. Stock. the signal arising from diffusing particles scales as Y = Q't. H.~ been ' employed to deduce features of W (V ) .7.1) can be analyzed by scaling the experimental autocorrelation functions according to X = Qt and subtracting data taken at one scattering angle from those taken at a different angle. Berne and R. J . Phys.3) is written in terms of the scaled variable X = Qt as (7. A useful parameter of motion can sometimes be obtained by examining the 5H '" B.3') we see that the swimming speed distribution can in principle be obtained by Fourier transformation as W ( V )=  2 71 v m!onr sin X V [ X l ' ( X ) ] d X . 21. the autocorrelation functions associated with locomotion (eiQ. Biopltys.3) is the Q dependence of the predicted autocorrelation function : if data taken at different scattering angles are plotted as a function X = Qt.7. Consequently. J .328 7. F.3) where W (V) is the distribution of swimming speeds of the scatterers. (7. Chen. W (V ) = 471V' s. J.7. 79 (1978). Hallett. (Orsay..7.
movements other than simple translation should also contribute to the light scattering spectrum. S . a ’”T.7.1)(7.7.Z(X)]lx+o. Noture (London) N ~ M Bi ’ d .~~. the halfdecay point of the autocorrelation function is found to deviate from that predicted for motile spheroids by an ever larger amount as the scattering angle is increased.43 Recent experimental data on Chlurnydornonas. J.5) Many assumptions have been employed in order to derive the relationships given in Eqs. coli bacteria. J . Schaefer.~ or’ example. Boon. though. Generally.3) are p r e d i ~ t e d . H.62 which from Eq. Various model calculations have been performed in which such spatial asymmetries are taken into account. R. . (7. by careful experimental design it should be possible to distinguish and quantitate certain complex movements of the scatterers. differences by a factor of two or greater might be observed.7. However. where only translational motions are r n e a s ~ r a b l e . 847 (1974). Unfortunately.7.7. MOTILITY MEASUREMENTS 329 initial decay of the autocorrelation function. that motion occurs parallel to the major axis of the scatterer). 253 (1973). R. J . Nossal. (7. and S. 35. and S. then significant deviations from simple Qt scaling as indicated by Eq. t). as they generally contain translating but perhaps wobbling scatterers.62a Because the dimensions of microorganisms are large compared with the wavelength of light.7. D. Nossdl and S.7. P. Hallett. some rotating or twiddling individuals. b3 64 ” R .~~ when a continuous ellipsoidal mass distribution is assumed for the scatterer. Perhaps the most severe is that of spherical symmetry. Nickel. J. Theoretical a n a l y s e ~ ~ ~ . Chapter 7. 164 (1974). ~ ~ indicate that wobbling motions and rotation should increase the rate of decay of Il(Q. Alpert. samples of microorganism such as flagellated bacteria are amenable to almost unlimited possibilities of data fitting.60 whereas a model of a prolate ellipsoid coated with a thin membrane leads to periodic deviations from Qt scaling. Biopl7j. Biophys. and B. 244. W. . exhibit scaling of the halfdecay point over a wide range of scattering angles.5).s.557 (1981). swimming microorganisms have the shapes of cylinders or oblate spheroids. ~ ~ F . G. H . For solid particles having the size and shape of E. (7. Nuture (London) 248. and they are so large that structural factors must be considered (cf. these perturbations are unimportant at very small scattering angles.3) is seen to be related to the rms swimming speed as (V2) = 1 0 00 V 2 W ( V ) d V= 6X’[1 . Racey. Chen. (7. Banks. The discrepancy between the autocorrelation functions for spherical and ellipsoidal particles increases with the size of the scatterers or degree of asymmetry. ~ ~ Thus. 14.5). If it is assumed that the direction of locomotion of a microorganism is strongly correlated with its orientation ( e g . Chen.
Sci. the more ambitious the applications. Attempts to correlate laser light scattering data with visual observations of sperm viability suggest that light scattering can form the basis of a rapid and objective assay. D. using laser light scattering as an assay for relative changes in motion may be more appropriate than employing it to determine absolute values of parameters associated with detailed movements. Verdugo. A particularly suitable application is the study of protoplasmic streaming in large algae67*68 and giant slime molds. 2 I2 ( I 977).^'. Nickel. 28. Blandau.6).373 (1976). and G. O n the other hand. B. 457 (1979). using techniques similar to those developed for measuring the speeds of charged particles responding to an electric field (Chapter 7.229 (1977). N. Biophys.. selfconsistent computerassisted data fitting gives reasonably good agreement with certain simple theoretical model^. R . Instrum. 17. Jouannet. Langley. Craig. P. Biuphj's. A. GaddumRose. I. Acla 444.Applications in Cell Biology Laser light scattering will probably receive increased use in studies of the dynamic behavior of biological cells and tissues. H. 893 (1976). F. R . and D. and D. one can determine the velocity distributions of cytoplasmic constituents quickly and objectively. V. H. 7).and P. V.254(1977).69770 Protoplasm contains inclusions ranging in size from that of mitochondria down to that of submicroscopic particles. R . Ware. Rui.330 7. Piddington. However. Newton. Thus. though. '' R. R . 1. 'OS. R. . Ross. P. Berge. P. 68 K .61 The motion of sperm cells through extracts of cervical mucus has also been BY orienting the sperm in narrow c a p i l l a r i e ~ . Ware. Acta 496. Langley. 47. However. Biochim. Unfortunately. K. 108 (1976). C. generally the more complex and uncertain will be the physical models needed to explain experimental observations. 16. several points must still be resolved concerning the interpretation of light scattering data from cells as large as spermatozoa. 71 1 (1975). '' W. Sattelle. Biophys.8.the ~ ~ entire swimming speed distribution could be determined from the Dopplershifted components of the spectrum (see Chapter 7. Biophys.66" 7. Inws/. particularly Fig. h9 R. LASER LIGHT SCATTERING fraction of resting or diffusing particles. G y n c w l .^^ One potentially significant biomedical use of laser light scattering is in studies of spermatozoan motility. David. and B. W. M.6. Sattelle. '"T. J. Muslacich and B. Hallctt. Hiochirn. Mustacich and B. B. Ford. J. Nuture (London) 252.8. DuBois. among which is the importance of head rotations as compared with translational motion. and members which collide with each other. Lee.
and assessing the extent of peripheral vascular disease. Quunr.. Illinois. Biophys. ‘’ R. Rowman. 1. c‘olcl Spring Ilurhor Symp. c‘. 248 (1977). the movements of which are considered to be the sum of a periodic ciliary oscillation and a modulation arising from the coordinated motions of clusters of cilia (“metachronal coordination”). ’‘ R. and A . Carlson. Giddon. Bonner and F.’F.9. Pli~siol. Ann.7. Haskell and F. Verdugo. F. cinematographic methods are expensive and cumbersome. Ins/rwiz. However. Nossal. B i d .C ~ . the theoretical analysis is rather complicated and several approximations have to be made. P. Gy./ i m . K. R. L. the theory gives a good representation of the prominent features of the measured autocorrelation function. Potential applications include detecting the onset of shock in patients confined in a criticalcare facility. 1258 (1980). and D.“ p. 37. R P P . B.72 A theoretical model of ciliary beat phenomena used to explain light scattering data72 trcats the ciliated epithelium as an oscillating rough surface. B. Bowen. Verdugo. Grn. S. 305 (1978). Ware. Bonncr. Lee and P. Nevertheless. ~~ 16. J . B / o p / i ~1. 53Y. R. F. D. BLOOD FLOW 33 1 Another interesting use of laser light scattering is as a probe of ciliary activity on the surfaces of cells and epithelia.8. Inc. . investigating the effectiveness of flow in skin transplants.633 (1975). Carlson.33. W. Even for this simplified model. 5 . Lee and P. 197X. I I I S (1976). Carlson. and generally cannot be used to study tissues in situ. Wunderlich. Bonner. in “Proceedings of the ElectroOprics Lascr ‘78 Conference.  ’? . determining blood flow in tissues chosen for biopsy. 389 (1972). R. R . 7Jr ” W. Fraser. Industrial and Scicntilic Conference Management.74“ 7. 27 (1981).Sci. 1. Compact instruments currently are being d e ~ e l o p e d ~ “ ’ ~ for specialized clinical applications which utilize flexible fiber optic probes ’’ W. Yoshino. 51. B i o n w l . D.9. Blood Flow Instruments based on laser light scattering can be designed to monitor local blood flow in a noninvasive manner. I . Lee and V e r d ~ g o ~have l. Chicago. B i ~ p h j .~~ used intensity correlation spectroscopy to measure the periodic frequency of ciliary beat in cells of the rabbit oviduct” and havc shown that their results agree with data obtained by phase microscopy and highspeed cinematography. Fujinie and S. D. A third instance where laser light scattering has been adopted for investigations of phenomena ofcellular physiology is in studies of resting and contracting This undertaking is especially challenging due to the complex structure of muscle tissue. 65. R . Folger. . Light scattering instruments based on fiber optics techniques can potentially be used for clinical investigation of ciliary activity in the respiratory tract. . and R . C.
) the structure The brackets indicate averaging over speeds. with velocities which are constant over distances large compared with the wavelength of light. even though light is seemingly incident upon the tissue from a specific direction. only the net path between a red cell and the fixed detector determines the phase shift of the scattered light. Stern.4) and (7. R. even though the precise value of the proportionality coefficient is hard to determine theoretically. Stein. Nossal. We need not consider the details of photon trajectories subsequent to interaction with the erythrocytes because. H441 (1977). Kciscr. do not interact with other moving targets (i. J . Consequently. D. Bowman.. before interacting with an erythrocyte. Opt. Thus. G. M. A m . by Eqs. 20. Bowman. J . Assume further that photons. P. 2097 (1981). P. R. having an effective radius a. Stern.78 This process is difficult to model analytically because of possible multiple scattering from the erythrocytes. Bowen. Photodiodes are used as detectors. E. Suppose that laser light is shined on a subject’s extremity (such as a finger) and detected as backscattered radiation. 79 M. and specially designed lowcost electronic analyzers are used to extract flow parameters from the data. Parma. W. and J .1) where the 0 integration accounts for all possible scattering angles. 77 18 . and scattering from surrounding tissue.sia/.~ ~ that changes in the width of the Dopplershifted spectrum can be correlated well with changes in flow. Chimosky. and ~ ( Q u is factor which. L. is given above in Eq. other blood cells) before emerging from the tissue. uncertain knowledge of the spatial pathways for flow through the vascular bed.3). D.5). assuming a fixed detector and 4n incident illumination (sin 8 is a geometric factor). Nonetheless. we assume that the scatterers are spherically symmetric. in the RayleighGans limit. Jr. F80 (1979). although several collisions with static tissue elements might occur.9. H . D. 7 R d R . R. various s t ~ d i e s ~ ~have *’~ indicated . and R.332 7. Stern. the intermediate scattering function can be written as (7. (Lundun)232. Nuiurc (London) 254. Phl~.y. M. photons make many collisions with the surrounding tissue and are randomly diffused.5. Boiiiier and R. A .5. For simplicity. D. One model which shows how the spectral bandwidth scales with flow rate is as follow^. Lappe. 56 (1974). R. D.~iol.^^^^^" Assume that. J .77. LASER LIGHT SCATTERING to collect light scattered from tissues such as skin or gums. (7. Osgood. once scattered by a red cell. (7. We also presume that the cells move in random directions in the capillary bed. L. at the time of the first interaction of a photon with an erythrocyte illumination from all directions is equally probable. L. P/i.e. App/. Holloway. Bowen. D. The motion of red cells in the microvasculature causes Doppler broadening of the incident light.7.. 231.
BenSira. Appl.(Mitrslritrqron.10.. + (7. Benedek.1) Y(X) = (7.5. 14. Examples are the lens and oitreous humor of the eye.9. either in situ or as reconstituted models. (7. if we set a = 0.ic.9. L = 4nnll'a and T = ( V2)li2t/$a. T.3) where ( V2)"' is the rootmeansquare value of the velocity.1av1.9.1 and numerically evaluate Eq. and C. blood flow in arteries of experimental animals has been measured with a fiberoptic catheter. S(. ' ~have shown that inelastic light scattering can be used to study blood flow noninvasively in human retinal vessels. (7. I . . we can express the result of the integration in terms of reduced variables of length and time. Using in Eq. E. which are.) 186. The equivalent relationship to the halfdecay of the power spectrum is ( V2)1'2 = 4.4). Opt. f1. A m w . (7.9. Tanaka. (7.9.1) the expressions given in Eqs.1. If the speed distribution is Gaussian. have the mechanical characteristics of amorphous solids or highly viscous fluids. Riva.*l Also. respectively. T. 830 (1974) " '* G. J . 68.C. Tanaka et ~ 1 1 . Riva. 189 (197. Feke and C.ricc.3). 7.9. Gels and Solutions of Fibrillous Proteins Various biological materials.7. extracts of cell '" T. (7. When 2uL2 is large (as for red blood cells) the resulting expression for Z ( t ) effectively becomes a function only of T. GELS AND SOLUTIONS OF FIBRILLOUS PROTEINS 333 A good analytical approximation to Eq. An instrument based on their prototype studies now is being used for clinical diagnosis. we find the halfdecay of the autocorrelation function to be related to the rms flow speed as ( V2)1'2 = 0.2) where CI = 0. Soc.5) which facilitates the integration in Eq. . example. (7. A more elaborate derivation also accounts for multiple Doppler scattering from moving blood and provides information on the manner in which spectral shifts depend on blood volume in addition to red cell velocity. 526 (1978).5).2) and (7. it then follows6o that the expression in the angular brackets is (sin[Q V t ] / [ QV t ] ) = eQ2(v2)'2i6.*2although the necessity for surgical intervention limits the usefulness of this particular procedure.71atQ:. I(t) = 2 4 2 ~ 1 T2). Opt. Tanaka and G.10.9.4) The halfdecay point of Z ( t ) is proportional to (V')'/' through the relationship t l i z = ($a/( V 2 ) 1 i z ) T l iFor z .9.
2) where p o is the equilibrium density. however. (For example.) Another advantage of certain light scattering techniques is that they can be used to study local properties of a sample. Thus. particularly if a sample is easily perturbed by the stresses imposed by conventional rheometers. cervical mucus. LASER LIGHT SCATTERING cytoplasm. . t). Clearly. Upon taking spatial Fourier transforms we find 6p(Q. 5151 (1973).10. t ) .1)  where U(r. In a continuum the deviation of the density p from local equilibrium is given by the continuity equation M. in which case light scattering fluctuations are linked to local displacements of material within the sample. t ) one first needs to assume a physical model. . Tanaka et considered the question of light scattering from a gel and took the *3 T.10. in essence they can be characterized as aqueous solvents containing a relatively high concentration of long polymeric molecules. long molecules tend to align in the stress field between the plates of a viscometer.2. P h y ~59. (7. Chew. and lubricants of joints such as sinovial fluid. Although these substances typically contain many constituents. L. t) = Ar. by Eq. In Chapter 7. It then follows that the diffraction pattern varies because of local changes in density. gels formed of fibrin and other blood constituents. Laser light scattering can be used as a probe of the viscoelastic properties of such materials. To compute U(r. (7. J . In general. Benedek. It is preferable to consider a gel or dense polymer solution as a continuum. t ) is the displacement vector of a differential volume element whose equilibrium position is at r. In certain instances the noninvasive quality of laser light scattering is essential.18’) we see that thc autocorrelation of photons would be sensitive to autocorrelations of various components of the Fourier transform of the displacement vector. t). and G .4 we discussed procedures for probing the motions of dilute molecules in solution. 0.3 34 7. We here assume that our sample is optically homogeneous on a length scale comparable to the wavelength of light. Tanaka. Hocker. B. In other instances the material can flow if it is subjected to steady stress. the more easily it is disturbed if classical rheological techniques are used for measurement. the less rigid the material. If the interaction between the polymer chains is sufficiently strong. and it exhibits viscous behavior. (7. whereas conventional rheological techniques probe bulk moduli. the material is resistant to deformation and has gellike characteristics. t)v U(r.ipoQ U(Q. physical models invoked for diffusing macromolecules are unsuitable for analysis of viscoelastic materials whose properties derive mainly from frequent strong interactions between polymer chains.
10. D. Eq. only the moduli appear in the term which multiplies the exponential. Fr. u ) . P. (7. S.971 (1977).10. K . Their assumed kinetic equation has the form83 pazu/at2 = p u + (K + $p)v(v. 86 .jau/at. J .5) where k is Boltzmann’s constant. and f .3) where K and p are taken to be the compressibility and shear moduli of the fiber network alone and f is a friction coefficient which is proportional to the viscosity of the gel liquid.4) Thus the photon autocorrelation function would decay with a time constant whose Q dependence is the same as for Brownian motion [cf. Candau. t ) ) = (WFY) exp{C(K + $p)/flQZt). Solution of this equation. (7. J . Fr. ~ ~ * ~ ~ although deviation from a single exponential decay also has been reported.4) is given asE3 (7. O>u.10. yielding’ the following expression for the autocorrelation of the component of the displacement which is parallel to Q: (U.3) and (7. and L the illuminated length of the scattering volume. Note that.)38.(Q. Hild. Hecht. Geissler and A. Thus they considered movements of a gel lattice against the fluid.7. (7.86 In these studies the driving forces for the displacements are thermal excitations. (Orsay. yields waves which propagate or are damped depending on the values of K. p.10. H e n . although the parameter which can be obtained from the decay of the autocorrelation is the ratio of the elastic moduli to the friction coefficient. GELS AND SOLUTIONS OF FIBRILLOUS PROTEINS 335 polymer lattice to move as an elastic continuum subject to frictional interactions with surrounding fluid. 955 (1978). The other terms in the expression have the same definitions as in Eqs. J.) 39. 190(1975). E. (Orsay. Marromolecuk~s8. Phys. M.10). assuming infinite spatial boundaries. Munch. and the term ( UZ(Q)’) is evaluated by thermodynamic argum e n t ~ The .(Q. The basic QZ dependence of the decay time has been verified in several l a b o r a t ~ r i e s . T the temperature.4. (7. L. and G .10. Thus. by measuring the intensity of scattered light and knowing instrumental and material H4 85 J.1)J but with a proportionality coefficient given as (K + $p)/f. Carlson. Phys. ~ ~field autocorrelation function for polarized scattering then derived from Eq.2. Nonpropagating modes are predicted for soft gels. p. (7. Wunand F.2.
10. Biol. 38. + " T. I A ." Significant deviations from an exponentially decaying form have been observed for the autocorrelation function. Macromo/oc. J . to examine the formation of cataracts in excised bovine lens. fh. Chylack. NOSSdi. . Mu/.Lett. in a somewhat analogous fashion. discrepancies between experimental results and the expression given by Eq. The technique involves driving a sample with a weak external mechanical field and relating obscrved resonances in the light scattering spectrum to the standingwave frequencies of gel displacements. RcL'. Nossal. F. 31 1 (1979). 1010 (1977).66 and it may be that a modification of the basic kinetic equation (7. D.' 9*90 Attempts have been made to use scattering schemes based on thermal excitation to study actin gels.771 (1977).10. Tdnaka. Ishimoto. (7. 50.1) should be used to describe such fluid like.vs. A. materials. 3105 (1979). B. Tanaka. T. Similarly. but heterogeneities in the sample have made it difficult to obtain consistent results..273 (1974). 89. This modulus pertains to the solvent and lattice acting together. Recently a light scattering scheme has been developed which yields values of the bulk shear modulus G. C. 139 (1975). Fraser.336 7. Jr. 95. LASER LIGHT SCATTERING parameters. This procedure has been used by Tanaka et aL8' to study phase transitions in polyacrylamide gels and. Gelman and R.4) have been observed in studies of the properties of cervical mucus. it closely approximates p for the lattice alone. L. . '" R.C. Ishimoto.7. but because of the low shear resistance of the solvent. and Ishiwata. though highly viscous.) 197. '' T. Phv. p p / . one can obtain values of K + $ p andf separately.irnce (Wrrrkiny!on.u/cs 12. Sc. D." A similar technique has been used by Geissler and Hecht" to study the concentration dependence of the compound modulus K $ p of various polyacrylamide samples. Carlson and A. and C. S . 8 9 R.
a few tenths of a milliliter of a 1 or 2 % solution can often produce interesting data. The technique. molecular size and shape. is called “smallangle’’ or “lowangle’’ scattering. the study of the diffuse scatter given by solutions of biological macromolecules. or sometimes simply “solution scattering. Observed under proper conditions. it is easy to prepare samples. therefore. and knowledge of some aspects of internal structure. is the topic of this article. is not a prerequisite. and amorphous solids. most of the macromolecular scatter is confined to regions close to the direction of the incident radiation. In biological experiments the scattering studied is the incremental contribution to the total scatter of a solution due to macromolecular solutes. Since macromolecules are large compared to the wavelength of the radiation generally used in such experiments (10100 versus 1 A). Inc All right5 of reproduction in m y form reserved ISBN 012 415962. Analysis of this contribution can lead to accurate estimates of molecular weight and volume.1.9 . Measurements of this kind have contributed significantly to our understanding of the structures of gases. VOL. SMALLANGLE SCATTERING TECHNIQUES FOR THE STUDY OF BIOLOGICAL MACROMOLECULES AND MACROMOLECULAR AGGREGATES By Peter B. All that is needed is a monodisperse solution of the molecule or macromolecular aggregate in question. Moore 8. the information obtained relates to the molecule as it exists free in solution. The variant of this experiment of most interest to biologists. diffuse scattering results. First. Thus the potential for artifacts is minimal and the 331 METHODS OF EXPERIMENTAL PHYSICS. the elastic component of this diffuse scatter can yield useful information about the structure of materials at the molecular level. Crystallization. liquids.” Smallangle scattering commends itself to the biophysicist for three reasons. for example. Introduction 8.1. The Nature of the Technique When shortwavelength radiation (A about 1 A) strikes amorphous materials. Second. sample sizes are small.8.1. In addition. 20 Copyright 0 1982 by Acdderni~P r e s .
The effort required is very large. data collection IS fast. the appeal of the micrograph is often overwhelming when compared with a model inferred from scattering data. There are undoubtedly many reasons for this. The completion of the splendid installation at Institut LaueLangevin in France in the early 1970s gave tremendous impetus to the field. a new kind of radiation detector has been introduced into the field. the positionsensitive detector. notably at the Julich. The smallangle field grew up using x radiation as its standard tool. Historical Comments Guinier's paper' on the measurement of the radius of gyration of particles in suspension from their diffuse scatter is a convenient landmark from which to date the birth of the smallangle field.1. when the scientific world was completing its recovery from World War 11. The attraction of crystallographic analyses of macromolecular structures lies in their richness of detail and accuracy. are widely used and become more effective every year. Harwell. by what is to many a nonintuitive process. Guinier. By the mid 1950s. and its use has significantly broadened the range of problems approachable by solution methods. electron microscopy and xray crystallography. paradoxically. (Poris) [5] 12. However misleading it may be. a new kind of radiation has become available for smallangle work. While this radiation is effective. and Brookhaven laboratories. . At the time of introduction. Phys. Although the method has always attracted adherents. Ann. neutron radiation has many advantages (see below). 8. smallangle techniques were virtually unique in offering a direct means for estimating the size and shape of biological macromolecules. thermal neutrons. 8. but historical factors have certainly played a role. Third. SMALLANGLE SCATTERING data correspondingly likely to be relevant physiologically. the competition.338 8.2. Yet. a few days often suffice. Detectors can be constructed to count ' A. two new techniques which could provide similar information had come of age. The interpretation of the molecular images generated by an electron microscope can be difficult. 161 (1939). but the pictures it produces are visual and direct. but the results cannot be equaled by any other technique. First. Second. the smallangle field is as vigorous now as it has ever been. The Sources of Current Interest Crystallography and microscopy. Four important innovations have contributed to its current strength.1. The first neutron facilities suitable for biological work came on line in the mid to late 1960s. it has never become widely practiced despite its virtues.3.
These devices replace the traditional singledetector. a synchrotron radiation source is a pulsed radiation source rather than a continuous one. There is a definite possibility that the unique properties of synchrotron radiation sources may engender some new applications of smallangle techniques. Among other distinctive features.1. In addition. in the past few years. Computers have also materially simplified the task of data collection. stepscan mode of data collection and increase the speed of data acquisition roughly 100fold. 8. . There are many steps in the analysis of smallangle data which require numerical processing beyond the capabilities of hand computation. Synchrotron radiation sources can provide x rays at all wavelengths useful in scattering experiments and at fluxes orders of magnitude higher than obtainable with conventional laboratory equipment. as is the case with ordinary xray generators.4. The experimental problems faced will be discussed in general terms. The applications of these methods will be illustrated from the recent literature. Purpose The objective of this article is to provide the reader with an introduction to the macromolecular smallangle scattering field. INTRODUCTION 339 either x rays or neutrons and to determine where in the sensitive volume counting events take place. They can be built to count all the scattering angles of interest in a smallangle experiment simultaneously. the influence of the computer has been felt in the smallangle field as in so many others. The widespread availability of computers has encouraged the development of new and powerful algorithms for processing smallangle data. There is hardly any physical technique that would not enjoy strong interest in response to stimuli such as these. The net result of these developments is that a variety of experiments which could hardly be considered using the equipment of 15 or 20 years ago are now practical. a new kind of ultrahighflux xray source has become available. This article will describe the kinds of information that can be obtained about a biological macromolecule or macromolecular aggregate by smallangle scattering using both x rays and neutrons.and twodimensional detectors are feasible.1. and their use is now routine. As a result the sophistication of data analysis in the smallangle field has improved and along with it the reliability of results obtained. the terms of trade between experimenter’s time expended and data acquired have improved 1001000fold. its potential is still under investigation.8. as will the approaches generally taken in data analysis. Third. Fourth. including timeresolved experiments. Both one.
” Oxford Univ. Much of the theory of the method was developed in the field’s early years with the result that the older reviews retain their utility to a degree which is unusual in the biochemical and biophysical literature. Beeman. W. 27 (1976). 141 243. Syracuse. Part C. Pilz. 1973. England. New York. B.I4 ’ A. ed. W.” Kynoch Press. 39.1. J.911 (1976). Fournet.” Wiley. Rev. James. 321 (1957). N. Prog. Phys. Rep. T. tthaca. “Theory ofThermal Neutron Scattering. Kaesberg. New York. l4 W.g.481 (1972). New York). Brumberger (ed. For example.Krdtky and I .340 8. ’‘ Neutron Diffraction. Academic Press. W. Q. “International Tables for XRay Crystallography. 1968. Anderegg. two symposium proceedings have appeared in the Journal of Applied Crystallography (1974. A rigorous theoretical development of the theory of thermal neutron scattering can be found in Marshall and Lovesey. Marshall and S. 1955.). ed. 11. Lonsdale. pp. Many other reviews can be cited which deal with the xray smallangle In addition. 1975. G. R. 27. Background information on xray techniques may be obtained from James’ and the International Tables for XRay Crystallography. E. W. Methods Enzymol. 0. (ed. P. “The Optical Principles of the Diffraction of Xrays. Pilz.Biophys. Biol. Q . 5. London and New York.5. H. New York. Pilz. Webb. 32.” Cornell Univ.” 3rd ed. Oxford Univ. “SmallAngle Scattering of XRays. Biophys. Schoenborn. Phys. Lovesey. The proceedings of a recent symposium dealing with all aspects of the application of neutron scattering to biology are a ~ a i l a b l eJacrot” . and S. Birmingham. London and New York. 1962. 9.12 Bacon13 has provided an excellent work for those wishing an introduction to neutron diffraction.Jacrot. F. H. Press. 1971. and M. Kumosinski. Krdtky and 1. Guinier and G. Brumberger’). I. ‘ ’ ‘” ’ . the proceedings of a number of symposia on smallangle topics have been published over the years (e. Bacon.~ has reviewed the neutron smallangle field. 39 (1978). P. Gordon & Breach.1978).Re[). Timasheff. the book by Guinier and Fournet2 remains invaluable despite the fact that it has reached its 25th anniversary. J . June 1965.). Earlier Reviews The smallangle field has been reviewed regularly over the years and a number of useful works are available. Brookhuwn Symp. 151 (1973). K. New York. 1967. Press (Clarendon). Leach. 8. W. Hundb. in “Physical Principles and Techniques of Protein Chemistry” (S.. B.. 0. “ SmallAngle XRay Scattering” (Proceedings of Conference on SmallAngle XRay Scattering.). SMALLANGLE SCATTERING and sufficient references provided so that an interested reader can complete his study of the field. Press. Pessen.
1). fi and fj are the scattering lengths of the two atoms appropriate for the type of radiation used. at a given R.] In the neutron literature.f.2. The Scattering Signal If the scatter given by a single isolated molecule could be observed over a period of time as it diffused rotationally. where there is no physical equivalent of electron density.8. Ann. It can be ignored in many cases or included infi(R) as a small correction factor. however.The Experimental Problem 8.1) describes the scatter given by gases at low pressure.2. 1 3I(R) is the number of photons (neutrons) scattered by a single molecule per steradian of solid angle per unit time. Equation (8. in Eq. For x rays. The scattering of the macromolecules in solution.1) by P. if the molecule is experiencing an incident radiation flux of one photon (neutron) per unit time. = (e2/mc2)J(R). (Leipziy) [4] 46. small scattering angles) can be regarded as a continuum having an average f per unit volume.Debye. r i j is the distance between the ith and jth atoms in the molecule and both sums are taken over all atoms. atomic scattering properties are tabulated directly as scattering lengths. will not be identical to that which they would give in uucuo. 809 (1915). where fi(R) is the atomic structure factor for atom i which can be thought of as the Fourier transform of the electron density distribution of that atom. THE EXPERIMENTAL PROBLEM 341 8. which is not zero." [The polarization correction will be very small for low scattering angles.1) usesfis. bi being the scattering length of the ith atom. (8.2. where molecules are well separated and behaving in an uncorrelated manner. which are measured relative to a vacuum which has an averagefper unit volume equal to zero. hencefi = h i . 1.1) Here R = (2 sin @/A. Flux is measured in photons (neutrons) per unit area per unit time. .2. The effect of being immersed in this continuum can be taken into account to a good first approximation by replacing each.2.1. It is also the correct basis for understanding macromolecular contributions to the scattering of a dilute solution. Solvent viewed at low resolution (i. where 26' is the angle between the incident and the scattered radiation and A the wavelength of the radiation (Fig. Equation (8. Phys..2.e.2. where the area is measured in the same units as the scattering lengths. a timeaveraged scatter I(R) would be measured which is directly related to its ~ t r u c t u r e: '~ (8. or scatteringlength density.
the partial specific volume of the solute. The experimental objective of virtually all macromolecular smallangle studies is to measure Z(R). 19 A.1) holds. Adv. 37. This subtraction is justified provided there are no correlations between the positions of the molecules of the solvent and those of the macromolecular solute. Monursh.2. are scaled to normalize for any differences in the total amount of radiation used in the collection of the two profiles and I ( R ) found as follows: (8.'6 If these positions do not correlate. the reverse operation is not possible: a knowledge of Z(R)will not give back the structure. The two measured profiles.3). Isol(R)and Zs0.the macromolecular contribution.2. reflected in the cylindrical symmetry of I ( R ) about the direction of the direct beam.'6p19 Equation (8. Bio/. Isolation of the Macromolecular Signal All components in a solution contribute to its scattering.u i p . problems can arise. is required. (8. '* S. (8. of the solution. Eisenberg and H.2) will hold.2).2) where u is the volume of solvent in an aliquot of volume u.2.342 8. The rotational averaging involved in the production of the signal. a number of lowerresolution aspects of the molecule's size and structure can be elucidated once Z(R) is known (see Chapter 8. Cohen. 849 (1975). although knowledge of a molecule's structure at atomic resolution enables one to compute f(R).1). 125. the scattered intensities due to solute and solvent will simply add to give the total measured signal. where uiis the volume made inacessible to solvent by the ith atom and p the solvent's average scatteringlength density. A sample of pure solvent and a sample of the solution are measured in the same apparatus under the same conditions. as is knowledge of concentration of the solute. The reader should recognize that. averaged over the time of observation. However. in order to carry out the subtraction indicated. 598 (1973) H. SMALLANGLE SCATTERING j .2. Hyman. Goodisman and H. Cheni. under conditions in which Eq. Mwrornolrculrs 8. Ser. Timasheff. Chem. is to be J . If solvent molecules bind to the macromolecules or organized regions of solvent exist around them. S. . therefore. N . Brumberger. Note that knowledge of 6. J . 'I' l7 . leads to the loss of all information about the relative orientations of the interatomic vectors whose lengths rij appear in Eq. Mol.2. 8."(R).2. (8. which are simply the counts registered by the detector at each scattering angle. with appropriately evaluated As. and Eq. 104. 327 (1973). The separation of macromolecular contributions from those of the solvent is usually done in a simpleminded manner.2. as is made clear below. 355 (1968).
and (iii) the type of measurement to be made. appropriately collimated beam of radiation directed at a specimen held in a fixed position. and a line drawn between the center of the beam’s intersection with the sample and the detector axis. and ( 6 ) detector. defined by the beam center. Its lowangle region will be well approximated as a plane.2. I . The macromolecules themselves may aggregate or otherwise correlate in solution. a sample holder. (2) collimatian slits. and a detector (Figs. (3) guard slits. hence its name. (8. The radiation scattered by the specimen is measured by the detector at some distance from it.1)does not hold for multiple scattering (see Beeman et d 3 ) . a monochromator. While it is not appropriate to discuss instrument design in great detail. (4)specimen. Critical components and positions in the instrument are numbered: ( I ) radiation source. some general comments are in order. Schematic diagram of a lowangle scattering apparatus. The plane of registration is more properly a surface of registration and is the region of space explored by the detector. a significant fraction of the radiation scattered by the average macromolecule will be scattered again by others before emerging from the sample. THE EXPERIMENTAL PROBLEM 343 regarded as a firstorder approximation for estimating I ( R ) from experimental data. the macromolecular contribution will be proportional to I(R). The scattering angle 20 is the angle between the optic axis of the instrument. In the dilute limit. (5) plane of registration. (ii) the kind of radiation source available.3.2.8. 4 5 FIG. . The purpose of these elements is to provide a monochromatic.2. The design of these elements is dictated by three factors: (i) the type of radiation being used.the profile for a single molecule. SmallAngle Apparatus: General Features All smallangle instruments consist of the same set of elements: a radiation source. Equation (8. 8. Distortions in Z(R) due to these effects can be dealt with by making measurements at several concentrations and extrapolating to zero concentration. 1 and 2). If the sample is thick. a collimator.1) be measured is that the sample be thin.2. The only condition besides diluteness which must be met by a sample in order that the scattering described by Eq.
The scattered radiation should then be detected using a device with an infinitesimally small acceptance aperture.344 8. Thls instrument has a monitor counter (thc cylindrical object on top or the flight path chamber at Its detector end) which measures backscatter from the beam stop. Failure to achieve perfect monochromatization of the beam has a similar effect. (4) sample holder. the ideal smallangle instrument should have a sourcemonochromatorcollimator combination which delivers a perfectly monochromatic. It was designed by D. The instrument depicted herc is in operation at Yale University. If the beam is a finite size. ( 5 ) flight path chamber. Johnson. In all cases the signal measured at some nominal scattering angle 28 will not be Z(28) but some kind of weighted average of 1(28) over a range of angles 28 f A. The two functions Z(28).2. The numbered elements are ( I ) xray tube. Engelman and constructed with the assistance of J . M . infinitely fine pencil of radiation which is directed through an infinitesimally thin specimen. the profile measured . and the weighted average of 1(28). and (6) positionsensitive detector. The reason this kind of geometry is desirable is that if the detector aperture is finite. The tubes connecting the source to the mirror and the mirror to the guard slits are all evacuated as is the fllght path chamber.4. The SmallAngle Compromise The principal problem in measuring diffuse scatter stems from the fact that the signal being measured is continuous. are different. (2) focusingmirror monochromator. SMALLANGLE SCATTERING FIG. In order to measure such a profile. Xray smallangle instrument equipped with a linear positionsensitive detector. given by Eq. even an ideal detector will be seeing scatter from particles whose angular relationships to the detector vary.2 . 8. (3) quard slit assembly. it will admit radiation scattered over a range of angles and at any given time will not measure the signal scattered at a single angle only.1).2. measured by instruments which deviate from the ideal. (8.
Rec. As a general rule. the larger the smallest angle at which measurement must be made is. An electron beam having an energy ofa few tens of kilovolts is directed against a metal target.2. Q. B. Stuhrmann. The interactions between the characteristics of the instrument being used and the structure of the material being studied. which is to say. on the structure of the molecule being studied. The size and shape of the beam pose a particularly difficult problem since it is usually highly desirable to measure I at angles very close to zero. . The practical problem is compounded by the fact that the signal of interest is often quite weak. The beam knocks electrons out of the lowlying orbitals of 2o H. (A full discussion of synchrotron sources and their possible application to smallangle work may be found in a recent review. 71 (1978). X Rays For the foreseeable future. most xray smallangle experiments will be done using conventional laboratory xray generators. the smaller the molecule.2. This instrumental distortion effect is called “slit smearing. the larger are the angular regions around zero where no measurements can be made.8. 8. as well as the additional constraints imposed by the purposes to which the data will be put. however. 11. Since small molecules scatter more weakly than large molecules on a weight for weight basis. Thus some degree of deviation between the ideal measured profile and that actually observed has to be accepted in order to make any measurement whatever. THE EXPERIMENTAL PROBLEM 345 is a distorted version of the profile desired. the smaller the magnitude of the correction which needs to be made. While computational correction of measured signals for instrument smearing effects a posteriori is possible (see Section 8. the collimated beam must have finite size.5. the detector used must have a finite aperture. as has always been the case. beam size can be adjusted upwards as the molecular weight of the material being studied falls so as to keep the data acquisition rate and the degree of slit smearing approximately constant. This situation is not entirely without hope. The larger the physical size of the beam. Biophys. the more accurate the final result is. as well as on the properties of the instrument making the measurement. it is obviously desirable to minimize smearing as far as possible by careful instrument design.2. and the monochromatization must deviate from perfection in order to ensure a practical rate of data acquisition. ensure that there is no single “best” instrument for smallangle purposes.”) These sources work by exciting the characteristic xray emission lines of the metallic elements.” Given the finite brightness of real radiation sources.19). The deviation of the measured from the ideal signal depends on the properties of the ideal signal.
illuminated area. metal foils will act as bandpass filters. Lonsdale. 1968. which leads in turn to high initial cost and maintenance problems.’l Of particular importance is the fact that the linear absorption coefficient for each metal shows a sharp discontinuity (its absorption edge) at a wavelength determined by its atomic number.). 111. . It has a high transmittance for Cu K. 73. W. lowintensity generator may more than balance the gains to be made in data collection rate by the purchase of a highintensity source. emission line (A = 1. SMALLANGLE SCATTERING the atoms. Birmingham.2. Where generators differ most significantly is in their brilliance. High intensity requires elaborate expedients to prevent the destruction of the target due to melting. passing radiation of wavelengths longer than the critical value and strongly absorbing radiation of shorter wavelengths. Kynoch Press. in “International Tables for XRay Crystallography” (K. There are a variety of generator designs on the market differing in size and shape of the. The radiation emerging from a nickel filter of appropriate thickness will be B. The refilling of these holes with electrons from higherlying orbitals gives rise to sharp emission lines. The other half shows up as a white background continuum which has a maximum cutoff energy equal to the electron beam energy. The simplest technique for monochromatization takes advantage of the fact that the linear absorption coefficient of metals for x rays is strongly wavelength dependent. which has a somewhat shorter wavelength and which is the second most important emission line from a copper target.6. nickel has an appropriately positioned absorption edge. The area of the target illuminated by the electron beam is the xray source. A variety of steps can be taken to “clean up” their output to the extent required for smallangle work. For Cu K. The result is that someone considering the acquisition of an xray smallangle apparatus should bear in mind the possibility that the savings in cost and ease of operation obtained by the purchase of a simple. the number of photons they emit per area of source. In general the source areas are 0. radiation. but strongly absorbs Cu K. The more brilliant the source. In a typical generator roughly half the emitted xray energy will be contained in these lines. The favorite target material for biological studies is copper. High brilliance is achieved by raising the amperage of the electron beam falling on the source. Vol. Thus near the absorption edge.54 A) is the one usually used. D. 8.11 mmz in area and rectangular in shape. and its K.346 8. Parrish. the faster the rate of data acquisition and/or the smaller the beam one can use is. radiation. ed. Monochromatization of X Rays Conventional xray sources have a partially monochromatic character due to the way they generate radiation. England.. Roberts and W.
325 (1943).. can provide an excellent source of virtually pure Cu K. Kratky. radiation. contributions and also be significantly reduced in shorterwavelength white radiation. 425 (1926). and divergence of the beam must be such that at P.'^ A useful degree of monochromatization can also be imposed by the detector.7.8. The ultimate expedient involves monochromatization by directing the beam onto a crystal. can be found el~ewhere. One measures the scattering profile first with one of the filters in place and again with the other filter in place and then takes the difference of the two profile^. shape. which include Cu K.. The radiation scattered in a strong Bragg reflection. The Cu K. will not reflect Cu K. or any of the other components in the beam of a shorter wavdength. but sufficient to contribute to making a scattering experiment effectively monochromatic. for example. A thorough discussion of the properties of monochromators of both kinds.2. contributions to a measured scattering profile can be more rigorously identified using the twofilter technique of Ross. Both mirrors and crystals can be bent to make the total reflection or Bragg reflection process focus the beam. Reo. one whose edge is at shorter wavelength than Cu K. 28. THE EXPERIMENTAL PROBLEM 347 effectively free of Cu K. The discriminating power of these detectors is not very high. both focusing and otherwise. 0. Witz. and all longerwavelength radiation from the source. The combination of nickel filters and detector energy discrimination often suffices. Xray beams can be totally reflected when they strike smooth surfaces (“mirrors”) at glancing angles of incidence. 22 23 . 2 4 J . A collimator often consists of a sequence of slits which shape the beam and control its angular divergence.. A A25. Both proportional detectors and scintillation detectors have energydiscriminating properties which allow them to “tune out” events due to radiation of energies different from that of Cu K. The purpose of the collimating system is to define the size and shape of the beam incident on the specimen. can then be used for scattering purposes. Nrr/urwissenrchaficn 31.’~ 8. A. The size. two further techniques can be employed. and the other whose edge is at a wavelength longer than Cu K. For applications where better monochromatization is required. A surface placed in a beam at the critical angle for Cu K. taken at the correct angle. The critical angle for total reflection is a function of wavelength.2. Acta CrjJstallogr. Collirnation Xray smallangle instruments stand or fall on the performance of their collimating systems. 30 (1969).22This technique employs two metals. Ross.Sect. Phys. The reflected rays.
348
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the plane of registration the detector can measure scatter at the smallest angles required without interference from the components of the beam that have been transmitted through the specimen without scattering. (Note that the transmitted beam is generally a large fraction of the total radiation incident in the specimen.) Conventional generators are not bright enough to permit one to collimate the beam to a round or square profile and achieve a geometry which permits measurement at the necessary low angles in an instrument of convenient size. Therefore most smallangle instruments use a beam with a narrow rectangular profile. This profile is convenient since most xray generators are set up to illuminate a long rectangular source which, viewed from a low angle, appears as a line. An appropriately oriented set of collimator slits will deliver a slitshaped beam at the specimen which receives contributions from the whole illuminated area of the source and is therefore as bright as possible. In addition, if a slitshaped beam is used, the detector can be brought very close to the beam center in its narrow direction; measurement can thus begin at very small angles. Slitshaped beams derived from conventional sources have sufficient numbers of photons in them to give good counting rates with ordinary specimens. The penalty paid for the count rate is serious slit smearing in the lowestangle regions of the measured scattering profile. Collimator design would appear to be a simple problem in geometric optics; indeed, this is the way it is usually approached. However, xray collimators never perform up to expectation. The problem is a phenomenon called parasitic scatter. The slit edges that are put in the xray beam to define its size and shape scatter x rays powerfully, regardless of their composition. The result is that instead of creating a beam with sharp, welldefined edges, a simple collimation system will produce a beam whose edges trail out over regions much larger than expected from geometric optics. The “sloppy” edges of the beam due to parasitic scatter have intensities which are large compared to the signal seen in solution scattering experiments; they cannot be ignored. There are basically two strategies for controlling parasitic scatter. The first and simplest is to mount the detector on a moveable arm whose axis is coincident with the position of the beam as it passes through the specimen. Between the specimen and the detector a second collimator is placed coaxial with the arm and restricting the detector’s field of view to the point where it only “sees” x rays scattered from the illuminated region of the specimen and not those scattered from slit edges.25 The second approach involves the use of auxiliary slits (or equivalent elements) called “guard slits.” These slits are placed between the sample and the collimator and are
W. W Hecman, in “SmallAngle XRay Scattering” (H. Brumberger, ed.), p. 197. Gordon & Brcach, New York, 1967.
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THE EXPERIMENTAL PROBLEM
349
positioned to intercept the parasitic scatter due to preceding elements in the collimator without themselves being illuminated by any part of the main beam. The first strategy is appropriate for devices that collect data using a single detector in the stepscan mode. The second can be used for singledetector instruments; it is also appropriate for devices that make use of positionsensitive detectors. It should be emphasized that control of parasitic scatter is probably the single most important aspect of the design and construction of a smallangle instrument. 8.2.8. Flight Path Samples are usually placed immediately following the guard slit, or the equivalent if guard slits per se are not used. The thinwalled glass capillaries used for conventional xray crystallography are adequate sample containers, although more elaborate sample holders can be used if necessary. Between the sample and the detector there will be a flight path. There are two problems which must be dealt with in this part of the instrument. The first is air scatter. Air scatters x rays like everything else. This scattering has two undesirable aspects. On the one hand it attenuates the signal seen at the detector, and on the other, air scatter of the portion of the beam which passes through the specimen can contribute substantially to background. The simplest solution to both problems is to evacuate the space between the sample and the detector. If for some reason the instrument cannot accommodate a vacuum, flushing of these spaces with helium will reduce the problem. The second issue to be considered is the undeflected beam. Both as a protection to the detector and for the safety of the people working in the area of the instrument it is essential to place a stop in the beam path between the sample and the detector. A small piece of lead placed in the appropriate position is adequate for this purpose. Obviously the beam stop should be as small as possible and positioned to shade the minimum angular region possible in the plane of registration. Placement of the stop inside the vacuum chamber prevents the air scatter that would occur if the beam were stopped outside. 8.2.9. Detection There are many ways to measure scattered xray intensities.26 Over the years the smallangle scattering fraternity has relied primarily on digital electronic counting devices of one kind or another, mainly because they give
26 K. Lonsdale, D. H. MacGillavry, H. J . Milledge, P. M. de Wolff, and W. Parrish, in "International Tables for Crystallography" (K. Lonsdale, ed.), Vol. 111, p. 133. Kynoch Press, Birmingham, England, 1968.
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direct numerical output. The traditional smallangle instrument is equipped with a single electronic counter which is supplied with some kind of aperture limiting the range of angles over which it can accept radiation. These apertures are often narrow slits of dimensions comparable to that of the beam at the plane of registration. The long dimension of the detector aperture is set parallel to the long axis of the beam profile and the detector scans the scattered radiation along an arc whose center is located at the specimen and whose periphery is tangent to the line perpendicular to the beam's long axis, passing through the beam center. In modern instruments the scanning operation is computer controlled. It has usually been the practice to use either a scintillation or a proportional counter in order to take advantage of their energy discrimination properties. Recently linear positionsensitive xray detectors have become availLinear xray detectors typically have sensitive areas which are roughly 5 mm high and 510 cm long. Spatial resolution in these detectors is of the order of 0.1 mm. Devices of this kind can be placed with their long axis along the line one would scan with a single detector. Their dimensions and resolutions are such that they will cover the entire angular range of interest with adequate resolution if they are placed at about the same distance from the sample as the single detector device they ordinarily replace. Twodimensional xray detectors can also be c o n s t r ~ c t e dThe . ~ ~positionsensitive detectors in common use are proportional counters which retain the energy discrimination properties characteristic of all devices of that kind. The gain in data acquisition rate obtained by the use of these detectors has already been mentioned. They have an additional virtue: fluctuations in beam intensity during the measurement of a single scattering profile will not influence the profile shape obtained. Fluctuations obviously do affect the profile obtained with a single detector.
8.2.10. Beam Monitors
In some experiments it is useful to be able to normalize data sets for diffcrences in the total flux passing through the specimen during the time of data collection, caused by instabilities in the source, for example. Accurate comparison of data sets, such as might be required when studying conformational changes in macromolecules, virtually requires such normalization. The way normalization is usually handled is to equip the instrument with an auxiliary counter which counts a small fraction of the incident or transmitted beam and accumulates a running total which is proportional to the
27
C. J . Borkowski and M . K . Kopp, R e f ) .Sri. Instrum. 39, 1515 (1968).
W. R. Kuhlmann, K. H . Lauterjung, B. Schimmer, and K. Sistemich, Nucl. Iiistrum. M c r h d r 40, 118 (1966). *' R. W. Hendrichs, J . Appl. Crvsralloyr. 1 I , 15 (1978).
2M
8.2.
THE EXPERIMENTAL PROBLEM
351
total number of photons that have been involved in a given experiment. This can be done by mounting the monitor counter so that it measures backscatter off the beam stop. A more elegant variation of this approach is to place a small crystal on the back of the beam stop and monitor one of its Bragg reflection^.^' In instruments using positionsensitive detectors a transmitting beam can be used. The stop is constructed to transmit a small fraction of the direct beam which leads to the presence of an attenuated image of the beam in the data accumulated by the detector. Its integrated area provides the required measure of relative intensity from experiment to experiment. This approach has the virtue that it compensates both for changes in source strength and for changes in detector sensitivity. Monitors have not been widely used in xray smallangle work in the past. Conventional laboratory xray sources, especially lowintensity xray sources, are so stable that the flux averaged over periods of minutes to hours will be quite reproducible from one experiment to the next, provided the equipment is maintained in a properly airconditioned environment.
8.2.11. Apparatus Designs
Scores of different xray smallangle instruments have been built and used over the years. For the purposes of discussion one might categorize these instruments (“cameras”) into several different classes according to the most striking aspects of their design. The following classes are described in the literature: (i) focusing crystal cameras,3133(ii) totalreflection camera^,^^,^^ (iii) the Kratky camera and its (iv) collimateddetector detector and (vi) c a m e r a ~ , ~ ~ *(v) ~ Opositionsensitive ,~’
3” 0. Kratky, it1 “SmallAngle XRay Scattering” (H. Brumberger, ed.), p. 63. Gordon & Breach, New York, 1967. 3 ’ V. Luzzati, Actci Cry.srullogr. 13, 939 (1960). 3 2 V. Luzzati, J. Witz, and R. Baro, J. Phys. (Paris), Suppl. 24, 141A (1963). .j3 H. Pessen, T. F. Kumosinski, S. N. Timashcff, R . R. Calhoun. Jr., and J. A. Connelly, A ~ uX .  R u y A M / . 13, 618 (1970). 34 W. Ehrenbergand A. Franks, Noture (London) 170, 1076 (1952). 3 s G. Damaschun, Exp. Trch. Phys. 13,224 (1965). 3 6 0. Kratky, Z. Elektrochem. 58, 49 (1954). 3 7 0. Kratky and Z. Skala, %. Elcktroclirm. 62, 73 (1958). ” G. Damaschun, G. Kley, and J . J. Mueller, Acrcr Phys. Austriaca 28, 233 (1968). 3 v R . W. Hendrichs, J . Appi. C‘r,:vrullo,qr. 3, 348 (1970). 4” H. P. Thomas. Z. Phys. 208, 338 (1967). 4 1 H. Wawra, Z . Nururfi)r.sch., C.: Binsci. 31C. 635 (1976). 42 . I . Schelten and R. W. Hendrichs, J. A p p / . Crysio//o,yr.8,421 (1975). 4 3 A . Tardieu, L. Mateu, C. Sardet, B. Weiss, V. Luzzati, L. Aggerbeck, and A. M. Scanu, J. M d . B i d . 101, 129 (1976).
352
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specialpurpose ame eras.^^,^^^ The designs cited are chosen to represent what can be done; this list of references is by no means all inclusive. References to earlier designs may be found in Guinier and Fournet.’ A few of the designs are available from commercial manufacturers as offtheshelf items. Satisfactory instruments can also be assembled on an optical bench using commercially available elements (e.g., detectors or monochromators) and some ingenuity (e.g., the instrument illustrated in Fig. 2). For someone wishing to set up an apparatus for measurements of this kind with minimum trouble, the purchase of a Kratky camera is probably advisable. The Kratky design has collimation properties that would be very hard for a firsttime designer and builder to match. It can be readily adapted for use with monochromators (see above) and positionsensitive detector^,^' and the analysis of the data it puts out is well understood.
8.2.12. Neutrons
A neutron in motion has wave properties and will act, under conditions where its wave properties are being expressed, as if it had a wavelength which can be calculated from its mass and velocity from the de Broglie relationship. It is easy to calculate the kinetic energy of a neutron whose de Broglie wavelength is in the neighborhood of 1 A. When this is done it is discovered that neutrons whose wavelengths are in that range have kinetic energies roughly equal to +kT, where T is approximately room t e m p e r a t ~ r e . Slow, ’ ~ or thermal, neutrons will be diffracted by materials in much the same way as x rays of the same wavelength. The only devices available today which can generate thermal neutrons at the flux levels required for biologicalstructure work are nuclear fission reactors. From the standpoint of a biophysicist the move from xray experiments to neutron experiments constitutes a move to a new world. An xray apparatus can be operated within the context of an ordinary laboratory as “private” equipment. Fission reactors on the other hand are facilities which because of their size and cost must be used on a shared basis by large groups of investigators. The scattering equipment which is attached to such devices to permit their use for structural studies is similarly large and costly. There are only a handful of research facilities of this kind in the whole world. A fission reactor consists of an assembly of uranium elements (the core) surrounded by a jacket full of a moderator substance, usually a fluid; this region is in turn surrounded by a heavy layer of radiation shielding for safety
44 V. Bonse and M. Hart, in “Small Angle XRay Scattering” (H. Brumberger, ed.), p. 121. Gordon & Breach, New York, 1967. 441 0. Kratky and H. Leopold, Makromoi. Chem. 133, 181 (1970). 4 5 D. Atkinson, personal communication.
8.2.
THE EXPERIMENTAL PROBLEM
353
reasons. Fission events in the core produce energetic neutrons in quantities beyond those needed to sustain the chain reaction. These excess neutrons escape into the moderator substance, where they lose kinetic energy by collision with moderator molecules. The neutrons equilibrated with the moderator will have the MaxwellBoltzmann distributions of energies appropriate for the temperature at which the moderator is maintained. In research reactors it is customary to keep the moderator at around room temperature. This yields a neutron energy distribution with a peak wavelength in the neighborhood of 1 A. Neutrons from the neutron “gas” which exist in the moderator region can be tapped for experimental purposes outside the reactor by insertion of a suitable pipe through the shielding. The thermal spectrum of the neutrons derived from the reactor is manipulable. In the past decade it has become apparent that for biological applications and some applications in chemistry and physics it would be desirable to have a distribution of neutron energies which peaks at longer (i.e., lowerenergy) wavelengths. This spectral shift is brought about by inserting a device into the moderator to produce a strongly refrigerated region within it. Such devices, known as “cold sources,” commonly operate near absolute zero, and neutrons taken from these refrigerated regions have an energy distribution which peaks in the neighborhood of 610 A. The design of neutron instruments, as compared to corresponding xray instruments, is dictated by the relatively low fluxes available from neutron sources. When the spectra given by the most powerful available neutron sources are measured, it is found that the neutron flux available per unit wavelength at any wavelength in the spectrum is orders of magnitude less than that available as Cu K, emission from a conventional laboratory xray ~ 0 u r c e . The l ~ probability of a scattering event occurring when a neutron or photon passes through a sample is given by the scattering cross sections of the material contained in the sample. (The total cross section for a macroIt turns o u t that the scattering cross sections for molecule is 4.n biological macromolecules are about the same for x rays and neutrons. It is clear then that one cannot simply transfer an xray camera to an appropriate reactor and hope to collect any data at all; the beam its collimating system would produce would be much too weak to produce data at a useful rate.
c.f’;.)
8.2.1 3. Monochromatization of Neutrons The neutron beam that comes out of a reactor beam core contains neutrons of all energies. In order to perform diffraction experiments it must first be monochromatized. Bragg reflection of the beam from a crystal is just as suitable a means of monochromatization for neutrons as it is for x rays; the same principles apply. A number of crystalline materials have been used
3 54
to be the favored
8.
SMALLANGLE SCATTERING
for this purpose in the past, but in recent years pyrolytic graphite has come Pyrolytic graphite surfaces have very high reflectivity; there is little beam intensity lost as a result of Bragg reflection. The surfaces used are artificially made and can be formed on curved surfaces, opening the possibility of focusing optics. In addition the surfaces have a mosaic spread which is also under control during manufacture. In general it is advisable to use pyrolytic graphite surfaces with as large a mosaic spread as possible to increase the bandwidth of the wavelength region reflected by the crystal into the collimating system. Reasonable smallangle measurements can often be made with beams whose wavelength distributions are quite broad (AL/A 0.1). The broader the band used, the greater the number of neutrons available for experimentation. For these devices the theories developed for the corresponding xray apparatus are fully t r a n ~ f e r r a b l e . ~ ~ Because the spectrum of the beam incident on such monochromators is white, it is necessary to place filters in the reflected beam to reduce contamination with radiation. A second method of monochromatization in common use is velocity selection.48Neutrons of wavelength in the angstrom range have velocities of the order of 1000 mjsec. This speed is low enough to permit neutrons to be sorted on the basis of velocity by mechanical means. A cylindrical rotor is placed with its axis parallel to the beam and set so that the beam is intercepted by its edge. Helical channels are provided along the cylinder’s outside wall and pitched so that when the cylinder rotates in an appropriate direction at an appropriate speed neutrons having the desired velocity will be able to follow the helical grooves without colliding with the walls. The width of the grooves controls the bandwidth of the velocity distribution passed by the selector, and its rate of rotation controls the average velocity and hence wavelength. Crystal monochromators are cheap, simple, and require no maintenance. They have three disadvantages, however. First, alterations of the wavelength at which a given piece of apparatus is operated require repositioning and realigning all the apparatus downstream from the monochromator. Given the size and weight of neutron instruments, adjustments of this kind can be a major undertaking. Second, the spacings of the crystals available sets significant limits on the maximum wavelength that can be obtained. (The planetoplane spacing used in pyrolytic graphite is about 6 A). Third, the monochromatization achieved using crystals is often “too good” for smallangle applications because of a smaller than optimal mosaic spread, which

47
R. W . Gould, S. R. Bates, and C. J . Sparks, Appl. Spcctrosc.22, 549 (1968). T. Kisteand K . Otnes, N u d . Instrum. Methods75, 197 (1969). 48 B. Buras, K. Mikke, B. Lebech, and J . Leciejewicz, Phys. Slurus Sdidi 11,567 (1965).
46
8.2.
THE EXPERIMENTAL PROBLEM
355
costs the experimenter usable flux. Some relief from both the spacing and bandwidth problems appears likely in the near future because of the deA multilayer monochromator velopment of multilayer monochromators.49~’0 is effectively a onedimensional synthetic crystal produced by depositing thin films of metals of alternating scattering lengths on an appropriate substrate. These Braggreflect incident neutrons just like a normal crystal. They have very large planetoplane spacings and their reflectivity can be controlled to permit them to reflect a very broad wavelength band, if required. Velocity selectors are costly to build. However, they can be adjusted over a wide range of wavelengths simply by changing the rotor speed. Such adjustments require no alterations in the optical track of the rest of the instrument. Their efficiency increases with the width of the wavelength band they pass.
8.2.14. Pulsed Reactors
The neutron sources discussed so far produce a steady output of thermal neutrons. Most of the experimental work done to date has made use of “dc” sources of this kind. However, some fission reactors and virtually all neutron sources which depend on particle accelerators (“spallation sources”) produce pulses of neutrons rather than a continuous stream. It seems likely that many of the highflux neutron sources built in the future will be this kind. Pulsed sources are best used with no monochromator. As a pulse of thermal neutrons travels through space, it spreads out in the direction of flight owing to differences in the velocity of the neutrons it contains. Its leading parts will consist of shortwavelength, highvelocity neutrons and its trailing portions will contain nothing but longwavelength, lowvelocity neutrons. The motion of the pulse monochromates it. There is a whole class of instruments called “timeofflight’’ instruments which exploit this property of neutron pulses. In this case one might place a detector to view the scatter given by the sample at a fixed angle and monitor the output of this detector as a function of time. A scan of the detector’s output in time corresponds to a scan in R as each pulse goes by since the neutrons producing the events seen at each time are constantly changing in 2 and hence R, since R = (2 sin @/A. It is possible to construct timeofflight instruments suitable for biological smallangle work. A description of the design and properties of such devices can be found elsewhere. ”
,’
4’) B. P. Schoenborn, D. L. D. Caspar, and 0. F. Kammerer, J . Appl. Crystallogr. 7, 508 ( I 974). A . M . Saxena and R. P. Schoenborn, Actn Cr.r.stu//ogr.,Sect. A A33, 805 (1977). L. Cser, Brookhrriw Symp. B i d . 27, VII3 (1976). D. F. R. Mildner, N u d . ftistrum. Met/iod.c 151, 29 (1978).
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3 56
8.
SMALLANGLE SCATTERING
8.2.15. General Design: Collimation
The primary fact about neutron sources is their low flux. If the flux an instrument can deliver is low, the only way a significant number of scattering events can be observed per unit time is to increase the amount of material placed in the beam. Some relief from the problem of low flux can be obtained by taking a broad cut from the thermal spectrum, as already mentioned. Relaxation of the degree of monochromatization of the beam will increase the flux entering the experiment. The penalty paid is an increase in smearing; the amount of broadening that can be tolerated depends on the type of data being collected. A second factor which assists the neutron experimenter is the low absorption coefficient of most biological materials for neutrons. Most of the apparent attenuation of a neutron beam as it passes through a sample results from highangle scattering, especially incoherent scattering if the sample is rich in hydrogen. This is in contrast with the xray situation, where much of the attenuation is due to the absorption of photons by the material in the sample. The way the cross sections for these kinds of events work out, samples of about 2 mm thickness can be tolerated for water solutions and thicknesses of 0.51 cm can be accommodated for materials suspended in D,O. The maximum tolerable specimen thickness for an xray experiment is about 1 mm. Thus, in a neutron experiment, one can place two to five times as much material in the beam as in an xray experiment, simply because of differences in sample thickness. The third and most important way the amount of radiated material is increased in neutron experiments is by setting up the collimating system to deliver a beam with a very large (by xray standards) crosssectional area. The size of the neutron source is set fundamentally by the diameter of the beam pipe through which the particles leave the reactor. Typical pipes deliver beams which are 2 x 2 cm. In order to take full advantage of that area, or some large fraction of it, the collimator must be very long. Large beams can be tolerated only if their angular divergence is small, and the only way angular divergence can bc controlled if the aperture areas are big is by placing successive slits at very large distances from each other. If angular divergence is not controlled, the direct beam at the plane of registration will have an angular size which forbids measurements at the low angles required for smallangle work. The second aspect of the apparatus which follows from the large beam is the requirement for a long flight path, to ensure that the angular area corresponding to the direct beam due to its crosssectional area at the final aperture is small compared to the contribution due to its angular divergence. The result of the operation of these factors is that neutron smallangle instruments are typically many meters from monochromator to detector,
8.2.
THE EXPERIMENTAL PROBLEM
357
whereas most xray instruments are of the order of half a meter. The weight and cost of the equipment goes up with size. Once the need for a physically large instrument is accepted, square or round beam profiles become practical. The use of such beam profiles means that the smearing effects due to beam size are roughly equal in all directions in the plane of registration. This has two practical consequences. First, it will be useful to collect data over the entire plane of registration rather than along one line, as is the case with slitbeam xray devices where the degree of slit smearing varies radically with direction in the plane of registration. Second, if the angular size of the beam is small, slit correction may often be close to negligible. A point which should be made about neutron collimators is that parasitic scatter tends to be much less of a problem in neutron optics than in xray optics. Slits made with cadmium absorb neutrons SO efficiently that parasitic scatter is negligible and thus collimation will be about as predicted from geometric optics. The net effect of the differences in design between neutron and xray equipment can be appreciated by considering the volume of solution illuminated by the beam. For x rays this volume is generally of the order of half a microliter; for neutrons it is commonly 100 microliters or greater. It is this difference in illuminated sample volume that makes neutron smallangle experiments feasible despite the low flux levels available.
8.2.16. Flight Path and Detection
The flight path in a neutron instrument is effectively a scaledup version of its xray counterpart. The flight path should be evacuated if possible, or failing that, flushed with dry helium in order to reduce air scatter. The path must also contain a beam stop composed of a strong neutron absorber, e.g., boron carbide. Detectors for neutrons consist of a chamber full of a gas including a strong neutronabsorbing species, such as loB as BF3, or 3He. A nuclear reaction takes place after the neutrons are absorbed by these atoms and the products of those reactions are i 0 n i ~ i n g . lThese ~ ionizing events are detected just as they would be for x rays. When the field started out, singledetector, stepscan instruments were commonly used. These have been rendered obsolete by newer twodimensional positionsensitive detectors.53 56 For instruments having round or square beams the scattered
J. L. Alberi, J. Fisher, V. Radeka, L. Rogers, ,and B. P. Schoenborn, IEEE Trans. Nucl. Sci. NS22, 255 (1975). 5 4 R. Allemand, J. Bourdel, E. Roudant, P. Convert, K. Ibel, J.P. Cotton, and B. Farnoux, N u d . Instrum. Merliorls 126, 29 (1975). '' J. Schelten, P. Schlect, W. Schmdtz, and A. Mayer, J . Bid. Chem. 247, 5436 (IY72). 5 6 R. Klessc and G. Kostorz, Crystallogr. Compur. Tech., Proc. Int. Summer Sch., 1975 p. 383 (1976).
53
358
8.
SMALLANGLE SCATTERING
profile will be cylindrically symmetric about the beam axis or nearly so. In this case an area detector acts like a piece of digital film. The signal ascribed to each scattering angle will be the average at locations on the detector face within an annular ring of appropriate radius and width. This mode of data collection insures that all neutrons scattered over the entire angular region of interest contribute to the measured data. The gain in data acquisition rate is roughly 1000fold compared with singledetector stepscan strategy. The importance of these detector developments in increasing the rate at which work can be done cannot be overstated. Monitors are routine in neutron instruments. Data collection often takes days; changes in reactor temperature, state of the fuel cycle, demand due to other users, etc., can all affect the flux available at a given wavelength. Such alterations can only be allowed for by use of a monitor.
8.2.17. Available Instruments
The instrumentation of a neutron beam port for scattering experiments is a major undertaking both because of the physical size and cost of the components required and because of the elaborate electronics often associated with such devices. Most of those doing biological experiments will find themselves traveling to a central facility to make use of an already existing, general purpose instrument on a shared basis. The properties of the instrument will usually have to be accepted as given. A number of instruments have been constructed for dc sources. Two closely related designs for simple, Soller slit cameras have been The possibility of constructing focusing cameras based on curved multilayers has been e ~ p l o r e d ,and ~~,~~ it seems likely that instruments of this kind will soon be available. Two large instruments based on velocityselector monochromatization and twodimensional positionsensitive detectors have been b ~ i l t . ' ~ ,An ~ ' instrument based on crystal monochromatization and a twodimensional position detector of high spatial resolution is available at Brookhaven.61 Timeofflight designs can be found in the references given in Section 8.2.14.
8.2.18. Absolute Intensity Measurements
For determination of molecular weights and volumes by both x rays and neutrons, it is useful to be able to place measured intensities on an absolute
A. C. Nunes, Nircl. Instrum. Methods 108, 189 (1973). B. C. G. Haywood and D. L. Worcester, J . Phys. E 6 , 568 (1973). 5 9 A. C. Nunes and G. Zaccai, Brookhuuen Symp. B i d . 27, VII49 (1976). 60 W. Schmatz, T. Springer, J. Schelten, and K. Ibel, J . Appl. Cr,ystal/o,qr. 7,96 (1974). 6 1 B. P. Schoenborn. J. Alberi, A. M . Saxcna, and J . Fisher, J . App/, Crysfulloyr. 11, 455
57
58
(1978).
Nicolaieff.367 (1961). (8.25. h3 h4 D. F~ * ~ which ~ ~ are ~ ~ . Luzzati. The thickness of the sample. H. 1274 (1965). Reo. the simplest being a resort to standards whose scattering can be calculated a priori.Instrum. Darnaschun and J. 691 (1963)." Provided the volume of the sample in the beam is also known. Z. and once again the absolute scale is set. A set of attenuators of known thickness can be prepared and beam intensity measured as a function of total attenuator thickness. 34. For scatterers for which I ( R ) is constant there is no slit smearing effect. What is meant by absolute scale in the smallangle field is the determination of what fraction of the incident beam is represented by a given measured intensity. the relationship between total sample cross section and measured intensity follows. Two other approaches have been used.) In such circumstances attenuation will not be logarithmic and accurate extrapolation to zero attenuation impossible. A semilog plot of the data permits extrapolation to the unattenuated intensity at zero thickness.13 Thus it gives a strong scattering profile which is virtually constant with angle.8.63 Examples of substances useful in this regard include sulfur hexafluoride and octafluorocyclobutane.2.rigorously ~ ~ monochromatized filters cannot be used because the linear absorption coefficient of matter for x rays is strongly wavelength dependent with short wavelength being more penetrating. 3. the total integrated beam intensity. which are gases of known structure25 whose scatter can be calculated using Eq. Weinberg. and A. What makes absolutescale measurements difficult in practice is that the intensity scattered in any direction by a typical specimen is orders of magnitude less than the intensity of the incident beam.O is excellent for these purposes. THE EXPERIMENTAL PROBLEM 359 scale. Lucite is a satisfactory attenuator. and the solid angle accepted by the detector suffice to set measurements on an absolute scale. J . Witz.1). just as is done with H 2 0 in the neutron field. Mol. J. Noturforsch. hence the observed I ( R ) is the true Z(R). Absolute scale is harder to establish for x rays. Thicknesses of several centimeters are required so that the preparation of a set of blocks of wellcharacterized thickness is easy. For an xray apparatus in which the beam is fully monochromatized (by Bragg reflection). L.2. A 20A.. (Such strong wavelength dependence is not seen with neutron attenuators. most detectors used for measuring scattered intensities would be damaged by exposure to the direct beam. absolute scale is reasonably easy to achieve. Almost all the scattering from water is incoherent scatter owing to its 'H content. A second method in wide use is to employ a standard scatterer and calibrate in terms of the scatter it gives. Sci. G. with the same detector used for measuring scattering profiles. The cross section of water is well known. Mueller. Foils of Cd can also be used. The second '' V. Bid. For neutrons. attenuaor ~ beams not tion with filters is ~ s e f ~ 1 . . Indeed.
315 (1972). 5 . What the analytical correction techniques attempt to do is to estimate what the data would have been if a detector of the same solid angle of acceptance as that actually used had measured the scatter given by the sample irradiated with a beam of the same intcgrated intensity as that used. on which estimates of molecular size and shape depend. h5 (8.qr. Chipman. De Marco. Kratky and his collaborators have used mechanical devices to attenuate the beam in order to estimate its in conjunction with the calibration of standards. Phjs. is given by the following expression: I(R . D. Appl. under conditions of ideal geometry and monochromatization. C'hcw~. ~ ' For either xray or neutron experiments. Passage through the specimen attenuates both x rays and neutron beams because of absorption and highangle scatter.2. the option is always open of determining molecular weights by comparison of unknowns with molecules of the same chemical type of known molecular weight. " B. Schmidt. Kratky. Kratky. Crjstullo. Wawra.2.3) '' 0.68 (1961). Further discussions of these problems may be found in Batterman et aL6* A useful theoretical critique of the various methods of determining absolute intensity has been provided by H e n d r i c h ~ . J . Pilz. J . 1655 (1978). Stahinger and 0. Batterman. 8. R. from which estimates of molecular weight and volume come. SMALLANGLE SCATTERING alternative is to prepare precalibrated secondary standards. I . Hendrichs. the curve actually measured. Slit smearing alters both the shape of a profile. h7 0. C h m . 179.4). the beam and the scattered radiation of interest are equally affected by attenuation. R. and its absolute magnitude. 11. Colloid Iriterfiice S(. I96 (1978). and J. It should be recognized that the beam intensity of interest in any scattering experiment is the intensity ujter passage through the specimen. C'rysrulloyr. .and ~ ~ a report has recently appeared on the accuracy with which profiles can be m e a ~ u r e d .19.i. J . The knowns in this case are used as secondary standards.122. 7" H . Data Correction Before data sets are analyzed consideration must be given to the impact of slit smearing (see Section 8. Monaish. J. Hence one always wishes to compare measured scatter with the at tcnuatedbeam intensity. The effect of smearing on the data can be thought of as a convolution. O ( R ) .360 8.24 (1966). W. Commission on Crystallographic Apparatus. W.R')W(R. It is easy to show that for low scattering angles. 981 (1963). and P. RPP. under equivalent experimental conditions.21. A p p l .2. R') dR'.94. In general it is advisable to attempt to correct for these effects computationally. M n k r o m d . J . Kratky and H .
Spencer.. 383 (1976). 7 3 J. R. ’’ 0. I975 p. A. includes some error. I n t . 7 7 M. One might single out for mention the iterative algorithm^. J . Crystullogr. 75 J. and (ii) a component due to the spectrum of the radiation used in the experiment. R’) the weighting function representing the averaging effects of the i n ~ t r u m e n t . Jones. Summer Sch. The problem is to find an estimate of I(R) in the face of these limitations in the information given. Proc. Actu Phvs. 1975 p. Waltcr. Damaschun. 210 (1971). Reo. London.2. ’’ G.as measured. R’) and O(R) are known. W ( R . The art of data correction is in a fairly satisfactory state at present. and G. What keeps the problem from being straightforward is the fact that O(R)is usually measured only at a finite number of points over a limited range of R. Tech. I . 618 (1931).W. Mueller. 11. Schelten and F. Comput.2. Ser.23. 362 (1976).79 Someone starting out in the field would be well advised to write the authors of the algorithms he finds appealing to obtain listings of the computer programs that have been written to implement them. Proc. Clatter. The first of these can either be calculated from the geometry of the beam collimator and the detector’s properties. 38. as seen by the detector.I..’~ method of G l a t t e ~ Deutsch . However. 7 4 0. In addition O(R). There are a number of techniques for solving such equations to obtain I(R). Aciu Crystullo. from counting statistics if nothing else. Austriucu 47.^^ the spline and the indirect transformation algorithm of Schelten and Ho~sfeld. Suc. Schmidt.. Phys.^^. 7 8 P. J . CrystoNoyr. J. In the past ten years a number of algorithms have been proposed which accept a reasonably wide range of scattering profiles and scattering geometries and find solutions which take the noise in the data into account. the amount of averaging varies with angle. F. CrystuIlo.yiulloyr. 147 ( 1 974). 16 (1938). Summrr Sch. Appl. 4. 191 (1967).~~ and L ~ b a n ’have ~ recently proposed a new method for dealing with slit height correction.8. Appl. What is required is the size and shape of the beam at the plane of registration. R. 98 (1978). I n / . ’ ~ W ” ~is generally a function of R . 83 (1977).yr. Deutsch and M. Lake. Hossfeld. THE EXPERIMENTAL PROBLEM 36 1 where I ( R ) is the ideal profile and W ( R . A 166. Cry. or measured directly. 87. Clatter. ’ and ~ some practical comparisons of algorithm performance have been performed. further progress may be expected in the future. the ideal algorithm is yet to be discovered.3) is an integral equation in which both W ( R . J. Proc. An extensive bibliography and discussion of techniques has been prepared by S ~ h m i d t . Kranold. Comput. In the case of neutron experiments. For xray experiments wavelength effects are usually small. Tech.R’) has two components: (i) a component due to the size and shape of the beam and the detector aperture. W. Luban. ” 72 . . the beam’s spectrum will usually be known and will be provided to the experimenter by those running the facility. Appl. R.yr. Equation (8. Crysiulkqr.
PO)262. and extrapolation to R = 0 follows.3. one obtains (Luzzati31. Criteria are discussed in Beeman et aL3) Guinier’ demonstrated that the lowestangle portions of the scattering curve of any molecule are Gaussian in shape. Kratky.. Defining fi = ( x i . SMALLANGLE SCATTERING 8.2. (8. or scattering length density of then taking the incident flux and experithe solvent. I(0)cannot be measured directly.3. Data Analysis Suppose that full data have been collected on the scattering of a given macromolecule in solution. Because of the direct beam.3. and li are measured in photons or neutrons per second. mental geometry into account.j&h.ys. 13. extrapolation to R = 0 is straightforward. Ii is the flux in the beam measured ufter its passage through the sample.3.1) at R = 0 becomes 2 = (Fh .1. 8. The interest in I(0) lies in its relationship to molecular weight and molecular volume. Prn. (The range of R over which data must be collected in order to permit extrapolation depends on the size of the molecule. provided data have been collected at R sufficiently close to zero. However.1) where N is the number of solute molecules contributing to the measured profile and I ( R ) the scatter of a single molecule as given by Eq. Z(0). Thus the lowestangle data plotted as ln[lexp(R)] versus R 2 will be linear. see Kratky and Pilz4) lexp(o) = (Ad/d’)ZiAtC(MW/NO)(p . Biophys.2.q.PoU) 3 where u = ui. The data are then used to construct a scattering profile corrected for concentration effects and slit smearing.is the total volume of the molecule inaccessible to solvent and p o the scatteringlength per unit volume. (8.362 8.2) Here I. 107 (1963).3162*80 Equation (8. A d is the area of the aperture of the detector and d the distance from the sample to xi *’ 0 . What information can be extracted from those data‘? In practice “full data” means that the incremental contribution of the macrornoleculc to the scattering of a solution has been measured at several solute concentrations and the data placed on an absolute scale. For such a corrected scattering profile lexp(R). Bioph. . Molecular Weight and Partial Specific Volume The first aspect of the corrected profile that can be examined with profit is its value at 20 = 0. Chem. (8. it will be true that 4 x p ( R )= N W .1).
3. Tanford. M W follows. po can be varied (see Section 8. Oxford. can be added to the solution. The scattering of solutions near R = 0 must be considered in thermodynamic terms. “Physical Chemistry of Macromolecules. its extension to xray or neutron scattering may be found in Eisenberg and Cohen” or Timasheff. While Eq. p. The partial specific volume of the solute must be measured. The xray volume will be that inaccessible to the solute used to manipulate po and will be a hydrated volume. Equation (8. 6 the partial specific volume of the solute. M W is the solute’s molecular weight. A is the area of the sample illuminated by the beam and t its thickness. it is necessary to be aware that its theoretical justification requires something more rigorous than Eq. and C its concentration (mass/volume). volumes much larger than that of a single molecule are being examined. the total number of neutrons or photons in the beam. The scatteringlength density of the solvent can be obtained from its chemical composition and density. Then MW = I. [Iex.OD. in “Electromagnetic Scattering” (M.1). The EinsteinDebye theory developed first for light scattering” is correct in this case as well. Note that the molecular volume emerging from a neutron study using H. Given the atomic composition of the molecule as obtained.3. In a neutron experiment po can be altered over a wide range by mixing D. for example.3. The scatteringlength density of the solute is generally obtained from knowledge of its atomic composition and 13. S. from its amino acid or base composition..12).3) where I = ZiA. it is the fluctuations in solute concentration within these volumes that give rise to the scattering seen. Pergamon.2) suggests a method for obtaining V from scattering data. ed.3) is often convenient. These values can be used to calculate M W [Eq.(0)d~No/A.3)]..3. (8. or sugars.If M W is known.3. 337. with the proviso about hydrogen exchange explained below (see Section 8. 82 ’’ C. (8. N o Avogadro’s number.O mixtures will be the anhydrous molecular volume. N. This procedure can be carried out on data that are not on an absolute scale but simply normalized for differences in transmitted beam. V follows.3.11).” . Timasheff.p o . 1963. When R 0.p0]262.3.). if 6 is known. Kerber.2 Thus the molecular weight value obtained from a lowangle experiment is a thermodynamic quantity.(0)/C]l’z is proportional to p ..8. 1961.3. DATA ANALYSIS 363 the detector. leading to a determination dependent only on scattering and atomic composition data. New York. (8. glycerol.3.8). 6 follows from p.ztC[p . In an xray experiment lowmolecularweight solutes of high electron density.’EsZ  Wiley. (8.O into the solvent (see Section 8. such as salts. Thus data collected on the same macromolecule at different pos will permit determination of the po for which p = p o .
.p0)V. Recognizing the thermodynamic nature of I(O).2.3). can be measured under the same conditions for the solutesolvent combination of interest and 6p/6C estimated from it (for details.364 8. the scattering curve of a macromolecule at low R is Gaussian in shape. Molecular Size and Shape: Radius of Gyration As already pointed out. Similar precautions are routine in the determination of molecular weight by other thermodynamic methods. Hyman and P. 1967. New York. It can be shown by expansion of Eq. in “SmallAngle XRay Scattering” ( H . Gordon & Breach. the change in scatteringlength density with change in solute concentration at constant temperature with the chemical potentials of all species except that of the macromolecule held constant. In practice the molecular weights obtained from xray or neutron forward scattering have been found to be of excellent accuracy. Thus the virial coefficients for the molecule can be obtained from such an analysis just as they can be derived from light scattering or osmotic pressure data. A. In practice hd/hC. 8. ed. they are equally necessary here. Under these conditions. 477. scatters almost entirely in the forward direction when observed with radiation of sinall A.po)V for use in Eq.). Additivity will not be observed if solute and solvent interact. (8. p. and use it in place of ( p .1) as a power series in 2nRr and considering the first two terms not only that the curve is Gaussian. Bruniberger. Vaughan.2. where d is the weight density of the solution. as they do with polyelectrolytcs. see Eisenberg and Cohen”). comparable to sedimentation equilibrium except for a few situations involving highly charged polyelectrolytes where the precautions mentioned above were not observed.3. Dust. (8. which is the bane of light scattering studies. SMALLANGLE SCATTERING In practice published values for 6 for macromolecules (within a given chemical type variation is small) are often used in estimating ( p . it can be understood that a study of Z(0) as a function of solute concentration will give information about the dependence of the chemical potential of the solute on concentration.2.3. 8 3 A.*3In principle one should measure Gp/GC. (8. The buffer sample whose scattering profile is used for background subtraction [Eq. This usage assumes that the scatteringlength density of the solution is the sum of the scatteringlength densities of both solute and solvent.2)] should be equilibrated by dialysis with the solute solution of interest. a correct molecular weight will be found. it does not interfere with this technique. different molecules differ only in the breadth of this Gaussian. The advantage of smallangle molecular weight determination over its light scattering analog is that sample preparation is much easier.
O solution will be close to the mechanical value for the molecule in question and provides a useful indication of the molecule's size.4) Hence the slope of the In Z(R) versus R 2 plot used for estimating Z(0) will be $rc2Rl.3. virtually every smallangle paper ever published includes radius of gyration estimates.) Radius of gyration is the sole fact about a macromolecule which can be deduced from its solution scattering profile without resort to ancillary data. is the radius of gyration of the molecule. Because this parameter is extractable from lowangle data in such a direct fashion. where R. When smallangle data are collected on such objects. where y i is the distance from thc ith atom to the center of mass ofthe scattering length of the particle. 124. . H4 G . Quantities Related to the Radius of Gyration Long. The value found by xray methods for a protein in water solution or determined by neutron scattering for a protein in D. Porod. The sums in all cases run over all atoms.^^.3. The scattering radius of gyration gets its name from its analogy with the mechanical radius of gyration. 125.^^ '' G .e. thin particles or thin plates will often have radii of gyration which are very large indeed. DATA ANALYSIS 365 but that the breadth of the Gaussian reports the molecule's radius of gyration. on the crosssectional distribution of scattering length in the case of long. Useful information can be obtained. Ko//oi&%. where T i is the vector from an arbitrary coordinate origin to the ith atom. 51 (1952).3. i.. or o n the transverse distribution in the case of flat plate^.3.9. Ko//oidZ.8. Furthermore. (The relationship between molecular structure and radius of gyration is considered at greater length in Section 8..' For small R.3. 83 (1951). Porod. 8. (8. radii can be obtained from data which are not on an absolute scale. I ( R ) = I(0) exp( $n2RiR2). thin objects. to the location r. it will often prove hard or impossible to measure those radii because of the narrowness of the central Gaussian. however.
Evaluation of M I L and the radius of gyration of the cross section for chromatinrelated structures has been reported by several gro~ps. Biopo/lwrrs 14. f. I. F E B S Lett. I. and P. M I L and diameter information are valuable parameters since they can constrain modelbuilding studies. S. The early work on pure DNA by Luzzati and collaboratorss6 and by Bram8’ is evaluated in the paper by Eisenberg and Cohen17 with reference to the polyelectrolyte problem alluded to above. University of Wisconsin. Madison (1968). L.3) in place of IexP(O)and an estimate of the molecular weight per unit length of the object will result. Once again a linear region will be discerned whose slope is 47t2R:. Saludgian. Luzzati. Federov. is the radius of gyration of the cross section of the object. Biochimiu57. have often been measured for elongated structures. V. These same techniques are useful for the analysis of far less elongated molecules. Bunemann. Vorobev. Thesis. The slope of this linear region will be 27c2RI where R.~ ’ and Bunemann9’ have recently described the alterations in DNA structure due to drug binding in a paper which includes a careful explanation of the data analysis used in MIL and R. If the density of the object is projected onto a plane normal to its long axis and the radius of gyration of the projected density calculated. however. Bram.e. For a uniform plate. We shall call this value “f(0)” for this purpose. 51. Ph. R. 1301 (1975). 64. 3 (1975). is the result.. Mathis. Note that the existence of a linear region in a ln[RZ(R)] versus R2 plot or in a In[R2f(R)] versus R 2 plot is diagnostic for a long. I133 (1975). is now the radius of gyration of the density of the plate projected onto a line normal to its surface. Aleksanyan. and they are hard to obtain by other means.D. Eur. S. 491 (1967). SMALLANGLE SCATTERING For long. The classic material for studies of this kind is DNA and its various complexes with histones and other protein (i. thin shape or a thin plate shape as the case may be. A . Biqiolymrrs 5. R7 88 8y ’’ ’‘I . Mass per unit length ( M I L ) and radius of gyration of the cross section. where R.. For platelike objects. Masson.366 8.89 (1976). Zipper and H. For the thin plate. J . Biochem. thin objects it will be observed that plots of ln[Rf(R)] versus R2 are linear over an appreciable range of R. similar use of 27tZ(O) will give an estimate of the molecular weight per unit area. P. (8. T 2 is the thickness of the plate. and B. A. The value of RI or R 2 1 found when the linear regions of one of these plots is extrapolated to R = 0 is useful. R . the analogous plot is ln[R2Z(R)] versus RZ. they materially assisted V.~~ Zipper . for the long rod 21(0)can be substituted into Eq. If measurements have been made on an absolute scale. investigations.3. The possibility of measurement of membrane thicknesses and mass per unit area by the method just suggested has seldom been taken advantage of. V. For indefinitely long structures like these. Bran]. chromatin). Tardieu. Sperling and A.
Debye and A.4. 8. Schlimme.=r c fifj sin 2nrR 2nrR and I ( R) = allr 2nrR c c fifj sin 2nrR rij=r 0 ’ Let us define a new function p ( r ) : where.8.one could equally well make a list of all atomic distances and collect these distances into groups of identical value. is written as a double sum over all atoms of terms of the form (sin x ) / x .92in their recent analysis of the tRNA structure from smallangle data. DATA ANALYSIS 367 Pilz et al. Pilz. Eur. 518 (1949).f. M. and E.1). 0. Thus the total information contained in a solution scattering 92 I. F.6) Note that knowledge of p ( r ) is equivalent to knowledge of Z(R). Pirenne.3.5) where p is the average scatteringlength density of the object. H . 401 (1970). Cramer. It follows from Eq. = (1 f i ) / N and N is the number of atoms in the molecule. 20. Often it is more convenient to consider p(r) a continuous function.3. the expression for Z(R). von der Haar.3. Phju. then where the sum is over all distances r. F. J . P. (8. 617 (1938)..5) that p(r) can be obtained from I(R ) by spherical Patterson i n v e r s i ~ n ~ * ~ ~ ~ ~ ~ : p(r) = P (J:RI(R) sin(2nRr) dR (8. Ann. (Lripziy) [ 5 ] 33. the two are Fourier mates. p ( r ) dr is the (weighted) number of vectors in the structure having lengths between Y and r + dr. 93 94 . J . Debye and M. Phys. Appl. Biochem.. P. Kratky.3. 15.2. The contribution of the group to Z(R) is simply all pairs r. then (8. Bueche. In computing I(R).3. Length Distributions Equation (8.
thc probability that a point a distance r from a given point in the structure will also be within the particle. rmaxrthe largest vector in the structure or its longest chord or maximum linear dimension. “ p ( r ) = 4nr2vy0(r). I(R)e.3. of course. The largeR data should show a linear trend. In practice this relationship is used by plotting the data as R41(R) versus R4.7) where v is the volume of the structure. this l/R4 dependence of Z(R) holds rigorously. not true of a real moleculc. but one in which all volume elements have the same scatteringlength density.8) For volumes of uniform density. where S is thc surface area of the molecular volume. nucleoproteins).5. For structurcs with gross fluctuations in scatteringlength density (e. 8. In fact it should be flat. The radius of gyration of the object can be obtained from the length distribution2: Ri = Jomr2p(r) dr/Jomp(r) d r . Characteristic Function Parameters From the definition of yo@) it is clear that it rcfers to a structure of the same shape and volume as the molecule being studied. d yo (r )/d r = S/4v at r = 0.. However.84385 From this relationship it can be shown that the trend of I(R)at large values of R must be given by: ’ Z(R) K p 2 S / 8 x 3 R 4 . (8. SMALLANGLE SCATTERING prqfile is the distrihirtion of interatomic vector lengths in the structure under study. The intercept of the trend line . For real molecules.$ a. provided background subtraction has been done perfectly and internal fluctuation contributions are not too severe.p will approximate the profile predicted by the characteristic function abstraction of the true molecule. For structurcs of uniform internal scatteringlength density. (8. Several intcrcsting aspects of yo(r) can be readily demonstrated. A function closely related to p ( r ) is P o r o d ’ ~characteristic ~ ~ ~ ~ ~ function” yo(r).368 8. can also be estimated. when the molecule is observed at low rcsolution such that R . when R gets large I(R) begins to reflect the inhomogeneities of the structure with respect to scatteringlength density and may dcviate from the 1/R4 trend. First.3. where a is the average distance between nearestneighbor atoms. averaged over all points.3.g. the trend may be significantly obscured. which is.
p)’u’. Without knowlege of the trend of the data this integration would be impossible since data are never taken at large R . of course.9) where ti is the molecular volume and Ap the difference in scatteringlength density between solvent and solute. (8.3. Once S and.10) The beauty of this relationship is that it permits u to be estimated from data which are not on an absolute scale since the scaling factors for I(0) and Q are identical. where choices between alternatives are made on the basis of other data. especially when R is large. B i d . models differing significantly in morphology can give rather similar scattering curves. Note that I ( 0 ) = (A. Mol. correspondingly. Molecular Shape In addition to permitting one to evaluate the parameters just described. Model building is not straightforward.3.3 The “best ellipsoid” 95 V. J . A . no hard rules can be given for how well a model must match the data to be useful. This being the case. One is then placed in the position of having to decide whether a deviation between measured and model data is significant or whether it simply reflects contributions due to internal variations within the true structure. Luzzati. on average. 8. the analysis of scattering data can lead to an estimate of the shape of the molecule which gives rise to it. Tardieu. Finally. T h ~ s ~ ~ . Stuhrmann. DATA ANALYSIS 369 at R = 0 yields an estimate of S. (8.3.3. I15 (1976) . and H. guided by the numerical information whose derivation is described abovc as well as by whatever information may be available from other sources. Mateu.6. L. This shape is then taken as a description of the molecule. Real molecules do not have uniform internal scattcringlength density so that even a correct model will predict a scattering profile which deviates from the data. Model building is. 101.8. ~ ~ : I(O)/Q = U. Porod’s invariant Q can be evaluated : Q = 4n /omRzI(R) dR = (AP)~u. one is at the mercy of their reliability. Procedures exist for comparing measured data to the profiles of ellipsoids of the same radius of gyration which differ in axial ratio. Unhappily. The simplest kind of model building is equivalent ellipsoid determination. B. ~ ~ . These shape estimates have traditionally been arrivcd at by a modelbuilding process whose goal is to discover the shape of uniform scatteringlength density whose (calculated) scatter most closely matches the data. the trend of the highangle data arc established.
7. Weinzierl. ~ ~ More elaborate efforts involve modeling in terms of assemblies of spheres and/or ellipsoids and explorations of more complex shapes. A. H. On the right is the ellipsoid model of Pilz rr a/. J . J.89(1969). J. A number of studies of this kind have been reported in the past few years. D. Damaschun. J. drawn to the same scale. S. Mueller. Rich. 33. London and New York. Hill. Kim. Science ( Washin.C. Thompson. Kim. Perhaps the most remarkable recent success was the determination of a shape for tRNA from solution scattering data by Pilz et uL9’ There is an extremely good correspondence between their model and the structure of tRNA determined . The equivalent ellipsoid models for E. F. Bielka.~ crystallographically several years afterwards (see Kratky and P i l ~ and The Pilz model was derived entirely from solution scattering Figure 3). M u / . [Rcproduced with permission from Kratky and Pilz. and M . J.96“ data. Acra Bid.9Z arrived at from smallangle analysis. Suddath. Bottger. coli ribosomes arrived at by Hill et dgSa from smallangle data have guided work in that area for many years. Recent studies of animal ribosomes should be equally p r ~ f i t a b l e .370 8. J. E. Biophys. 44. rather than a Y shape as indicated by electron microscopy. however. Comparison of the smallangle model of yeast tRNAPheand its crystallographic structure. is a view of the Same molecule as deduced from xray crystallographic data. Rev.96”The trace of the polynucleotide backbone is shown. SMALLANGLE SCATTERING FIG. . W. Quigley. Ger. 817 95a (1974). McPherson. On the left. Bid.285 (1973). Sneden. Men. Press.92. L. and A. Studies on Gtype immunoglobulins in solution demonstrated a T shape for the molecule in solution. J.qton. D.] so found constitutes an unsophisticated model for a macromolecule whose main virtue is that its limitations are well understood by physical chemists. and J . Anderegg. 3.D. “’ G. a finding recent crystallographic W.39( 1978). J . “ A comparison of xray smallangle scattering results t o crystal structure analysis and other physical techniques in the field of biological macromolecules. Two other recent modelbuilding studies by the same group were guided electron microscopic results.)179. 11. with its principal dimensions indicated.” Q. Such models can be useful. H. Published by Cambridge Univ. G .
it will be true that the maxima in the profile have real phases and alternate in sign.4998 (1973). and J . "I3 C. Mol. J . J. Gladkikh. 13. I. 417 (1977). Spherical Objects If an object is spherical or nearly so.3. Contrast variation methods. DATA ANALYSIS 371 results have tended to A less useful effort was that made on . M . Mol. Witz.638 (1965). Biochem. B i d . Biochemistry 12. Biol. the lowresolution data from viruses can be analyzed in a more sophisticated manner than is normal in the lowangle field. Fishbach. Thus the intensities measured in solution can be inverted to give the scatteringlength density of the object as a function of distance from the object's center. Kozlov. the model the DNAdependent RNA polymerase from E . 124. Mol. M. its radial density distribution. l o ' B . Chauvin. For objects of this kind the rotationally averaged intensity profile observed by solution scattering is the same as the intensity profile not averaged by rotation. The reader should appreciate that direct statements about the distribution of material in an object result from smallangle data in no other case. but at low resolution they are effectively spherical. Jacrot. Anderegg. A.. Nature (London) 266. S. 42. Rubassay. Jack and S. Pilz. Ogievetskaya. Both of~this ~ ' ~kind ~ have been carried out xray l o o and neutron e x p e r i r n e n t ~ ' ~ 97 97n I. The problems of extending such an analysis to resolutions higher than those where strict spherical symmetry hold have been discussed by Jack and Harrison. and M . In addition. Licht. The classic biological spherical scatters are the small isometric viruses (e. A. Nezlin. P. 28. J . Z. C. C.Krdtky. FEES Lett.205 (1972). Harrison. Kratky. These viruses are in fact icosahedral in symmetry.3.. Sela.g. see Kratky and Pilz4). and D. 457 (1969). can be used to decide where the different chemical components of the virus reside. i. Bid.e. '" I A. L. Thus I(R)"* = I F ( R )1. Chauvin. J.'"' Viruses are complexes of protein and nucleic acid which sometimes include lipid. 641 (1978). Cser. and J. W. but also there will be secondary maxima at higher angles at predictable positions. 0. ~ this ~ case. 99 F.3. J . 99. C. A. Pilz. c ~ l iIn building may have been biased by spurious electron microscopic results. Jacrot. Mol. this fact will be readily apparent from its solution scattering profile. Eur. J . IS (1975)."' Because of their quasispherical symmetry. 0. M. discussed in the next sections. A. Ostanevich. I . and Yu. 8. Witz. In most cases phase assignments can be made.7. Harrison. Bid. Harrison.8. and B. M. R. Fishbach et for review. l o o S. 283 (1976). . where I F(R)I is the modulus of the Fourier transform of the object's scatteringlength density distribution. 68. Some of the more complex modelbuilding studies in macromolecular aggregates are discussed below (Section 8.16). Not only will the ellipsoid of axial ratio unity fit its lowangle data best.
from which R . In fact. and of the effect of variation in solvent scatteringlength density on scattering profiles. The Dependence of Radius of Gyration on Contrast In rcal molecules significant fluctuations in p exist which can affect the radius of gyration manifested by the molecule under any set of experimental “I4 H .. Even the initial slope of a Guinier plot of I(R). internal fluctuations will average out. Appl. the intensity of the scattering signal they produce.p o .. this prediction does not hold. 8..3. 173 (1974). thc argument being that for small enough R . Ap = p . and (ii) toward a characterization of the internal structure of macromolecules and macromolecular aggregates. In the classic analysis. Cry. B. . however.e. The prediction it makes is that scattering profiles collected on the same molecule in solvents of different po will differ only in amplitude.372 8. J . Contrast may also exist between different regions within the same molecule. The contrast Ap between a molecule and its environment is simply the difference between its average scatteringlength density p and that of the environment (e.8.via/ln~r. In the past 15 years there has been a significant shift of emphasis in both theory and practice towards consideration of the influence of density fluctuations within a molecule on its scattering behavior. i.3. In fact this approximation is a good deal less useful than often thought. Contrast is a concept used abovc without calling it by name. i. 8. Stuhrmann. Contrast During the early years smallangle scattering studies mainly concerned themselves with determination of molecular parameters of the kinds discussed above with the object of discovering the shape and dimensions of the object of uniform scattering density which best approximates the molecule of interest.g.9. solvent). a molecule is regarded as a shape with a constant 0.lo4 It follows that a correct estimate of a molecule’s shapc from its solution scattering profile may not result unless thesc factors are taken into account. is determined. These developments have led in two directions: (i) toward better solutions of the classical problems of molecular size and shape. The significance of contrast lies in the fact that the visibility of chemically identifiable regions within a scattering specimen. is proportional to Ap2. not in shape. p o . varies significantly with po in molecules whose internal density fluctuations are as modest as those of a globular protein.e. 7. SMALLANGLE SCATTERING and have provided useful information on the arrangement of the different chemical species in small viruses.
Kirste. 67 (1977). 5547 (1977).g. Phyc.3. 105. D. N<irl.3. 1"' D. Z .vs.112: P = (w. V fi = ( Jompl(F)Fd r ) 2 . I"" I"' . Fr. (8. J. Mattice. Bcnoit and C . is also the radius of gyration of the uniform solid the size and shape of the whole molecule. 105 (1972). and R. B i d .'the volume of the molecular shape. Mateu et a1.11) where R is the radius of gyration observed when the contrast is Ap. and R . 524 (1960). P. P. B. M. 57. M. Biopolymers 16. B. Luzzati. Cricliton. (Orsuy. and A. For an object composed of two regions of uniform but different scatteringlength density p1 and p . 247 (1965). Benoit. fhy. one can show that R2 = R. ' O R H.P/Ap2.12) where a will be recognized as the second moment ofthe fluctuations in density relative to the center of gravity of the uniform shape and /I the square of the . A satisfactory theory for these effects has been developed by considering macromolecules as having an average scatteringlength density ji on top of which is superimposed a fluctuation in scatteringlength density p ' ( r ) . Wippler. A. L. A. + V2P2)/(U. 56. Ci. vectors from the center of the uniform solid for the whole object to the centers of the two regions whose volumes are v1 and u2111. K .383 (1976). Luzzati. R . Moore. R. " " L. lo') J. and V. Mol. Thus for such an object /I gives information about the separation of the centers of mass of the two regions and c1 reports primarily their differences in radius of gyration. H. Stuhrrnann. Chim.A. 335 (1967). e. Sc. Ci. 905 (1975). Tardieu. R. Cotton and H. ' I ' C. J. . J .8. Chcnz. DATA ANALYSIS 373 conditions (see. Pro(. B.u first moment of these f l u c t u a t i ~ n s ~ ~ l"o~ ~is .i. Sardet. Carpenter and W. Bid.S. Kirste. (8. H. Z . and ? . B. Stuhrinaiin and R. Artid. The sum of the two functions p(F) = p + p'(F) then describes the scatteringlength density of the whole molecule..) 36. M. 70. a =I JoWp'(?)(r)' dr. Phys. Engelman. J .Z + U/Ap . Ph. Substituting this expression into the definition of radius of gyration. the radius of gyration of the object at infinite contrast. and H. 46. J.r. Parfait.. H . Tardieu. Mol. J . are thc radii of gyration of the two regions considered separately and F. R. U. Aggerbeck. Scanu. V . Stuhrmann and R. + u 2 ) . Chem.3.. Koch. Haas. 74. L.1°5). . Mateu. the following expressions hold: where R .
14) where Z(R)is the observed data.1 0.. One obvious point in favor of such a decomposition is that Zc(R).3. Dependence of the Extended Scattering Profile o n Contrast In studying the full scattering profile of a molecule.. 8.1) using this premise.(R) is the profile given by a uniform volume of the size and shape of the molecule.1). (Wiesbaden)72. ~ ~ *l ~ ~ ” ~three ’ ” com3 The ponents of I ( R ) are its “characteristic functions. . etc. absolute scale is set since these two parameters allow one to predict the absolute scatter expected from a given specimen in a specified incident flux. like virtually everything else connected with smallangle profiles. Absolute Scale and Contrast Variation Luzzati et ~ 1 have . .” If data are collected at several Ap. Phy. When Z(R) is developed from Eq.like R. solid angles.1 1. A p is a calculable quantity once the contrast match point is known. and the constant term I . 8. The contrast match point can be established from data which are not on an absolute scale (see Section 8. Plots of Q/Z(O) versus l/Ap2 should be linear. (8. where u is the volume of the molecular shape. Stuhrmann. SMALLANGLE SCATTERING 8.3.(R) is the scatter due to the internal fluctuations within the molecule. it is found that: (8. 177 (1970).2. Z. I. the same decomposition of the molecule’s scatteringlength density distribution into constant and fluctuation parts which leads to Eq. depends on contrast and is affected by scatteringlength density fluctuations within the molecule under investigation.7.11) retains its utility..3. As already observed (Section 8. (8. and the value at l/Ap2 = 0 should be l / u .. Chem. It is clearly the profile to use as a basis for shape determination. relates to the shape of uniform density most closely related to the molecule’s structure.374 8. However. one can fit the values observed for each R with a quadratic expression in A p with secondorder coefficient I . AII the classical methods for the analysis of scattering profiles are rigorously valid for data of this kind. Thus contrast variation combined with a consideration of Porod’s invariant leads both to a setting of absolute scale and a value for particle molecular weight independent of measurements of beam intensities. Z.5).3. all of which are sources of uncertainty in ordinary 11) H. using the chemical composition and density of the solvent. and ZJR) the component due to interference between shape and fluctuation s ~ a t t e r .3.3. . once u and Ap are known. recently ~ ~ pointed out that Porod’s invariant Q. u determination likewise does not require scaled data. firstorder coefficient I.
1 +9.01 +4.0 +8. @ 1975 by Annual Reviews. Table I gives some representative values for p for normal 'H materials and their 'H counterparts.'3 The m). with permission. Atztzual Review of Biophysics and Bioengineering.8. are Hrich substances.54 +7.36 $8.74 F (1 F = +6. Engelman and P. Neutrons versus X Rays As has been emphasized repeatedly in the past. whereas that of 'H is scattering length of 'H is 3.2 + 14. .3 +12. * Neutron scattering length densities are calculated assuming that all exchangeable hydrogen positions are fully deuterated.4 + 16. Moore. Since biological macromolecules. A practical application of these ideas to the analysis of the structure of lipoproteins from smallangle data has been given by Tardieu et al.3 +12. TABLE I.3.2 + 14.4 +16.0 +8. Neutron and XRay Scattering Length Densities for Biological Materials" Substance Fully H substituted H2O protein nucleic acid fatty acid carbohydrate Fully D substituted Neutronb X ray 0.07 +9.89 +8.43 8.55 +3. not to mention water.44 +6. B. DATA ANALYSIS 375 absolutescale determinations.12. Inc.11 +4. Volume 4. from D. M.3.27 +6.1 D2O protein nucleic acid fatty acid carbohydrate " Reproduced.44 0.67 F. large alterations in p can be brought about by substitution of 'H and 'H with minimal effects on the chemical and physical properties of substances in question.g the primary reason for studying biological structures using neutron radiation is the sensitivity of neutrons to 'H and the large difference in b between 'H and 2H.
’ l4 who collected contrast variation data on the same object using both D. by use of solvents which are H 2 0 / D 2 0 mixtures. glucose) can be used to raise solvent densities into the region of ppro. therefore.) 8. opening the possibility of experimental manipulation of the distribution of densities within a macromolecule.95 In this connection it should be noted that the D 2 0 / H . M. Crichton. including the condition of contrast match. the scatteringlength density of the macromolecule. and R.. A wider range can be explored with salts. . the range of contrasts available is limited. R. Mol. 0 method for po adjustment is not ideal. Manipulations of this kind lead to isomorphous replacement experiments for the study of internal structure. Parfait. J . The Hydrogen Exchange Problem In most contrast variation experiments it is the scatteringlength density of the solvent. J . the chemical and physical properties of substances inevitably alter with changes in electron density. Carbohydrate materials (e.g. Moreover. It is not surprising. (An interesting illustration of the difference between the anhydrous and hydrated volumes of a macromolecule was recently given by Stuhrmann and his collaborators. The volume explored by addition of smallmolecule solutes is the hydrated volume.O is included in the solvent.. Also. and do not perturb its structure. Ibel. Koch. these positions deuterate. SMALLANGLE SCATTERING Inspection of Table I reveals that pHZO is less than and pDZ0 greater than the scattering length of any ‘H biological material. glycerol.3. but their presence in high molarity is often incompatible with the requirement that a monodisperse solution be examined. The advent of neutrons for study of biological structure has stimulated interest in contrast techniques enormously.but can never produce solutions such that po = pRNA. Alteration of p in an xray experiment corresponds to alteration of electron density. K . H. which is manipulated. p o . B. Stuhrmann. B i d . When D. 203 (1978). Haas.O and deuterated glucose as the contrast modifying agents. 119. Unfortunately. R. A p = 0. The problem is that all biological macromolecules include significant numbers of hydrogens which exchange at appreciable rates with water protons. that contrast variation has not been popular in the past in xray solution work.376 8. An “ideal” experiment is one in which alterations in po do not effect p. J. p of a macromolecule can be altered over a wide range in a nontraumatic manner by isotopic labeling.13. Lipids cannot be contrast matched in an aqueous environment since pI < pHZ0. Thus a wide range of Aps for protonated macromolecules can be explored. raising p. The 114 H.
93. The difference should be due to exchange. 41. If a macromolecule can be obtained both in deuterated and protonated form. J . Ado. no perturbation will be seen. the great majority of the labile hydrogens do ( . the C8 proton in guanine) the rates of exchange are negligible at neutral pH and room temperature. .' 5 . [Eq. 288 (1966).1l)] will include contributions due to the distribution of exchangeable protons.. I L L ( P.g. do exchange. within experimental error. The radius of gyration found at infinite contrast. determined by solution physicalchemical methods. Engelman.. 255 (1975). DATA ANALYSIS 377 exchange process has a big enough effect on p that it can be followed quite accurately by measuring Z(0) as a function of time following the mixing of a protein in water. Hvidt and S. S.903 (1972). Mnl. Teitelbaum. In the few situations in biological small molecules related to macromolecules where proximity effects labilize Cbonded H (e.J.3. P. D. Bid. Downes. First."2~"7 Firm estimates for molecular weight and volume can be obtained provided the extent of exchange is properly taken into account. Ibel and H. for example. Annu. ' I 7 K . 101 (1975). and H . Mol. the molecule in question can be deuterated. knowledge of its monomer composition leads not only to an accurate overall atomic composition.75 % or more).104 As a general rule. 1 In order to make full use of neutron data. Prolein C'hcm. it is important to know what fraction of the potentially exchangeable hydrogens d o in fact exchange. and B. N . If they are distributed uniformly through the molecule's volume. which can then be evaluated. 21. the perturbation can be significant. then comparison of the density match points for the two forms gives both 0 and the fraction of the protons undergoing exchange. R.'0. V . but also to an accurate estimate of thc total number of hydrogens that can exchange. 91. Biol.' The formalism for the perturbations exchange in neutron contrast variation experiments has been worked out to some extent. In most cases. (8. Englander. If the distribution is nonuniform. Not all chemically labile hydrogens in a macromolecule will exchange.' Second. Nielsen. therefore.. owing to steric constraints imposed by molecular folding. assuming no exchange and that value compared to po at contrast match. Moore. W. The justification for this approach is that in many cases V determined from neutron contrast matching experiments for molecules where the exchange level was known in advance has been identical to V determined by classical methods. in small organic molecules hydrogens bonded to C are nonexchangeable and those bonded to 0. N. 0. M. Biochem. can be combined with chemical composition data to predict p . W. Schoenborn. Rea. tritium exchange methods can be used. B. with an aliquot of D20. There are a number of ways to obtain this information. Stuhrmann. etc.3. In a biological macromolecule. however.' 1 7 * ' Third. S. B. 01 will be altered only if the center of gravity of the exchange distribution differs from that of the ' ' '' '' 'Is I" A.8.
H 2 0 / D 2 0 contrast variation would be the perfect answer for many smallangle problems. Radii of gyration observed will differ with radiation and give information about the issues in question. Contrast Variation in Practice: Radius of Gyration The simplest contrast variation experiment is the measurement of radius of gyration as a function of contrast. and light differ significantly. e. Ap can then be calculated for all other solvent conditions from 'Iy lZo 1. They are not large enough to upset the qualitative validity of the results of neutron contrast variation experiments. Federov. They should be regarded more as a source of frustration in that were they not present. N. Serdyuk. 0 composition. t?rookhac>enSymp. the radii of gyration of the regions occupied by the two species.A. and an estimate of the separation of the centers of mass of the two regions. 11. and (iii) use of several types of radiation to measure the same The operation of the first two kinds of experiment is fairly obvious from what has already been said.' l 8. Thus the internal contrast between chemical species within a particle will change with radiation type as will the contrast of the whole particle with its surrounding solvent. Srrdyuk and 9. neutrons."^ From the solvent composition at which Ap = 0.g. and fl will be unaltered. Tardieu et (ii) solute density manipulation"2. in the ideal case the characteristic function for the uniform volume. 27. A straightline relationship will be observed for homogeneous sample^. This is a particularly useful experiment when applied to structures which are aggregates of two kinds of material differing significantly in p. for which many examples may be cited (for review. and the changes are different for each species. A p for each data set is evaluated from the observed dependence of [l(O)/C] l i 2 on solvent composition. see Jacrot". rather than merely a very good one. Three experimental approaches to this kind of information have been used in the past: (i) solvent density manipulation. The third type of contrast variation experiment deserves further comment. The scattering length densities of different chemical species vary according to the type of radiation with which they are studied.3. pis estimated in absolute terms with due allowance for exchange.645 (1973) 1. Biol. J . will now correspond to a volume of nonuniform p whose nonuniformities relate to the distribution of exchangeable protons. It exploits the fact that the physics of the scattering of x rays. Sci.378 8. It should be emphasized that in practice exchange effects are not overwhelming. . N . 8.. The now classic neutron solvent variation method involves measurement of the radius of gyration of the molecule of interest in solvents of different D 2 0 / H .1V49 (1976).'" I . Polym. SMALLANGLE SCATTERING whole structure. For such an object a contrast variation experiment should yield the radius of gyration of the whole structure. .14.
C .112 Neutron contrast variation studies have been done on a variety of twophase structures including ferritin. Nulure (London)253. and K. Ibel. and IF. Boseley.'30'3 1 and fibrinogen.e. Bid. There are a number of ways of plotting the data to evaluate their trends. Baudy. and IF. 102. D. Kitzes.!7 will only be as good as the available information about the volumes occupied by the phases in situ. G. L. Stuhrmann. Biochum. B. Baudy. Biochm. G . If a is positive. S. R. DATA ANALYSIS 3 79 knowledge of solvent composition and density. J. M. 177 (1978)..3. H. Biophys. The quality of these estimates can be greatly improved by studying the structure in a variety of deuteriumlabeled forms. B. Ibel. 1043 (1975). Brookhuren Symp. 13. i.' 22' 2 7 ribosomes.A. Osborne. Koch. Thus the quantitative estimation of R . Proc. 27. J. Biophys. Stuhrmann. Worcester. Yeager. are obtained from the plot by picking off the values for R at the contrasts where phase 2 and phase 1. at I/Ap = 0 is R:. Commun. and E. P. M u / . Bram. P. CeN 10. 4. M . 143 (1976). 13 in situ) as well as values for R . 139 (1977).8. ' I J J . 1113 (1976).l [see Eq. MichelVillaz. K. 123. (8. M. p curvature will always be concave down. Knedle. Pardon. Biol. Bram. Such an investigation can lead to accurate estimation of the volume fraction occupied by each phase (i. J. Chabre.p o . Richards. and P. Acud.S. . 1 3 ' H. and M.3.. Vastel. Crichton. Bram. Rex Commun.I3' "I H. 2163 (1975). Sci. R.. 0. and R. E.Biol. and R.e. R . . B.. and A . Baudy. 72. Talchell. M. Le Pault. and B. S. J .U. and B. Van Holde. 100. R. Sardet. E. J . P. G.Petersen. J. B. K. J . J. Sci. Ibel. high p regions in the particle must lie towards its exterior. H. Res. G . ''* H . In general. P. prior knowledge of chemical compositions and partial volumes is precise enough so that the chemical identity of the high and low density regions is certain. 2275 (1978). S . K. M . J. Haas. Jacrot. Bradbury. respectively." ' nucleosomes and other chromatin related materials. De Wolf. Lc Pault. A nonzero p implies a lack of coincidence in the positions of the centers of mass of the high and low density regions within the particle. B. 1 3 " M . are contrast matched. and K. Biol. Marguerie and H. 72. Aerrd. Baldwin. 391 (1976).  YzJ. P. P. A . Baldwin. M. The slope of the plot I/Ap = 0 gives a and its curvature 7 !. and R . H. Haas. F. ButlerBrowne. R. R. K. In these situations the qualitative statements that can be made about their relative distributions in the aggregate from an analysis of the type described are unambiguous.. Wooley. U. S. Stuhrmann. Mo/. Bradbury.' 1 2 ~ 1 2 8 * 1 2 9rhodopsin in detergent micelles. Suau. B. Ibel. Beaudry. Crichton. . if a is negative. in "two phase" aggregates. 2379 (1976). Mol. Hjelm. G . Ap = p . 176 (1976). Proc. Nut/. M. Nail. Hermann. J . Perhaps the The intercept most convenient is to plot R 2 versus l/Ap (see St~hrmann"~). C. 399 ( I 976).2. Parfait. 72. Nuckic Acids Res. the opposite is true. U . Koch. 245 (1975). J .13)] from a and . D. Nucluic A d s RPS. P. P. GrunbergManago. 12Y P. .
Recent extension of such studies to 70 S ribosomal couples in which one subunit is deuterium labeled has led to determination of the intersubunit distance and evidence that couple formation is accompanied by little conformational change on the part of the individual subunits. Kostner. some extensive xray studies have also been undertaken on twophase systems. Laggner. A . 1 3 4 R. IV3 (1976). Biuphys. With the increase in interest in contrast variation methods. 13'" H. 251 (1978). B.6. Stuhrrnann. Mueller. J . W . R. Mech.'~ a~review ? ' ~ ~ see Laggner of lipoproteins have been i n v e ~ t i g a t e d ~ ~ * ' ~ ~ and Mueller' 3 7 ) . Zc(R). Contrast Variation in Practice: Extended Scattering The analysis of contrast variation data sets into characteristic functions is still a relatively new method. B i d . Srruc. J . Biochrrn. more deductive approach has been suggested by S t ~ h r m a n n ' ~ which ~ " requires I. Kratky. P. Mueller. Glatter. I"' K. SMALLANGLE SCATTERING The nucleosome studies alluded to are notable in providing direct physical evidence for the now widely accepted view that the basic subunit ofchromatin structure is DNA wound around the outside of a histone core particle. Jacrot. J. Haas. 4. 2421 ( I 977). is the best possible input into a shape analysis.73 (1978). Biophys. Laggner. Res. P. Q . Stuhrmann Brookhawn Symp.'33 Giege et have done an interesting study on the formation of complexes between tRNA and tRNA ligase by contrastmatching the protein.r. and recently the rhodopsindetergent system has been and its comstudied by these same means. 213 (1974). Laggner and K.(R) as its input. Thierry. Moras. and G. G . with protein tending to form the exterior surface of the particle. Holasek. . D. the shape scattering profile or at least an approximation to it. B. Crichton. R. Giege. The ribosome studies have produced the only data available on the relative distributions of bulk protein and RNA in these structures (see also Serdyuk'"). FEBS Lett. Shapes can bc derived from i .3. The 30 S subunit structure has been recognized as a reasonably homogeneous mixture of RNA and protein. 4. Under these conditions it was possible t o show that at low ligase concentrations a 2 tRNA: 1 ligase complex forms. 11. Recently. Kostner. ' 35 K. B. 82.Glatter.3. and H. and 0. As already emphasized.'" The paper of Tardieu et panion theory paper95 contain a particularly detailed exposition of the analysis of two phase systems. Eur.C. 8. R. H.380 8. 0. The conccpt behind the approach is to regard the density distribution ' 3 3 M. 0. Parlhit. and G. 40. however. Koch. while the 50 S subunit shows considerable segregation of the two species. a second. ( R ) by model building just as described in Section 8. Mueller.15. A number (for . J. Zaccai. Nucleic Acids Res. 27. 371 (1978). 1 3 7 p .
DATA ANALYSIS 38 1 of a molecule as the sum of sphericalharmonic density w a ~ e s .I3' as have the subunits of the E . Sect. Investigations of the arrangement of the two phases can hinge either on naturally existing differences in scatteringlength density or on artificially created distinctions such as can be produced by deuteration. B. B. If the object in question has uniform internal scatteringlength density. Second.'~* for which a shape quite close to that of the crystal structure was obtained.S. multienzyme complexes. ' ~ ~ A~ ' ~ ' ~ ' unique decomposition into spherical harmonics exists for any object. At some level of description many of these can be regarded as twophase problems and approached by contrast variation as already indicated. Sluhrmann and H. Only a few structures have been dealt with in this way. Fuesa. nucleosomes and other chromatinrelated structures. the Z. Natl. 13' . Macromolecular Aggregates: Synthesis Methods In the past decade an increasing amount of lowangle work has been done on macromolecular aggregates.'37aTakingthese into account one can deduce the coefficients from the data within certain limitations and can then calculate models. For a discussion of the impact of these problems see Stuhrmann et 8.A 26. considerable restrictions are placedon thevaluesthesecoefficientsmayhave.8. and its solution scattering profile directly reflects the magnitudes of the coefficients of this expansion. Koch. Stuhrmann.3. which are affected by exchange. Acra Crystallqr.. Sci. One of the first was ly~ozyme. R. as is true for the Z.(R) obtained corresponds to an object of nonuniform density. B. R.'i2~"8 For structures with more than two subunits. and lipoproteins. ribosomes. J. Acta Crystuiiogr. Proc.A. viruses. most of the data used have been neutron data. but its limitations are still unclear. coli ribosome. and R. H. The 50 S structure'39 obtained resembles the shape the object appears to have in the electron microscope. 1 3 " H. Haas. 2316 (1977). J. In this class of structure one would include multisubunit enzymes. A A32. M. Ibel. For an object undergoing exchange.3. the impact of error in the input data on the final result has not been worked out. Parfait..(R) object. H. The results of this kind of analysis are intriguing." Synthesis H. Crichton. There are two problems with the analyses done so far. Acud.16. Stuhrrnann. contrary to the assumption of uniform density which guides the choice of coefficients for harmonics in model building. these methods are not readily applicable and other means must be used.297 (1970). K.67 (1976). Two methods have evolved for studying multisubunit structures which might be described as (i) "synthesis" and (ii) "distance finding. 74. First. U. Fibrinogen has also been analyzed. SCTI.
Mecl. The analysis performed on haemocyanin. 'The ~ ~ work of Stockel et on the hemoglobin of Tuhfex can also be cited. Eur. Witters. l ~ ~ ' 4 1 G . Actci B i d . of the ternary complex. and R. Purschel. H . . using extended scattering curves. is the fraction of the total scattering length of complex relative to solvent contributed by trypsin. Biochem. Berger. Mayer. B. 309 ( I 968). separately and the R. J . is to determine the disposition of the subunits within the aggregate. Model building is then done in which the objective is to match the whole structure's scattering profile by assembling aggregates of its substructures. Acru B i d .3. (8.15) wheref. 14' G . Damaschun and H. 37. J . in either case. Ger. whereas distance finding comes into its own for structures containing single copies of several different kinds of subunits. C'rysfaNoqr. and so work up to a description of the whole complex in terms of subunit radii of gyration and locations based on relative distances. Eur. R.12)]. A. Med. and for two subunit systems gives results equivalent to those obtainable by contrast variation [Eq. M.479 (1978). The parallelaxis theorem is the proper formula for combining radii of gyration. f 2 the corresponding quantity for the inhibitor. Damaschun. V. Ger. Fichtner. 21. and D. Stockel. P. These two techniques are thus suitable for the analysis of different kinds of structures. but even this approach is of limited a p p 1 i ~ a b i l i t y . One could imagine a third protein in a complex with the other two. The objective of these analyses. P. J.79 (1977). 193 (1973). ' 4 3 P. Biochrm. V.865 (1968). Appl. by Pilz and colleagues is a fine example of ~ y n t h e s i s . Purschel. 11. which is an aggregate of 200 subunits.382 8. 80. and their distance apart on the complex inferred using the parallelaxis theorem: (8. It is clear that an important premise of such an analysis is that the shapes of subunits and subassemblies do not change during degradation and/or reassembly of the complete structure. Lontie. It should be noted that deuteration of subunits followed by appropriate contrast matching offers the possibility of testing the assumption that subunit radii of gyration are unaltered by complex formation. 21. A. and J. SMALLANGLE SCATTERING is the only approach possible for aggregates containing many copies of only one or two subunits. Pilz. Onecan also study the shapes of the subunits and those of the subcomplexes they may form. J . Moore. Langer. as well as that of the whole aggregate. 14* J. and A the distance between their centers of mass. Engelman. A simple example of synthesis would be the work of Damaschun and his R s were meacolleagues on the trypsintrypsin inhibitor sured for the two molecules separately and for their complex. Keller. Reich. I.3. G . and R. measure its R .
Mol. N . Monutslz. ( R ) and Z2(R). DATA ANALYSIS 383 8.574( 1970). Thus measurement of I. W. Hoppe. Akad. W.and for the unlabeled structure I(R).(R) = CI12W) +w 1 .een repeatedly pointed out theoretically and demonstrated in model system^. d .C I l W + I2(R)1 = F. K .. wliere. + D. + + C + D + E + F. Sosfenov. J. Macromolecular Aggregates: Distance Finding Over the years the possibility of measuring distances between points within a large structure by xray solution scattering has b.17 . I. 1. A.3.'^^'^^ The points in question are labeled with heavymetal atoms by chemical means and solution scattering profiles obtained for the doubly labeled species I . 78. 10. Then Z12(R) =A B Il(R)= A + B 12(R)= A + C I(R) = A. F is a damped sinusoidal ripple whose nodes occur at regular intervals R = n/2r12 in R. 2(R). Nauk S S S R 190. lsr. Chum. B. both of the possible singly labeled species I . and f2 are the scattering lengths of the heavy atoms and the sums run over the unlabeled atoms. Hoppe. Feigin. Thus if profiles are normalized to equal numbers of molecules and total flux. 581 (1973). 76. Worthmann. Let F= 2f1f2 sin 271Rrl 271Rr.3. .(R) leads to determination of r I 2 . C/zmi. Dokl. the distance between the two labeled points. Vainshtein. 263 (1947).f. + E. and L. Kratky and W. F will be recognized as the interference contribution in the scatter of the doubly labeled structure due to the presence of the two heavy atoms. 145 146 1 4 ' 1 4 ' 0.8. 321 (1972). B i d .
72. Hoppe. the subtraction will still yield l x ( R ) . Sci. A . E. L. Langer. M.. Some comments about data interpretation are in order for these experiments. i. Bid. which is a foursubunit structure. AcuJ. and P. Is" . Engelman. D. B i d . U . J . the practical applications of the distance method to macromolecules have all involved the mapping of subunit locations by the neutron version of the experiment rather than its xray counterpart. ~ ~ l ileading . Simultaneously with these developments it was observed that this labeling strategy was likely to work using deuterium as a label for whole subunits within a macromolecular aggregate and. Katz. D. ~ to ~ the ~ determination * ~ ~ of ~ the ~ placement of 9 of its 21 protein subunits. Ibel. W . two systems of considerable biological importance have been worked on. B.e. 595 (1979). neutron radiation. Stockel. over 30 distances have been measured within the 30 S ribosomal subunit of E . Moore. So far. Moore. M. B. I51 P. and the same done with the two singly labeled samples at equally high concentration. The intermolecular vectors that make normal solution scattering measurements useless at high sample concentrations will be the same in both samples and their contributions will cancel when the difference is taken. M. Mol. Schindler. I x ( R ) D. So far this requirement has limited work to structures from bacterial sources.463 (1978). When whole subunits are labeled rather than simple points. Heumann. The possibility was envisioned of mapping the positions of subunits within aggregates by triangulation based on distances. J . it must be possible to disassemble it into its subunits and cause it to reassemble.3888 (1972). has been fully analyzed. D. W . Moore. I . The structure must come from an organism which will grow in the presence of high concentrations of D. J . B. May. ' pointed ~* out a unique feature of this type of measurement. Mu/. Engelman and P. Engclman. A. J . 12. of course. Proc.O. R. S . In addition. A p j ~ / C'r)~s/d/ogr. If the doubly labeled and unlabeled samples are mixed in equivalent amounts. At the time of this writing. and P. the DNAdependent RNA polymerase. only biosynthesis leads to levels of D incorporation into macromolecules sufficiently high to make labeling useful. No//. . Zillig. another difficult step. Cjeka.384 8. SMALLANGLE SCATTERING H ~ p p e ' ~ ' . Strell. the structure must be reconstitutable. 2. J . Crespi. 149 The signaltonoise ratio for such an experiment is 10 to 100 times better than in its xray analogue. namely that it can be carried out with specimens at very high concentration. and K . the profile of the mixed samples obtained at high concentration. H. J .I5l The results are generally in good agreement with other data available on these structures. 134. the ribosome and thc DNAdependent RNA polymerase. 119. in order that the required placement of deuterated subunits be achieved. The biochemical requirements for such measurements are stringent. 176 (1979). Nevertheless. H.
B. lSZa J.. where R . just as in the twoatom case. First. respectively. . This function looks superficially like a damped sinusoidal ripple. 112.3. I. M.'~' 8. and D. being influenced by subunit shapes and relative orientations.B..qr. Alternatively..s/u/lo.3. H and 'H. Engelman. r = Jomip(r) dr. Moore. Moore and E. The periodicity of its ripples is irregular.'^^" 5 1 For large structures like the ribosome this relationship can be used as the basis for solving sets of distance data both for subunit positions and radii of gyration. are the scattering lengths of . The possibility of analyzing such data for subunit shape and relative orientation has been pointed out. Solution Scattering Studies on Molecules of Known Structure So far we have discussed smallangle methods primarily from the viewpoint of an investigator seeking initial information about the shape and properties of a molecule of unknown structure. and R . one can make use of the fact that 7 . . and the sums run over all deuteriums in subunit 1 and all deuteriums in subunit 2. Two methods for obtaining distances commend themselves.I. Bid. A. = Joa)r2p(r) dr = d f 2 + R: + R:. and h. p(r) in this case will be the length distribution of all vectors joining d in the two subunits.8. $1. P. Schoenborn. but no general procedure has yet been di~covered. and dl. Langer.(R) can be inverted to obtain its corresponding p(r). Although this is the most common P. the distance between subunit centers of mass. Mol. C'rj. contrastmatching out the protonated parts and measuring the necessary Ris directly.Appl. will be 5 2 : d. 199 (1977). but is not. B. Weinsiein. 12. 321 (1979).' For less complex structures the possibility exists of deuterating single subunits.18. becomes (for neutrons) DATA ANALYSIS 385 where b. are the radii of gyration of the two subunits as they are configured in sit^. P.
701 (1973). M . Carpenter. A number of investigators have suggested approaches for making these calculations.qr. The purposes of such studies have been several: (i) as benchmark investigations to establish the validity of smallangle analysis. Gordon & Breach. Tech. Soler. The favorite objects for benchmark studies have been hemoglobin and myoglobin. lnt. 81 (1977).2. New York. A. and (iii) there is the solvent problem. K . Surnmw Sch. p. A critical step in such investigations is the computation of an expected scattering curve from the crystallographic data. it is perhaps not surprising that calculated radii of gyration commonly differ from measured values by 0. This is not a trivial computation for a number of reasons: (i) Eq.0 A in 25 A. The lysozyme work of Stuhrmann and Fuess’ 3 8 has been described ’ ’’ H. Kayushina. L. Shchedrin. 1975. 1967. and (ii) xray and neutron results on the same molecule are consistent. Is’ Yu. with lysozyme coming in a distant third. Myoglobin has been investigated by Beeman” and Stuhrmann. ed. 15‘ W. (ii) surface polar residues are often poorly located in a crystallographic structure because of intrinsic disorder. In general the results obtained have led to the conclusions that (i) the crystallographic structures of proteins are very close to those found for the same proteins in solution. in “SmallAngle XRay Scattering” (H. the highresolution solution scattering curve shows much less structure than anticipated from the crystal structures. This suggests that myoglobin in solution may be a more flexible structure than it appears to be in the crystalline statc. C‘omput. as is the case with proteins. Brumberger. Kristallngrufiya 18.51. they have also been used from time to time to study macromolecules whose structures are known at high resolution.’ 53156 Given the problems to be overcome in such a calculation. Biopolvtners 16. The last is particularly difficult because the structure of the solvent at the surface of the molecule will affect its apparent size and the shape of the boundary between the molecule and the solvent.). (ii) to study the correspondence between the crystallographic structure of a molecule and its configuration in solution. In the latter investigation the myoglobin characteristic functions were determined and the intriguing finding made that although the fit of calculated to observed scatter is good at low and intermediate angles. Mattice and D. (8. Robbin. R . and B. These problems lead to an interaction between goals (i) and (ii) of the preceding paragraph. L. SMALLANGLE SCATTERING application of these techniques.1) is cumbersome to apply for molecules in which the number of atoms 2 1000. C.386 8. Cryslollo. Proc. 267. . and (iii) to investigate conformational changes connected with ligand binding or alterations in environmental conditions. A . 376 (1976). 1 5 4 J. Feigin. Watson. L.
H o p p e ~ Srj. Both xray and neutron et ~ 1 . Conrad. Eitr. and H. I"' H. A . and F. Sttjckel. Mayer. R . Kirchauser. B i o p h ~ ~ 69. Eicher. '('' G. Chrm. PIz~~siol. and Y . P. data have been c o l l e ~ t e d ~ ~ (see ~ ~ also ~ ~Damaschun . W. Mucller. Kratky. "'I R. Moshe. 1216 (1970). 351. Mateu. Schneider. Durchschlag. Mayer. and F. 0. 1499 ( 1970). I"' J. Durchschlag. Scherm. 184 (1972). 0. Chrm. H.'68 Efforts have been made to develop an ultrasensitive difference method for detecting small changes. 231 (1969). they also demonstrated a substantial alteration in radius of gyration of hemoglobin upon oxygenation and dcoxygenation. J .3. Pilz. A. B i d . Hq~prSiylc. 1. Schwaiger. H. Mayer. S. ~ . Mayer. and J . 47.8. Damaschun. Bioclirm. A . j ) 1436 (1978). Schneider. Biocheni.I7' The point is that for molecules of known structure. Mayer. Kirschner. and G. and M. Puchwein. The idea of using smallangle methods to monitor conformation changes is likely to be a significant application of the method in the long term. 30. V. Physiol. A . 350. Eur. and K . J. Phj. J . 19. 87. G. H. 845 (1969). Kugler. J. 151 ' Is" . ' ~ ~The ) . ~ name ~ a few other systems.V.' ~ ~ neutron data are significant because they are among the first ever obtained on a protein. K righaum and F. 41. Zinke. H.ler's Z. R. Mol. Schmatz. Krigbaum and Kugler' 5 7 have also examined lysozyme by xray methods as well as chymotrypsin and chymotrypsinogen.225 (1969). In addition to hemoglobin. 85 (1978). W. Schuster. I h " 1. 350. Malnig. Zipper and H. HoppeSeyler's Z. A. The change observed is once again close to that anticipated from crystal data. Simon. most notably by the Julich group. 7 5 3 5 (1977). 71. "" H . V. 851 (1969). Kaiser. other studies of the same kind have been done on lipoprotein^'^^ tRNA. P. I h h 1.'659'6h and a n t i b o d i e ~ to . Schmatz. W . l b 8 P. . 9 (1971). K. 119 (1978). von der Haar. Small changes in structure have also been observed in malate dehydrogenase upon substrate binding. Luzzati. 27 (1974). H . Ninio. B i d . J . and R. W. Schneider. J . Biophys. Biochem. 217 (1972). 41. In addition to verifying the possibility of collecting neutron data and showing the close correspondence between the solution and crystal structures. J . Damaschun. Sfud. have demonstrated a small volume change in yeast Durchschlag et glyceraldehyde3phosphate dehydrogenase as a function of saturation with NAD. There have been a whole series of solution scattering experiments done on hemoglobin. Bid.169 and a useful critique of the information one can derive from studies of changes has becn prepared. V. J. R. Schelten. Eur. Thomas. Stud. Biochiw.Kratky. Bioc~hmistry 9. Mol. ""' J. T. Schelten. G i r .. Ruchpaul. B i ~ d ~ m i s t r 17.r'sZ. Cl7rm. J. I h Z R . Conrad. Schmatz. Vogel.sio/. Hossfeld. Mol. I h 4 L. Purschel. Camejo. DATA ANALYSIS 387 above. and R. B.
Lagerkvist. 1. B i d . The formation of complexes between macromolecules can also be usefully followed by smallangle methods. In electron microscopy there are always problems of visibility of molecular edges. Folkhard. Mol. R . It will undoubtedly become common to test hypotheses about the conformational properties of macromolecules by smallangle scattering. In addition to the parameters listed in Table I1 there are a number of more esoteric quantities which can be measured2 but arc not described above for reasons of space. Biochern. 63 (1975). On the other hand. which limit its accuracy in this regard. and V. J . Smallangle data. J . ’’ 8. etc. SMALLANGLE SCATTERING minor alterations in smallangle scatter which in the absence of other information might appear totally mysterious are often interpretable. 59. B. R . Stoefiler. it should be remembered that one of small angle’s primary virtues is that it characterizes a molecule’s structure as it exists free in solution. Rymo.1. 153 (1973). of course. In the case of aggregates it is sometimes possible to go beyond this “whole molecule” description to specify how its subunits are arranged. 0. L. Bid. 77. In the case of uncharacterized macromolecules it is clear that smallangle techniques can lead to a rather complete characterization in terms of size and overall shape.4.4. Garrett. whatever it is. W . The resolution of the description produced by a smallangle analysis corresponds roughly to the best electron microscopy can do with favorable macromolecular specimens. 394 (1975). One of the failings of spectroscopic methods for studying these kinds of changes has always been the difficulty of relating alterations in spectral properties to alterations in structure.388 8. Mul. Concluding Remarks 8. This R. The strength of the smallangle approach lies in the accuracy of the size and dimensional information it produces. Osterberg. and G . although the smallangle data must necessarily bc consistent with the correct shape.. Finally. its shape is more likely to be identified correctly from electron microscopic data than from smallangle results. 99. ”’ . if a molecule has a highly unusual shape. Pilz. J . G r r . Kratky. distortions during sample preparation. Summary Table I1 briefly summarizes the most important kinds of information one can extract from smallangle data on macromolecules or macromolecular aggregates. SjBberg. Osterbcrg. report structure directly and offer unique opportunities for studying the subtleties of macromolecular action in solution.
molecular volume 3. Its correspondence with the molecule’s chemistry verifies the correctness of the analysis. molecular weight 2. however. lowresolution picture of the internal distribution of high and low scatteringlength density regions 0. position of the centers o f mass of subunits 2. Critique Lowangle scattering. subunit radii of gyration 3. A wellcollected solution data set might permit one to specify a length distribution as 15 or 20 independent numbers. may raise that number severalfold more.2. No comparable internal control is available for the models deduced from smallangle data.q.yaiesonly I . molecular shape. The numerical data given by smallangle experiments constitute solid information. must be approached with caution. Principal Molecular Parameters Deducible from SmallAngle DaVa A. and the effective lumping of vectors into classes according to length does the same for the scatteringlength information. Models deduced for molecular shapes. as equivalent ellipsoid or better I. Internal labeling methods. CONCLUDING REMARKS 389 TABLE 11. distribution of interatomic distances 5 . There is an additional .qre. subunit shapes and orientations fact makes smallangle techniques useful as a way of testing models and assessing conformational changes. surface area 6 . especially the more complex ones. 8. which involve large increases in experimental effort.8. In a crystallographic study the covalent structure of the molecule under investigation eventually emerges.4. is a lowresolution technique.4. All maruomolecules I. A typical macromolecule contains 103105 atoms and requires 3n . radius of gyration 4. The rotational averaging inescapably connected with solution experiments results in the loss of much of the coordinate information otherwise available in the singlemolecule diffraction pattern. as already emphasized. In any case nothing approaching a full description of a macromolecule is ever possible. a failing this technique shares with other lowresolution methods. A fullcontrast series might increase the number of specifiable parameters by three.6 coordinates and n scattering lengths for its full description. Macromokcuior a.
The question must then be asked: under what circumstances can the limited information available be made to yield a biochemically significant result? Four kinds of profitable situations can be recognized. have contributed to this work by furthering my education in smallangle scattering: D. P. It is conceivable that synchrotron radiation sources and highspeed detectors may prove of great value for investigations of this kind. Luzzati. First. SMALLANGLE SCATTERING shortcoming common to all lowresolution techniques: most biochemical problems ultimately require structural information at atomic resolution. The ribosome is a prime example of such an object. there arc a number of large biological structures which are simply not suitable for crystallography at this time. Third. or both. Smallangle methods can rapidly yield a molecular weight and good estimates of size and shape. Engelman. Second. It is in this last area that one anticipates the most growth in the years to come. both consciously and unconsciously. and V. The fourth opportunity is the study of conformational change and binding interactions of wellunderstood structures. smallangle methods are a sensitive means for examining the relationship between solution and crystal structures for biological macromolecules. for example.390 8. This work was supported by a grant from the National Science Foundation (PCM7810361). traditionally valuable information. Acknowledgments 1 wish to acknowledge my indebtedness to those who. The validity of these shapes as a description of the molecules in solution can be examined by smallangle means and by virtually no other. Many macromolecules have very eccentric shapes when viewed. For some of these it is also true that lowresolution information is relevant to the kinds of questions biochemists are asking about them. and in such situations the means and the ends are well balanced. Similarly. in the electron microscope. . a smallangle opportunity exists at the very outset of an investigation of a new biological macromolecule. M. for reasons of size or lack ofcrystals. B. Schoenborn. smallangle methods have no rival as a means for testing models for structures.
39 1 METHODS OF EXPERIMENTAL PHYSICS. a second objective of this paper will be to indicate areas where there is still a need for continued development of experimental methods of a physical nature in the biophysical applications of electron microscopy. Among the requirements that must be met are that the specimens should be very thin. “Introduction to Electron Microscopy. The purpose of this article is first to describe some of the current developments in methods of electron microscopy. Hall. Highresolution images can only be obtained.9. Such is by no means the case. Heidenrcich. Therefore. McGrawHill. finally. New York. 1964. and particularly for the biophysicist.” Wiley (Interscience). 1966.” 2nd ed. ELECTRON MICROSCOPY By Robert M. Glaeser 9. New York. however. placing much of the emphasis on the new methods that have been developed for molecular structure determinaation. VOL. lnc. the development of new experimental methods of electron microscopy that are suitable for molecular structure determination and the development of new physical methods that are suitable for application to special problems in cell biology still represent lively fields of research. D. ’ 20 Copyright @ 1982 hy Academic Press. C. that the specimen structure should not change during the period of electron irradiation while the image is being recorded. provided that the specimen materials are prepared in a suitable form. The electron microscope is an optical instrument l S zwhich permits (but does not guarantee) the direct visual observation of structure at very high resolution. ISRN 0124759629 . that they can be placed into a vacuum of lop6 Torr or better. In the field of electron microscopy there still remain many challenges for the experimental physicist. Electron Microscopy as a Tool for Structure Determination The electron microscope has by now become such a routine tool for the cell biologist that one might suppose that electron microscopy had long ago passed out of the realm of experimental physics. As will be seen. “Fundamentals of Transmission Electron Microscopy.1. these experimental methods are in many cases still at an incomplete stage of development. and. E. All rightr ofrepruduction in any form reserved. R.
5 A. because the electron optical lens aberrations cannot be corrected in the same way as is possible in light optical systems. It is the spherical and chromatic aberration of the objective lens and not the electron wavelength which limit the resolution. the depth of field works out to be approximately 165 A. under proper conditions of instrumental operation. in all cases the images so obtained are frequently subject to ambiguous or even misguided interpretation. In practice the resolution experimentally obtained with biological specimens is limited in most cases to approximately 2530 A. Improved “resolution” can also be obtained with tilted (nonaxial) illumination and in the darkfield image mode. Electron microscopes are also commercially available which operate at an energy of 1 MeV (A = 0. At this energy the electron has a relativistic wavelength of A = 0. but depends upon the resolution at which the image is being examined. and a variety of research instruments going to energies as high as 3 MeV have been in operation for several years. then the depth of field is given by the expression AZ = d2/2A. the contrast transfer function continues with appreciable magnitude out beyond the point of the first zero. The instrumental resolving power is best specified in terms of the resolution at which the contrast transfer function of the instrument has its first zero. = 1. One limitation on the specimen thickness is given by the depth of field of the microscope. ELECTRON MICROSCOPY Electron microscopes have traditionally been designed to be operated at an energy of 100 keV. This sort of specimen thickness is not a limiting factor for biological applications if the image resolution is not as good as 3.5 A. and an electron energy of 100 keV. but it is important to have this factor in mind when going for very high resolution. A3)”4. If the objective aperture is optimized for the resolution that is of interest. where C. or worse. The depth of field is. This limitation in the practical results is due to numerous different factors which may be of varying relative importance according to the specific problem under . At 100 keV and with a realistic value C. and it is prudent to assume that the limit of interpretable contrast is given by the expression f(C. In some microscopes.392 9. this resolution limit works out to be about 3. Iis the electron wavelength. Such highvoltage electron microscopes were initially designed to make it possible to work with thicker specimens than can be used at 100 keV.2 A.5 8. where d is the resolution. However. not just a single value. This very short wavelength is two orders of magnitude smaller than the actual resolution limit of the electron microscope. It has only recently been the case that specially designed highvoltage electron microscopes have begun to surpass 100keV instruments in terms of improved instrumental resolving power. of course.0087 A). For 100keV instruments this resolution limit is approximately 3. under conditions of optimal defocus. is the coefficient of spherical aberration and . For a resolution of 3.037 A.0 mm.
Overcoming these technical difficulties represents an area of active research development at the present time. A second factor which can limit the resolution attainable with biological materials is the serious problem of radiation damage. Protein denaturation and other types of destruction of the specimen material can be expected to occur whenever the preparation methods require chemical fixation and dehydration. This damage is an inescapable consequence of the inelastic scattering events that are produced by a fraction of the electrons that pass through the specimen. This information is essentially of a spectroscopic nature. cause the inelastic electrons to be scattered out to quite wide angles. not to mention possible embedding and sectioning. This limitation is not directly due to any instrumental factors.9. that is to say. the . With sufficiently thin specimens and careful control of the radiation exposure. The effect is particularly troublesome in the case of thick specimens. First. A VERSATILE TOOL FOR STRUCTURE DETERMINATION 393 investigation. The inelastically scattered electrons in themselves carry a great deal of useful information about the object structure. where the spread in energy loss values can be very great and where multiple scattering events involving one or more elastic processes. one must remember that the spatial resolution is once again limited by radiation damage. together with one or more inelastic processes. hydrated specimens. There is no reason. the energy spread produced in the transmitted electron beam may become large enough that chromatic aberration can become a significant limiting factor in determining the resolution. in principle. However. numerous technicaI difficulties still stand in the way of this achievement. When there are a large number of inelastic scattered electrons.1. However. In discussing the information available in the images produced with inelastically scattered electrons. a resolution of approximately 1015 A is possible on certain stained and unstained biological specimens. there may be the problem of poor specimen preservation. provided that the electron exposure is kept extremely low and provided that extensive spatial averaging of the image is used in order to compensate for the shotnoise fluctuations produced in the image at low electron exposures. The imaging of inelastically scattered electrons therefore provides structural information that is not obtainable from the images produced by elastically scattered electrons. Even higher resolution can be achieved on unstained. the examination of the electron energy loss spectrum at each point in the specimen allows one to carry out spectroscopic observations with extremely small volumes of saecimen material and to record differences in the spectrum from point to point. but rather is indirectly a result of the dual requirements for having thin specimens and for placing these specimens in the vacuum of the microscope. why the spatial averaging technique cannot be used to obtain experimental results down to the instrumental limit of resolution.
a great deal of physics and electron optics must still be worked out in order that “filter lenses” can be regarded as practical devices in conventional microscopes. It is true that an energy filter lens can. In terms of instrumental approaches. it can be said that inelastic electron images arc best obtained with a scanning transmission electron microscope equipped with a suitable energy loss spectrometer. For a resolution in the 35 range it is still necessary to use a dedicated scanning transmission electron microscope (STEM) which has been built exclusively for operation in the STEM mode. Opt& 41. At the same time. In fact. which may in turn impose a severe limitation on the attainment of high resolution in the image. 92 (1974) . The preponderent number of inelastic scattering events occur at energy losses of 350 eV. ELECTRON MICROSCOPY resolution limitations that are caused by radiation damage have not yet been so carefully analyzed in the case of images produced with inelastically scattered electrons as is the case for elastically scattered electron images. But while such highloss events are quite well localized. P. As a result. for example. in principle. If a resolution of only 2030 A is desired. be developed for the normal imaging mode in the transmission electron microscope.3 Higherresolution images can in principle be obtained with electrons that have suffered much larger energy losses. Other areas of major instrumental improvement also deserve mention. Rose. there also remains considerable need and opportunity for improvement in the design of spectrometers for scanning transmission electron microscopes. The continued development and refinement of highvoltage electron microscopes offers a promising direction for the achievement of higher resolving power. provide the localization of the “chromophore” to a resolution better than 1020 A. highvoltage electrons have a distinct advantage in overcoming the multiple scattering processes that can seriously confound image interpretation. energy losses that would be produced in the excitation of innershell electrons. The reduced frequency of such highloss events greatly reduces the signaltonoise ratio in the measurements. An alternative direction to take for improving instruM. Isaacson. the instrumental features needed for operation in the scanning transmission mode with energy filtering can be provided as an attachment to a conventional transmission electron microscope. the cross section for such events is smaller by about a factor of a thousand than is the case for the lowerenergyloss events. and H . images produced with these inelastically scattered electrons cannot. However. Langmore. in principle. J. The scattering events are “delocalized” in the sense that excitation and ionization of a molecule can occur quite easily due to the passing of an incident electron at some distance away from the “target” molecule. The amount of delocalization of such excitations is in the range of 1020 A.394 9.
therefore. one which can be easily corrected for provided that the image formation occurs under conditions of very high coherence. on the basis of considerable experimental experience. their experimental realization remains still as a great challenge. In this view of the origin of image contrast. we shall restrict our attention to a description of images that are formed by the elastically scattered electrons. IMAGE FORMATION IN THE ELECTRON MICROSCOPE 395 mental resolution is the development of devices for correcting lens aberrations. Although the theoretical possibilities have been quite clearly defined for some time. one can now say.2. Fortunately. In particular. the resolution limitation normally associated with the spherical aberration of the objective lens is. where the specimen is thicker or has .1. in fact. the effects that can be seen in the image intensity are the same as if the electrons had actually been absorbed in the specimen. and in the subsequent discussion of contrast transfer function theory and methods of threedimensional reconstruction. The theoretical justification for leaving out the inelastically scattered electrons in the description of “normal” image formation in electron microscopy has not been well developed in the literature. A Simplified Picture: The Mass Thickness Approximation The earliest ideas of image contrast in electron microscope studies with biological materials were based upon the idea that a fraction of electrons are always scattered at angles sufficiently wide that they are stopped by the objective aperture. That the question is complex and cannot be easily treated in general terms is in itself not a sufficient excuse to avoid a discussion of image formation of inelastically scattered electrons. we shall proceed to “forget about” the inelastically scattered electrons. it should be mentioned that the resolution limitations of presentday electron microscopes could be quite directly overcome by the use of image processing techniques. that the theory of image formation by elastically scattered electrons provides an excellent representation of the actual image intensities that one normally records in transmission electron microscopy.3. The use of images formed by inelastically scattered electrons will be dealt with again in Section 9. Thus.2.9. On this basis. The experimental task confronted in this case is therefore to provide an electron source of high brightness and unusually small energy spread for use in the conventional transmission electron microscope. 9. Image Formation in the Electron Microscope In discussing image formation in the electron microscope. 9.2. Finally. only a systematic error.7.
” The electron image intensity is thus expected to obey a law that is identical to Beer’s law in spectroscopy or the exponential attenuation law in the absorption of xray radiation.396 9.. The conditions of validity of the massdensity approximation described above have been carefully and accurately described by Zeitler and Bahr in terms of the specimen thickness for which the approximations apply in materials of different atomic number. there is more scattering and therefore more “absorption. F. the exponential function can be expanded to give I(x) Z Io[1 . Zeitler and G. In this limiting case. in the sense that the angular dependence of the scattering intensity should be the incoherent sum of the individual atomic scattering intensities. z) is the mass density at each point in the object and z is the direction of propagation of the electron beam.k p ’ ( ~ ) ] .12. it should be stressed that the derivation assumes that the object structure is amorphous. Cell RL‘s. The parameter k is an instrumentdependent constant which depends upon the objective aperture semiangle and the incident electron energy. p(x) = k I p(x. ~ who derived the result that.pL(x)I. z ) d z = kp’(x).” such that phase contrast image effects are negligible. According to the approximations of Zeitler and Bahr. Exp. In this expression the effective absorption coefficient p(x) is proportional to the projection of the mass density in the direction of propagation of the electron beam. Other possible sources of intensity variation in the transmitted electrons. Bahr. the image intensity at a point x in the image is given by w= 10 expc . In addition. 44 (1957) . where p(x. such as E. ELECTRON MICROSCOPY greater density. where x is a vector in the plane of the image. if the specimen is sufficiently thin and the mass density is correspondingly small. Clearly. This whole idea of “ pseudoabsorption” or scattering contrast was translated into terms of practical use to biologists by Zeitler and B a h ~ . that is. but which does not depend upon the properties of the specimen itself. the derivation implicitly assumes that the image is taken in the “infocus condition. Furthermore. the image intensity would vary exponentially with the local “absorption coefficient. more precisely. under suitable conditions. a linear function of the projected mass density of the structure. the image intensity can be seen to be a linear function of the projection of the structure.” and the image is correspondingly darker.
the assumption of incoherent scattering and lack of phase contrast effects means that the “massdensity approximation is limited to rather lowresolution detail and “closetofocus” imaging conditions. The wave optical approach to image formation automatically takes into consideration the effects of lens aberrations. In practice. Glaeser. A m Crystulloyr. it is still necessary for us to neglect the effects of inelastically scattered electrons. M. K. which tends now to be more fashionably referred to as “Fourier optics” (see.(x) = Yo(x)[l  (i/hc&V‘(x)]. Glaeser.. and the “absorption”ofe1ectronsby the objective aperture. even under conditions when its derivation is clearly no longer valid. The ”Weak Phase Object” Model A more rigorous treatment of image formation in the electron microscope must proceed from a physical optics point of view. particularly at very high resolution. J . R. Goodman’ or Glaeser6). Goodman. can be written as the product of the incident wave Yoand a transmission function which is linear in the scattering potentiad (see. Under these conditions. for example. “Introduction 10 Fourier Optics. M. V’(x) is the projection of the Coulomb potential within the object in the direction of propagation of the incident beam.” McGrawHill.9. 94 (1978). Nevertheless.2. New York. automatically includes the “mass thickness effect” described above. (Oxfi. Sect. are also neglected. this picture of image contrast is useful for the average cell biologist. making use of AbbC‘s “diffraction theory” of image formation. However. Jap and R. as well as all other factors that may affect image contrast by the elastically scattered electrons. that is. ” 9. B. ’ J . However. 77 (1979). tt is Planck’s constant divided by 2n. B is the relativistic electron velocity divided by c. c is the velocity of light. and e is the electron charge. the transmitted wave Y. As a first step in describing the images formed by elastically scattered electrons we shall make the assumption that the electron wavelength is vanishingly small and that the intensity of the scattered portion of the wave is very much smaller than the intensity of the unscattered wave.2. 1968. image defocus. the massdensity approximation is not at all suitable for modern applications of the electron microscope in the area of molecular structure analysis.2. Microsc. In the equation above. This treatment. . therefore. IMAGE FORMATION IN THE ELECTRON MICROSCOPE 397 inelastic scattering processes. Jap and Glaeser7): Y.rd)17. W. for example. A A34.
2. the function A(s) is unity in the open part of the aperture and zero in the opaque part of the aperture.” y ( s ) incorporates the effects of lens aberrations and defocus. if we make the approximation of perfect coherence.2. Situations where the scattering is too strong for the weak phase object approximation to apply. At present there are two effects that are of concern to us. These problems include the following: 1. on the one hand. If we further assume that the incident wave is a plane wave.398 9. 9. h(x) = 9‘{exp[ . Complications A number of complications can arise that make the weak phase object approximation an invalid description of image formation. and 9lindicates the inverse Fourier transform. be regarded as the failure of the “zerowavelength” approximation.3. that is.3 below. Before proceeding to that discussion. A more detailed and explicit discussion of the very useful weak phase object case is given in Chapter 9. coherent . Situations when the effects of inelastic scattering cannot be ignored. so far as a description of the elastically scattered. Such a situation can. where s is a vector in Fourier space and is called a “spatial frequency. where the * symbol indicates the convolution product (integral) and the function h(x) is the inverse Fourier transform of the function describing the effects of wave aberrations and the objective aperture. ELECTRON MICROSCOPY The approximations described in the paragraph above are commonly known in the field of highresolution electron microscopy as the “weak phase object ” approximation. The first is the fact that the electrons that are inelastically scattered may just as well have been totally absorbed. More explicitly. however. which can occur even as the wave propagates through the specimen. These strong scattering situations are effectively the same as those which would lead us to declare the failure of the first Born approximation. then the application of AbbC’s diffraction theory of image formation5 leads to the simple result that the image wave Y i is given by Yi(x) = [l  (i/hcfl)eV’(x)] * h(x).i y ( s ) ] A ( s ) } . At the same time it is also useful to recognize that the problem in this case is essentially a problem of inadequate depth of field in the image forming processes. 3. Situations in which the specimen thickness is so great that we can no longer ignore Fresnel diffraction effects. we pause to introduce some of the complications that can invalidate the approximations which we have so far employed.
Y. and many of them continue down to the image plane. A rigorous derivation of its validity.(x) = exp[(  i/hc/?)eV‘(x)]. but pass through it. This nonlinear description of the transmittance function of the object is known in the electron microscope literature as the “strong phase object” approximation. The scattered and unscattered electrons then proceed to be transmitted through the remainder of the specimen with no further interaction. where a small fraction are scattered.’ albeit in quite a different context. One can then picture the electrons as penetrating without interaction to a certain depth in the specimen. we find that the transmitted wave retains a simple but no longer linear relationship to the projected Coulomb potential.9. intensity) variations in the wave transmitted through the object. in the sense that the transmitted wave has constant intensity and there are no amplitude (i.. I . Schiff. It should L. leads to rather considerable complications in the description of image formation. Rev. but instead the transmitted wave must be written as the sum of waves transmitted through different layers of the object. was first given by Schiff. IMAGE FORMATION IN THE ELECTRON MICROSCOPE 399 wave is concerned. The simplest case to discuss is one where the weak phase object approximation is valid for a thin slice of material. Assuming once again that the incident electrons are described as a plane wave. at each level of the specimen. The final transmitted wave can no longer be written as the product of the incident wave and a transmittance function of the object. we can consider what happens if we keep the zerowavelength approximation but allow strong interaction and failure of the first Born approximation to occur. where they form their own separate image intensity. Relaxation of the zerowavelength approximation. Phys. . At the same time the inelastically scattered electrons are not truly absorbed in the specimen.” This simple model is described here not with any recommendation of its accuracy or validity. which means essentially the admission of wave propagation and Fresnel diffraction within the specimen. As a first step. The relationship between the strong phase object approximation and the weak phase object approximation discussed above is obvious upon expansion of the exponential to first order in the projected Coulomb potential. 103. using the summation of the infinite Born series under the stationary phase approximation. but only to give a first idea of the type of complication that is involved under conditions where the zerowavelength approximation is no longer valid.2.e. It is worth pointing out that in the exponential approximation the specimen is still a perfect phase object. each such transmitted wave contributes to image formation with its own value of “image defocus.443 (1956).
e. a trial and error approach can be taken. it is quite impossible to work backward to find out what the object structure is.’ and this description is commonly referred to as the multislice dynamical theory of electron transmission through moderately thin specimens. However. ‘J M Cowiey and A F Moodie. for an object of known structure. ELECTRON MICROSCOPY perhaps be said that the difficulty in this case has to do not so much with our being able to calculate what the transmitted wave would be. where it serves as the next “incident wave. and therefore what the image intensity would be. this is an easy task for modern digital computers. provided that the slice is thin enough.(x) is the wave function entering the nth splice and Exactly the same expression for the transmitted wave function can also be derived on the basis of the Feynman path integral formulation of quantum mechanics.” This idea is expressed mathematically by where ‘Y. A fairly rigorous description of the transmitted wave requires not only that one take into consideration the Fresnel propagation (i. Rather. defocus) of the individual transmitted waves. Of course. But it has the great disadvantage that it is no longer possible to describe the transmitted wave as a product of the incident wave and a simple function representing the transmission function of the object. Such a description of the transmitted wave was first given by Cowley and Moodie. The wave that is transmitted through one slice is then imagined to propagate by Fresnel diffraction to the next slice. but also requires that allowance be given for a scattered wave to subsequently interact with and be further scattered by the specimen as it continues to propagate to the lower surface of the specimen. the difficulty is that if we are given only the final image intensity. where different threedimensional models of the object structure can be used to calculate the expected image intensity. propagation through individual. this trial and error approach can become so formidably long and expensive as to be impossible to use as a practical method. A t /o Crv~talloyr 10. In the CowleyMoodie multislice formulation.400 9. 609 (1957) .’ The CowleyMoodie multislice formulation is sufficiently rigorous for any practical application. The path integral formulation has the advantage of showing clearly what portions of the electronspecimen interaction are taken into consideration and what portions are neglected. very thin slices is approximated by the phase object approximation..
3.” That is.3. contrary to the results in the simple phase object approximation. under appropriate conditions. we show that the image intensity is related to the object structure. for a given periodicity or “spatial frequency. the wave aberration can be described by the function . Since there can be amplitude variations in the transmitted wave. Contrast Transfer Function Theory In this chapter we treat the process of image formation in the electron microscope according to the Fourieroptical theory of “linear systems. We also discuss at some length the restrictions on the imaging conditions under which linear transfer theory may be applied with validity. of the same peroidicity. introduces a further important effect in our description of the transmitted wave which does not appear in the zerowavelength. CONTRAST TRANSFER FUNCTION THEORY 401 Fresnel propagation and spreading of the electron wave front.1. where s is a general spatial frequency. and at the end of the chapter describe some of the complications that may be encountered in practice when the strict conditions of valid use are not observed. ” 9.3.9. This “Fourier optics” treatment of contrast in the electron microscope is essential to an understanding of the focusdependent contrast effects that are seen in highresolution applications. The performance of a linear system is most elegantly described in terms of the “transfer function” of the system.” is related to a sinusoidal variation. For nearly all situations of practical interest in electron microscopy. in the transmittance function of the object. within the finite thickness of the specimen. 9. of which the spherical aberration effect is the primary example. It is therefore important to keep in mind that amplitude variations in the elastically transmitted wave can arise from Fresnel propagation effects as well as from inelastic scattering effects. The phase distortion y(s) is due in the first instance to any sort of wave aberration associated with the lens. “infocus” imaging conditions. “pure phase object approximation. The power of the transfer function lies in the fact that it tells us how a sinusoidal variation in the image intensity. This effect arises from the fact that the intensity of the transmitted wave is no longer constant in space when Fresnel diffraction is included. by a set of linear equations.Origin of Phase Contrast in Electron Microscopy Phase contrast effects in electron microscopy are generated as a result of the phase distortion function exp[iy(s)]. these can be faithfully imaged under ideal.
is the coI is the efficient of spherical aberration. we have assumed that we can ignore the curvature of the Ewald sphere and that the Fourier transform of the transmitted wave satisfies Friedel symmetry. involves nothing more than looking at the Fouriertransform representation of the image intensity.k. We have further assumed that the objective aperture is axially symmetric.i y ( s ) ] A ( s ) } .. electron wavelength. This. In this equation. p(s) is the Fourier transform of the projected scattering potential.(x) = [I . where k is the scattered wave vector. Our next task is to consider the image intensity as a linear superposition of many different sinusoidal functions. and we have invoked the convolution theorem of Fourier transforms to obtain the The Ewald sphere is the locus of all vectors s such that 2ns = k . if we assume that the specimen is a weak phase object.s is the complex conjugate of the scattered wave at f s . that the wave propagation vector is parallel to the optical axis. C. We thus have I(x) z 1 . we have dropped from further consideration the quadratic image terms. ELECTRON MICROSCOPY where s is the spatial frequency vector and s its magnitude.t These last two assumptions are already guaranteed by the approximations that we have had to make in describing the transmitted wave in terms of the projection of the structure.i/hc/3)eV’(x)] It is important to realize that certain approximations and special conditions have been embedded in the procedure used to obtain the expression for the image wave function above.(2e/hcfl)P’(s) sin y(s)~(s). Finally. This is consistcnt with the fact that quadratic terms in the object potential were already neglected when approximating the object’s transmittance function by the linear expansion of exp[( . First. we obtain i(s) = 6(s) . The wave function in the image plane is given by Yy. Taking the Fourier transform of the righthand side of the equation for the image intensity.[(2/tic&V‘(x)] * . and .F’(sin[y(s)]A(s)}.(i/hcp)eV’(x)]* 9‘(exp[ . of course. AZ is the instrumental defocus. we have assumed that the illumination is axial.402 9. The image intensity is obtained simply by squaring the image wave function. In the spirit of the weak phase object approximation. i(s) is the Fourier transform of the image intensity. Friedel symmetry applies when the scattered wave at . . that is.
We thus find that the various sinusoidal components of the object structure are each transferred with an individual modulation of contrast. of the same period.3. from instabilities in the accelerating voltage.3. the function sin[y(s)] is commonly referred to as the “phase contrast” transfer function in electron microscopy. the superposition being weighted according to the distribution of energies and angles of incidence. In the image intensity we find that the amplitude of each sinusoidal (Fourier) component of the potential is modulated by an amount 2 sin[y(s)].2. onetoone relationship between any one Fourier component of the image intensity and a Fourier component. Although this does not directly involve the incident electrons. the amplitude undergoes a change in sign whenever the function sin[y(s)] goes through zero. The energy spread can arise from the physics of the emission process at the filament (source). The effect that partial coherence will have on the transfer function can be derived from the perfectly coherent case by considering the linear superposition of image intensities. and depending upon the defocus value. Thus a finite energy spread in the illuminating electrons must lead to a corresponding reduction in the degree of coherence. The spatial coherence is determined primarily by the angular convergence (or divergence) of the illumination. which is associated with ripple in the objectivelens current. of the projected potential. From this result we can see that there is a simple. Temporal coherence is determined primarily by the monochromaticity of the illumination. the effect of focus instability in the objective lens. the effect that objectivelens current instability has on the image is indistinguishable from the effect of a corresponding energy spread in the incident beam (given a perfectly stable objectivelens current). For these reasons. The finite size of the primary electron source is obviously a factor in determining the divergence. and from electronelectron interactions at points where a beam crossover may be formed. each calculated for the appropriate wavelength and propagation vector. but also of importance is the condenserlens aperture size and the conditions of focus of the condenser lens that are used. and that. CONTRAST TRANSFER FUNCTION THEORY 403 result that the convolution product in real space is converted to a direct multiplication in Fourier space. in the factors leading to degradation of temporal coherence. It is also appropriate to include. . furthermore. 9. In the real case. several factors can reduce both the degree of temporal coherence and the degree of spatial coherence. The Envelope Function: Partial Coherence The result given in the previous section for the weak phase object is valid only under conditions of perfectly coherent illumination.9. some sinusoidal components may even be transferred with reverse contrast.
In practice this approach to the image restoration problem leads to In ” J . Frank. and depends also upon the defocus value. Frank. is retrieved from the observed image intensity I(x). ELECTRON MICROSCOPY In the case in which the object’s transmittance function is approximated by the weak phase object approximation. highresolution electron microscope has values close to unity out to a resolution of 45 A.3.3.’l Under normal circumstances the envelope function of a modern. one must correct for the fact that the envelope function is not always equal to unity. Stated more specifically. In addition. so we have suppressed the aperture function. R.1) above. we need only obtain the Fourier transform P’(s).3. and thus lacks the correct “average value” of V’(x). where . in addition. 519 (1973). But examining Eq. Optik 38. Wade and J . (9. this approach cannot be used where A(s) = 0. H. That is. image restoration is the process by which one corrects for the systematic errors that exist in the image due to the fact that the function sin[y(s)] is not always equal to one and does not always have the correct sign. it has been shown that the effect of partial coherence can be represented as a multiplicative envelop function according to The envelope function E ( s ) takes on values between 0 and 1.(2e/hcp)P’(s) = i(s)/[sin y ( s ) ] ~ ( s ) .3. 9. Now it is clear that in order to obtain the projected potential V’(x).404 9.2) Needless to say. Image Restoration The term “image restoration is used here to refer to the process by which the projection of the object structure. we can see that a formal solution to the restoration problem for all spatial frequencies except s = 0 would be to divide the Fourier transform of the image intensity by the product sin[y(s)]E(s). Optrk 49. the formal solution is given by V’(x) = 9“P’(s)]. At very large defocus values. (9.’O. the envelope function may also affect lowerresolution details. 81 (1977). The exact functional form of the envelope function depends upon a detailed description of the factors influencing the partial temporal and spatial coherence. V’(x). .3. the function V ’ ( x ) obtained according to Eq.2) excludes any zerofrequency (constant) term. (9.
in the latter case the problem being that the envelop function has become too small. the envelope function is not very sensitive to the value of the spherical aberration coefficient. provided that one is prepared to engage in the image restoration task. it is worthwhile to insert some numbers into the analytic functions' representing the envelope function. at the same time sin[y(s)] will be continuing to undergo oscillations. such that the product sin[y(s)]E(s) is sufficiently close to unity at all spatial frequencies in one or another micrograph.5 A) that is actually realized in presentday electron microscopes. And finally. and the coefficient of chromatic aberration normally has a value not smaller than 1.5 eV. we find that the envelope function has decreased to a value of 0. one can always find another defocus value such that I sin[y(s)] I is close to unity.3. Using these figures.and intermediateresolution regions. This resolution limitation is in fairly good agreement with the practical limit for useful image restoration ( . each taken with a different defocus. The practical limit on the illumination angle for conditions of iniaging at high magnification is approximately l o p 3 rad for short exposures and rad for long exposures.3. However. It is enlightening to see that the envelope function is the real villain when it comes to considering the image resolution that can be obtained. At low spatial frequencies. at very high resolution the envelope function must gradually tend to zero.5 mm. The main limitation to image restoration will then occur either at low resolution or at very high resolution. . while at the same time the envelope depends quite strongly upon the chromatic aberration coefficient and the energy spread. At intermediate spatial frequencies. while. that if 1 sin[y(s)] 1 is close to zero in an image taken with one defocus value. sin[y(s)] can oscillate.9. It is commonly supposed. A typical value for the energy spread of a 100keV microscope is approximately 1. depending upon the value of defocus. at a resolution of 2. that the objectivelens spherical abberation is the main factor limiting image resolution. '. at the spatial frequency ofconcern.35 A. and not without justification. CONTRAST TRANSFER FUNCTION THEORY 405 noise amplification whenever the product sin[y(s)]E(s) becomes rather close to zero. of course. therefore.25 at a spatial frequency of 0.that is. In this way it is possible to patch together data from many different images. particularly for moderate values of defocus. we can take advantage of the fact that I sin[y(s)] I takes on values close to one for different spatial frequencies. The use of transfer function theory therefore teaches us that the real limit to image resolution is associated with the energy spread and the chromatic aberration effect and not really with the spherical aberration effect. In the low. It follows. sin[y(s)] can never take on appreciable values except for very large defocus. This condition can arise in three regions of the Fourier spectrum of the object.8 A. By way of a practical example.
since the phase contrast term is characterized by one transfer function while the amplitude contrast term is .2p(x) * 9(cos[y(s)]}.3).3. The same analysis as was used in the previous section leads to the result that the image intensity is given by I(x) = 1 . However. In this case the object transmittance function becomes T(x) = 1 . mixed phase and amplitude object is once again easily derived by the procedures above.3. According to Eq. Complications The discussion has dealt thus far with the electron microscope contrast transfer function for the special case of a specimen which satisfies the defining conditions for a weak phase object. The result of such analysis is that the Fourier transform of the resulting image intensity is given by i(s) = 6(s) . (9.p(x). where T(x) is the transmitted wave function when the incident wave is a plane wave. A slightly more complicated case than the weak phase object is obtained by admitting a weak amplitude variation in the transmitted wave. The image intensity for a weak. the result remains exceedingly simple and convenient to use. it is no longer possible to represent the Fourier transform of the image intensity as the product of the Fourier transform of the object function and a phaseandamplitude “modulating function. in contrast to the weak phase object where the transfer function is sin[y(s)]. but still making the assumption of weak scattering.” Nevertheless.iq(x) .4. we obtain i(s) = 6(s) . Once again taking the Fourier transform of both sides of the equation. in addition to a weak phase variation.2 ~ [ q ( x ) ]sin[y(s)] .3) which is the linear combination of the phase contrast and amplitude contrast effects.3.406 9. To understand the effect that the amplitude variation has in the contrast transfer that is obtained in the image. ELECTRON MICROSCOPY 9. We note now that an essential property of the concept of a transfer function has been lost in this description of the relationship between the image intensity and the object structure.2 ~ [ p ( x )cos[y(s>]. (9. Thus for a pure amplitude object the contrast transfer function is found to becos[y(s)]. it is convenient to consider first the case of a pureamplitude object. it is not at all uncommon that one wishes to work with specimens that do not accurately satisfy the conditions of the weak phase object approximation.2F[p(x)] cos[y(s)].
Klug. For all but a few spatial frequencies. CONTRAST TRANSFER FUNCTION THEORY 407 characterized by another transfer function. (9. The analysis of Erickson and Klug might have been an overly simplified approximation of the complete problem.Let us make the further. but for which the weak phase and amplitude object provides an accurate representation. More general cases are easily evaluated by the same sort of analysis. Trans. Soc. London. Thus the relative proportions of phase and amplitude contrast. As can be easily seen from Eq. P.9. is the possible difference in the phase origin between the phaseobject Fourier coefficients and the amplitudeobject Fourier coefficients.3. The mechanism for carrying out image restoration remains as clear and straightforward as it is in the case of the pure phase object. Philos. There is no good theoretical indication as to whether one might expect to find a wide range of types of specimens for which the weak phase object approximation fails. even though theoretical considerations might not encourage one to accept its validity.' In this work the relative weights of the phase contrast and the amplitude contrast terms were allowed to vary continuously at each spatial frequency. were used to determine whether the contrast transfer could be correctly predicted at other defocus values. the magnitude of the Fourier transform of the image intensity will not vary with changes in defocus. the experimental fit was found to be moderately good. let us suppose that at a particular spatial frequency the Fourier transform of p(x) is 90" out of phase with the Fourier transform of r](x). it seems quite likely that an envelope function expression similar to that obtained in the case of the weak phase object would continue to be a valid expression for use in the case of the mixed object. Nevertheless. in the sense that these authors sought only to determine the magnitude of the Fourier coefficients of the phase object and amplitude object terms in the transmittance function.Erickson and A. however. These relative weights were then obtained from a series of micrographs taken at different values of defocus.105 (1971). The conditions under which the weak phase and amplitude object might be a valid representation of the object transmittance function have not yet been carefully explored in the literature. However. B261. R. which could be obtained from just two micrographs. there is some experimental evidence that the mixed object representation is a practical representation. A fairly extensive analysis of the defocusdependent contrast transfer characteristics of uranylacetatestained catalase was carried out by Erickson and Klug. To illustrate this point.3. assumption that the two Fourier coefficients have equal magnitude. A third parameter. l2 H . which perhaps should not be ignored. perhaps pathological. Ser.3). . The question of the effects of partial temporal and spatial coherence in the case of the mixed weak phase and weak amplitude object have not yet received attention in the literature.
. it is not possible to use a central beam stop to produce a darkfield image. One further complication merits brief mention at this point. We have so far neglected the effects of the quadratic terms obtained when squaring the image wave function in order to obtain the image intensity.2. must necessarily be of the same general form as the quadratic terms we normally wish to eliminate from our description of the contrast transfer theory. ELECTRON MICROSCOPY The effects of the other complications.(s) is the Fourier transform of Id(x). are not so easily discussed in terms of contrast transfer function theory. In practical electron microscopy.3. one cannot possibly hold to the assumption that the magnitude of the scattered wave is negligibly small in comparison to the unscattered wave. the coherent darkfield image.4). the darkfield image intensity. which have been described in Section 9. which is applicable in the case of perfectly coherent illumination. The effect that the quadratic terms have on the Fourier transform of the image intensity is to add an expression of the form (9. One can only say that it is prudent to be aware of the existence of these complications and to consider effects such as Fresnel diffraction within the finite thickness of the specimen and dynamical interaction of the electrons with the specimen as sources of possible discrepancy between the obtained experimental results on the one hand and the mathematical representation of contrast transfer function theory on the other.408 9. The interesting point that is worth emphasizing in this connection is the fact that the “new” function generated by admission of the quadratic terms is identical to the darkfield image intensity that would be obtained by blocking the unscattered beam. however obtained. In those cases whcre the image contrast is quite large. and the asterisk indicates the autocorrelation product (integral). (9. In these cases we might truly expect to find nonnegligible contributions from the quadratic image terms.3. Inclusion of the quadratic terms necessarily prevents the use of a transfer function in the description of the image intensity. As may be clearly seen from the autocorrelation (selfconvolution) representation in Eq. This result follows directly from the application of the convolution theorem of Fourier transforms.3. Nevertheless. Darkfield images are usually produced with either a displaced aperture or tilted illumination. the operation of squaring the image wave function generates spurious coefficients for spatial frequencies representing all possible combinations of differences of the true spatial fequencies of the object. under coherent brightfield illumination conditions. These are actually identical to the darkfield image intensity that would be produced by stopping out the central.4) where i. unscattered beam in the objective lens’s back focal plane.
the conditions of . Fourier (Crystallographic) Methods The Fourier transform methods of threedimensional reconstruction are in many respects similar to the xray crystallographic methods of macromolecule structure analysis. to recover the threedimensional Fourier transform of the object. That the structural phases can be obtained from the images is a direct result of the fact that.1. The conditions needed to provide this linear relationship are. This class of methods has a great kinship to the welldeveloped methods of macromolecular crystallography. the Fourier transform provides the easiest way of obtaining the correct weighting of the different spatial frequency components in the “filtered back projection method of threedimensional reconstruction. One can distinguish two general classes of methods for threedimensional reconstruction. the phrase “threedimensional reconstruction refers to the task of obtaining a threedimensional model of the object from many different twodimensional projections. of course. There is no need in electron microscope structure analysis to obtain phases by the use of isomorphous heavyatom derivatives of the native structure. of course. under suitable conditions. There exist many difficulties in the task of threedimensional reconstruction. One important difference. first. One class of methods attempts. which is still fundamentally a realspace” method.9. each obtained at a different tilt angle of the specimen. the Fourier transform would still be used during the steps of twodimensional image restoration . as long as the image resolution extends to sufficiently high spatial frequencies. the image intensity is a linear function of the projection of the object’s structure. More precisely. The second class of methods works directly in real space and does not resort to the use of Fourier transforms except for steps in which the Fourier transform might present a particular convenience in the correction of systematic errors. and many different approaches have been taken in an attempt to overcome these difficulties.4.4.4. ” ” “ 9. As an example.Dimensional Reconstruction The expression “threedimensional reconstruction” is used here to refer to the class of methods in electron microscope (EM) structure analysis by which information obtained from several different EM images is used to construct a threedimensional model of the sturcture of the object. Three. as another example. is contained in the fact that the structural phases are obtained directly from images in electron microscopy. after which the threedimensional structure is obtained directly by a threedimensional inverse Fourier transform. THREEDIMENSIONAL RECONSTRUCTION 409 9.
One of the most favorable instances in which symmetry facilitates a threedimensional reconstruction is provided in the case of cylindrical or helical structures. Thus. each taken at a different angle. central sections through the threedimensional Fourier transform. 01. S. The net result is that the Fourier transform of one image gives a twodimensional slice through the threedimensional Fourier transform of the whole object.410 9. In the latter case. The “crystallographic” Fourier transform method described above is completely general in nature and approach. if we have many different projections of the object. whereas in the case of numerical calculations both the magnitude and the phase can be computed. 2 where P(S. it has some special advantages in those cases when the object has some form of known symmetry. ELECTRON MICROSCOPY validity for the weak phase object approximation. we do not have the usual problem encountered when a diffraction pattern is produced physically by some form of radiation. In addition. Once the threedimensional Fourier transform P(s) has been computed. Y) m. Such structures are assembled from identical subunits (e. then by computing the Fourier transforms of each of them we can obtain many different twodimensional..g. only the modulus of the Fourier transform can be obtained from experimental measurements. that is. it is a straightforward matter to obtain the threedimensional Coulomb potential by inverse Fourier transformation. proteins). Thus a single projection of a cylindrical or helical structure contains as much information . S. The projection theorem thus states that the Fourier transform of a projection is a central section of the threedimensional Fourier transform of the object.. The idea of the Fourier methods of threedimensional reconstruction is then to obtain a sufficient number of different central sections that they will “fill in” all of the threedimensional Fourier transform of the object. which are arranged in a repetitive pattern around and along a cylindrical (helical) axis.. Considering for the moment only one repeating segment of the structure. it is evident that a single projection in a direction perpendicular to the axis will be composed of a superposition of many different.) is the threedimensional Fourier transform of the object potential. For a weak phase object we need only to apply the projection theorem of the Fourier transform to obtain the result ‘F2DW. independent projections of the individual subunits.. Since we assume here that the Fourier transform of the image intensity can be calculated numerically.s.
As a consequence. Narure (London) 217. For this reason it has been necessary to do such work only with stained. T. l4 l5 . Klug. 226. Bid. 87. 107. A severe limitation on the resolution that can be obtained in threedimensional reconstructions of individual objects necessarily accompanies the fact that a large number of successive micrographs must be taken of the same object. 50. T. J . as was first demonstrated by DeRosier and Klug13 in their study of the structure of the T4 bacteriophage tail. A. including the stackeddisk protein of tobacco mosaic virus. Klug. E. Amos. 1978. Moore. Biol. J . p. B261. Crepeau. Dykes. dehydrated specimens. Philos. ed. l 9 G.). 66. A. ’’D. R. J .Ser.221 (1971). 271 (1972). Crowther. J. R. H. Klug. DeRosier. In the case of a helical structure with a large number of subunits per repeat. The method of helical reconstruction has now been applied to a wide variety of biological specimens. and S .’ and helical fibers of proteins such as glutamine synthetase’* and sickle cell hemoglobin. P. DeRosier.279 (1970). its own “tilting stage” in the electron microscope. Finch.506 (1978). Highresolution threedimensional reconstructions can only be achieved at the present time if the specimen can be obtained in the form of rather large.9. Sor. P. Nature (London) 272. the amount of biological information that can be obtained is rather limited. 60. l 6 H. and this approach is not to be recommended.4. a single projection is sufficient to give a good threedimensional reconstruction. l 9 Other symmetries besides helical symmetry can also be of great help in recovering the threedimensional Fourier transform from a limited number of “views” or projections. asymmetric unit.'^ microtubules. Cell Biol. and A. Nature. Edelstein. J . in “Electron Microscopy 1978” (J.I. Huxley. 130 (1968). Mol. and all cylindrical (nonhelical) structures require two or more independent projections with a known angle of rotation about the symmetry axis. a single projection again provides many different views of the identical protein subunits that lie at the surface of the virus. J. Mol. Slayter. N. L. H . In the case of the icosahedral (“spherical”) viruses. Lake and H. J. one for each time the tilt angle is changed. Sturgess. and D. Frey. T. 2 1 R. A. . A cylindrical or helical structure is. D. A. Helical structures with only a few subunits per repeat. G. in effect. Microscopical Society of Canada. twodimensional periodic arrays of the fundamental. 641 (1974). London. Truns. S. J. Mol. 20 R.I4 ribosome^. Vol.20*2’ A limited amount of work has also been done on the use of Fourier methods for threedimensional reconstruction of individual macromolecules. DeRosier and A.’6 myosindecorated actin filaments. Toronto. Unwin and A. 42 (1 970). in which case the object lacks any special symmetry relationships.Crowther. P. THREEDIMENSIONAL RECONSTRUCTION 41 1 about the basic subunit as could be obtained from a large number of equivalent projections of a single subunit. Erickson. Biol. I l l . 153 (1974).B. M. J.
photosynthetic bacterium Hulobucterium hulobium. Steven Hayward. Henderson and P.28 (1975) . (Courtesy of Dr. Nutitre (London)257.) The requirement for twodimensional arrays exists strictly to overcome the limitations arising from radiation damage. Unwin.22) (h) Linedrawing idealization of the model with the cytoplasmic and extracellular surfaces identified. Bacteriorhodopsin is a membrane protein of the halophilic. asymmetric objects as far as the threedimensional reconstruction problem is concerned. ~ and ’ by Henderson rt (a) Structural model built from the 3D contour maps. Bacteriorhodopsin is particularly well suited for electron microscope structure analysis since the protein naturally 22 R. Seven rhelical rodsareshown accordingto themodel published by Henderson and Unwin. (From Hendcrson and Unwin. N. ELECTRON MICROSCOPY CYTOPLASMIC SIDE EXTRACELLULAR SIDE lb) FIG. The present state of the art in the area of threedimensional reconstruction in highresolution clcctron microscopy is represented by the work of Hendcrson and Unwin” on the structure of bacteriorhodopsin.” while the identification o f the cytoplasmic and extracellular surfaces o f the protein was carried out indepcndcntly by Hayward et ~ 1 . T. I . The requirement for twodimensional arrays has nothing to d o with providing additional crystallographic symmetry. This protein acts as a photondriven proton pump and thereby establishes a pH gradient across the cell membrane when the bacterium is exposed to light.5. These limitations are described in greater detail in Chapter 9. and objects with twodimcnsional redundancy must be treated in the same way as general.412 9. Threedimensional inodcl of the structure of bacteriorhodopsin at about 7A resulution.
M . and S. Inverse Fourier transform of the sampled data yielded a threedimensional of the structure of the protein with a resolution model. 1. The threedimensional Fourier transform could then be “filled out” from twodimensional central sections over all of reciprocal space except for a “hollow cone” of semiangle equal to 30” about the direction perpendicular to the membrane plane. R .S. The factors by which the various methods might differ from one another include (i) the computer time and costs required for completion of the threedimensional reconstruction. DirectSpace Methods The Fourier transform (crystallographic) method is by no means the only one by which threedimensional reconstruction can be accomplished. Natl. and K. and which appear to span the full thickness of the membrane. 24 R. which can be biochemically isolated and which are referred to as “purple membrane. J . Hayward. There exist quite a number of other approaches which also represent formal solutions to the problem of obtaining a threedimensional model from many different twodimensional projections. (ii) the uniqueness of the solution that might be obtained. and whether realspace methods are better than the Fourier transform method are questions which have sometimes been the subject of very hot debate.4. Proc. B i d . The basic idea of the back projection is to project the density of ” 2 3 S. 9. D. Whytock. A. Whether one realspace method is better than anothcr. Glaeser.3. S. (iii) the stability of the reconstruction procedure with respect to experimental noise and errors in the measurements and (iv) the ability of the method to achieve the correct solution when one has available only “incomplete data.9. Jubb.2.” These membrane patches occur in pieces as large as one micron in diameter which contain more than lo4 protein molecules per patch. 123. THREEDIMENSIONAL RECONSTRUCTION 413 occurs in twodimensional crystalline arrays.A. A. In subsequent work Henderson and Unwin” collected image data over a range of tilt angles of f 6 0 ” from the normal. B. Unwin and Henderson first demonstrated that twodimensional projections of the structure could be obtained from electron microscope images out to a resolution of about 7 A. Sci.4320 (1978). shown in Fig. U. Grdno.4.2’24 of approximately 7 A in the plane of the membrane and approximately 14 A perpendicular to the plane of the membrane. One class of directspace reconstruction methods is built upon the idea of producing a “back projection from many different twodimensional projections. 75. Fisher. Mul.259 (1978). Acad. J. .4. Henderson.” This last problem will be treated at further length in Section 9. From these studies it was learned that the bacteriorhodopsin molecule has seven ahelical regions of polypeptide chain that run nearly perpendicular to the plane of the membrane.
This simple modification of the back projection procedure is referred to as the “filtered back projection. The unwanted convolution is easily removed by computing the Fourier transform of the naive back projection. parallel sections through which are everywhere identical to the original projection.’s. J . while unwanted density makes only a general background underlying the actual object.25The optimization of the model to the experimental projections can also be set up as a ” 25 P. It can be shown in a straightforward way that the naive back projection approach produces a function which is actually the convolution product of the object structure and the inverse Fourier transform of 1. 105 (1972) . Another complication is that the theorem regarding the pointspread function (that is. When the back projections from many different images are superimposed.414 9. Thus a single projection is used to generate a threedimensional density function in real space. Another class of realspace threedimensional reconstruction methods is based on what one might almost call trial and error methods of fitting an object structure to the experimentally obtained projections. One algorithm allows for the simultaneous optimization of the density values to all projections in each iterative step. ELECTRON MICROSCOPY the object backward along a straight line perpendicular to the plane of the projection itself. Complications to the filtered back projection are encountered first in handling the abrupt cutoff at the edge of the “filter function. Gilbert. becomes invalid if the projections are not equally spaced. Biol. and therefore have a certain approach in common with the reciprocalspace refinement methods that are now becoming commonplace in xray crystallography of macromolecules. Theor.” as must be done in any real. Because this background level is rather high. numerical implementation of the approach. The basic approach to the threedimensional reconstruction problem can be described as follows: Pointwise density values of a model structure are permitted to vary so as to achieve a best fit between the calculated projections of the model structure and the actual (experimental) projections that have been obtained from electron micrographs. 36. and if they are not very “dense” in reciprocal space. the use of the simple back projection method is not a very satisfactory approach to the threedimensional reconstruction problem. the true features of the object are reinforced at points of intersection. These approaches represent essentially the “refinement of a realspace model.” It is to be emphasized that the filtered back projection approach is still philosophically a realspace method. and then carrying out an inverse Fourier transformation. and the use of the Fourier transform to execute the spatial filtering is only incidental to the philosophy of the approach. multiplying this Fourier transform by s. A common approach to this method is to vary the density parameters of the model in an iterative procedure. the inverse Fourier transform of l/s).
no study has yet been published on the suitability of the maximumentropy method for threedimensional reconstruction in electron microscopy. within experimental errors.4. .~’ The rationale in this case is to find the set of “’quantified” density values m iin real space which serve to maximize the sum s= c mi ln(mi). A formal solution of the problem is obtained by application of Cramer’s rule or by other. to the observed summation of density values in individual columns. giving in this case the leastsquares solution. Nuture (London) 251. as unknowns. THREEDIMENSIONAL RECONSTRUCTION 415 leastsquares fitting problem which can then be solved by any of the variety of methods that are possible for the solution of leastsquares problems. more rapid methods of matrix inversion. Perhaps the most direct approach to threedimensional reconstruction in real space would be the formal solution of the simultaneous linear equations that relate the pointwise density values. A. with the observed data. to be more attractive than they really have a right to be. to some extent. Gull and G . that is. At this time.490 (1974) ” S . The first difficulty has to do with the size of the matrices that are involved 2 6 R. Matrix inversion solutions are also formally available when the linear equations are overdetermined. Daniell. to the observed projections. It is always possible that such a favorable result could actually be the case. There are two difficulties confronted by the approach of directly solving the linear equations. numerical simulations by Crowther and K1ug26have shown that the simultaneous iterative reconstruction procedure does not lead to as accurate a threedimensional reconstruction from the data as does the Fourier method. F. The lack of a rigorous analytical foundation might tend to seduce some investigators into believing that the realspace model fitting approaches are less troubled by such difficulties than is the case for the Fourier. when the tilt views are equally spaced in angle. J. A rather special version of the realspace model fitting procedures is the socalled maximumentropy te~hnique. This is particularly true when noise has been added to otherwise perfect data. Their sensitivity to noise and their sensitivity to problems that might arise when the projections are not obtained at equally spaced tilt angles are not easily analyzed except through numerical simulations. Klug.9. Nature (London) 272. In fact. The directspace model refinement procedures may appear. I while giving projections of the object structure that are consistent. The model fitting procedures have no rigorous mathematical foundation by which one might evaluate the limitations of the methods. Crowther and A . but it must be said that there is little mathematical justification to believe that it is true. crystallographic approach. 686 (1978).
4. It is always possible to compute an inverse Fourier transform of just the data accessible to experimental measurement.4. As already described in Section 9. but at the same time there will necessarily be some differences between the reconstructed object and the original object. The effect of being able to work with only a limited range of views is to produce a threedimensional reconstruction in which the resolution is not equally good in i l l directions. the Fourier transform of each twodimensional projection corresponds to a twodimensional central section in reciprocal space. We elaborate on this point in more detail in the next section. Thus the experimentally obtained data are related to the full threedimensional . 60”. since the extension of electronmicroscopic structural studies to atomic resolution might not be possible without an adequate solution.1. The Hollow Cone Problem The task of threedimensional reconstruction in electron microscopy faces a very special problem which is due to the fact that projections of the structure can only be obtained over a limited range of tilt angles. ELECTRON MICROSCOPY for problems of interest in highresolution structure analysis. for example. the matrix inverse can easily be very poorly conditioned. crystallographic method of threedimensional reconstruction. as is illustrated in Fig. 9. for example. The reason why we refer to this difficulty as the “hollow cone problem” is most clearly seen in the context of the Fourier. 2. The object structure retrieved in this way will certainly have some relationship to the real object structure. But by the samc token the limited range of tilt angles make a conical section of reciprocal space inaccessible to direct experimental observation. The precise nature of the relationship can be seen by application of the convolution theorem. The consequence of poor conditioning is that the presence of the least amount of experimental error makes it quite worthless to compute the matrix inverse.3. in the case of helical or cylindrical objects. The size of the corresponding matrix equation makes the numerical calculations in the formal solution quite formidable.416 9. even if one has an unlimited amount of free computer time. In addition. The existence of a hollow cone in the sampled data is therefore a general problem in threedimensional reconstructions which can be avoided only in those happy instances when the object itself has some additional symmetry as. The need to find an optimal solution to this problem is very great. It therefore follows that a continuous distribution of projections at different angles in real space serves to fill out continuously the threedimensional Fourier transform of the object over the entire range of angles that can be realized in real space. It would not be at all uncommon to expect as many as a thousand simultaneous linear equations.
Extrapolation is traditionally known to be a very refractory mathematical problem. the convolution product (integral) of the real object structure and a threedimensional pointspread function. The inverse Fourier transform of this product is. From this equation it is quite clear that the resolution in the z direction must be substantially degraded by the threedimensional pointspread function a(r). Thus we have 9[t b . unfortunately. on the left. The method most commonly used for interpolation and extrapolation in electron microscopy has involved the representation of the sampled Fourier transform in terms of cylindrical Bessel functions for helical objects. Thus we have tbs(s) = p(’(s)A(s). Fourier transform by a product between the real Fourier transform and a threedimensional “aperture function. Thc hollow cone problem is illustrated by the fact that.2.” The threedimensional aperturc function A(s) is unity everywhere within the domain of observation and is zero within the region of the hollow cone.4. The success of interpolation and extrapolation procedures in solving the hollow cone problem is. by the use of the convolution theorem. and in terms of spherical Bessel functions for general .[ ~ ( s ) ] = V(r) * a(r). The diagrams should be interpreted as figures of revolution about the vertical axis.[ P(s)] * F. to a family of “central sections” (planes) over the same angular range in Fourier space. The primary effect of the hollow cone can therefore be seen. The limited range ofviews leavesacone about the vertical axis in Fourier space which is inaccessible to experimental incasurement. THREEDIMENSIONAL RECONSTRUCTION Fourier Space 417 Real Space An arbitrarily large number of different Fourier coefficients known to arbitrary accuracy within a restricted domain of reciprocal space projections within a reslricted domain of tilt angles FIG. and :!ie application to the threedimensional reconstruction problem is no exception to that generalization. of course. to result in a loss of resolution in the z direction.9. ( s ) ] = 9. on the right. rather limited. a family of projections obtained over a limited angular range (say +60”)in real space corresponds.
Zeitler. within the context of the Fourier crystallographic method of reconstruction. This limitation is particularly severe when one wishes to work with hydrated.5. Other “basis function” methods are certainly to be admitted as candidates for interpolation and extrapolation. London. A. A. Klug and R . R. where D is the “diameter” of the object under investigation. ELECTRON MICROSCOPY objects. Organic materials in general undergo considerable changes in structure and properties as a result of exposure to very large doses or fluxes of electron irradiation. a formal evaluation of the effects of the hollow cone problem in the case of threedimensional reconstruction by directspace methods has apparently not been dealt with in analytical terms in a way that is equivalent to the state of analysis for the Fourier crystallographic methods. 396 (1974). D. unstained biological materials. The limitations of the safe application of FourierBessel reconstruction have been described in some This analysis has given the general rule of thumb that the highest isotropic resolution d that can be obtained with m equally spaced projections is given by d K 2D/m. A . For unstained hydrated specimens the object 2* 29 30 R . without question. . Nor has there been any effort to apply any compensations or corrections. The incident electron beam must. ’’ 9. be considered to be a flux of ionizing radiation. the fundamental limitation in the overall effort to obtain highresolution images of biological materials. and there is therefore no way to escape the damage that must occur as a result of prolonged exposure to such radiation. Crowther.Soc.418 9. to the original data before the realspace threedimensional reconstruction procedures are started.and A . Nurure (London) 238. The question of what basis set would be optimal for the reconstruction problem has been addressed by Zeitler. In addition. such as interpolation and extrapolation. With stained biological materials it is true that a stable specimen can normally be obtained which provides biologically significant information at the level of 2550 A resolution. E. Proc. Radiation Damage Radiation damage is. Crowther. J. Klug. A general assumption seems to run through the literature of realspace threedimensional reconstructions. Optik 39. Evidence on this point is not yet compelling and is confined mainly to numerical simulations of the effects caused by using data that are obtained with unequal projection angles. DeRosier.435 (1972). that these methods are not sensitive to the same problems felt by the Fourier crystallographic method.319(1970). Ser. after all. A 317. and biological materials are no exception.30 The hollow cone problem of threedimensional reconstruction cannot be really said to have yet been solved as well as it might be.
However. since for lowatomicnumber elements a Kshell ionization is most commonly followed by a second ionization through the Auger process. and R. E. even in the more radiationresistant aromatic materials. Even higher resolution can be obtained. G . and the limitations on meaningful resolution at very high exposures are far worse than for stained specimens. object detail as small as 2030 A can be imaged in noncrystalline objects. collective molecular excitations. and this invariably results in some form of molecular damage. RADIATION DAMAGE 419 is far less stable to electron irradiation. Dietrich. In nonconductors the “collective” excited state decays rather rapidly into individual. The innershell electron excitations. 31 I. The molecule therefore winds up with a double vacancy in the valence electron orbitals.9. the theory of such collective losses becomes relatively simple. The Kshell ionizations are often ignored in discussions of radiation damage since their cfoss section is again quite small in comparison to the cross sections for other inelastic scattering events. also deserve some special attention. Weyl. F. 9. On the other hand. The collective excitations can be regarded phenomenologically as having their origin in the imaginary part of the dielectric constant. the Kshell excitations can be particularly damaging processes. which for organic materials means Kshell ionizations. Knapek. in the frozen hydrated state. Fox. statistically noise images in the periodic array. have a far smaller probability or cross section and can generally be ignored. Socalled knockon collisions. it is not infrequent that the decay of the collective excitation results in multiple ionizations. and the radiation damage problem can be further circumvented.1.5. all within a close distance of one another. Heide. . if periodic specimens are imaged with very low electron exposures and if spatial averaging techniques are then used to superimpose the individual.3I The inelastic scattering processes can be classified as individual molecular excitations. 185 (1978).5. and the inelastic scattering processes are then referred to as “plasmon” excitations. if great care is taken with the microscopy and if a minimumexposure technique is used. In the case of metals and other conductors. localized molecular excitations. and innershell ionizations. Radiation Physics and Radiation Chemistry The problem with radiation damage in organic materials arises primarily from the inelastic scattering of the incident electrons. which are elastic scattering processes in which an atom is torn out of its molecular context by a headon collision with the incident electron. Since the collective losses often involve as much as 3050 eV. Ultramicroscopy 3. valence electron ionizations. that is. H. they are a kind of “dielectric loss” process that is characteristic of the material as a whole and cannot be understood as inelastic scattering processes involving only a single molecule.
the molecule is bound to separate into two pieces. transmitted electrons normally shows three main regions of interest. . which can be formed by neutral molecule capture of secondary electrons. In addition. ELECTRON MICROSCOPY The spectrum of energy losses exhibited by the inelastically scattered. there normally is a broad. however. Since this timc is on the order of sec. This resistance can be ascribed to the delocalized nature of the molecular orbitals of the valence electrons in aromatic materials. many bondscission events can occur either as the result of direct excitation or as the result of recombination of an electron with a positive molecular ion. In the region from zero. is manifest in the electron energy loss spectrum at cncrgies slightly above the Kshell edge. V. 55. It can be taken as a reliable rule that nearly every molecular ionization will result in some form of bond disruption in biological molecules. The Kshell edges exhibit an interesting fine structure which is characteristic of the chemical bonding and molecular structure of the parent molecule. Rodhint.to 10eV energy losses. featureless absorption curve which shows little difference from one organic material to another. The third region of interest of the energy loss spectrum is due to the Kshell ionization processes. As a result. electronically excited state. For completeness.5 to 20 eV as being dominated by individual molecular ionizations. while the higherenergyloss region is dominated by collective excitations. it is worth pointing out that aromatic materials are highly resistant to effects of radiation damage. This broad peak represents the superposition of individual molecular ionization processes and collective excitation processes. which is also a characteristic of highresolution xray absorption spectra.Johnson. an extended fine structure. the features of the energy loss spectrum in this region can be well correlated with the known electronic absorption spectrum of the molecule. Crewc. and A. Res. the inelastic scattering processes are primarily due to valence shell excitation and possibly ionization. in the time that it takes for the nuclei to travel 1 or 2 A. there is virtually no hope of the ionized molecule capturing an clectron from its environment and reconstituting itself into a stable. are frequently unstable to bond scission as well. Negative molecular ions. Thus. Many highly excited (neutral) molecule electronic states are also characterized by purely repulsive potentialenergy curves. Approximately 90 % of the inelastic scattering events occur within this general region of energy On the whole one can think of the lowerenergyloss region from . 205 (1973). Isaacson. D. The delocalization of the valence electrons M. One reason for this is simply the fact that removal of one of the valence electrons from a molccule results in a purely repulsive potentialcnergy curve for the vibrational motion of the nuclei. which occur at energies of 300400 eV for organic materials.420 9. In the region from 10 to 40 or 50 eV.
the excited state may move about until it lands at a particularly labile or easily dissociated bond. which is by nature a quantummechanical phenomenon. Thus a repulsive intramolecular potential may not occur in aromatic materials as easily as it does in the case of aliphatic materials. Thus there are G (parent molecule) values to describe the disappearance of the original molecule and G (product) values that describe . For our purposes. at which time the energy may be dissipated as heat. Thus two effects of resonance energy transfer are (i) an increased amount of fluorescence from aromatic moities in the specimen. and there is no simple scheme or generalization that can describe all of the effects that can occur. In most systems there is a large number of different pathways that can be taken by the secondary products.9. where the loss of the valence electron is confined to a single chemical bond.” The effect of resonance energy transfer. labile bonds is seen which is much larger than can be accounted for by random direct hits at those particular sites within the molecule. through bond rupture. The excited state thus “hops” around from place to place. which can be seen both in complex biological macromolecules and in simpler chemical systems that are “doped” with small molecules that can serve as an “energy sink. Alternatively. which is due to the fact that aromatic groups tend to be traps or sinks for excited states created elsewhere in the molecule. and in this process the excitation energy may eventually find a somewhat lower energy state and become trapped at that location. This empirical approach is based upon the extensive published studies of radiation chemistry in which the disappearance of parent molecules and the appearance of a variety of product molecules have been measured. The processes of radiation damage are by no means over with once the primary steps of molecular ionization and bond rupture have occurred. which in some cases can go through a considerable sequence of steps involving many neighboring molecules. it is sufficient to take an empirical approach in order to understand the expected consequences of electronbeaminduced radiation damage. These primary steps lead to the formation of a variety of molecular ions and molecular radicals which have enhanced chemical reactivity with other molecules in their environment. RADIATION DAMAGE 42 1 in aromatic molecules serves to distribute the loss of chemical bonding that accompanies ionization over the entire molecular network. Also worth mentioning is the phenomenon of resonance energy transfer. These products therefore engage in a host of secondary chemical reactions.5. The results of empirical studies in radiation chemistry are traditionally presented in terms of the “ G value” for the particular reaction being considered. is that energy absorbed by one part of the molecule can be transferred to other parts ofthe molecule or even to other neighboring molecules. and (ii) a large cross section for the rupture of specific.
5. While it is true that these G values can differ depending upon the quality and type of radiation involved. While none of these factors are such as to make the situation seem tremendously more encouraging in electron microscopy than would be inferred from previous radiation chemical studies. Rohrlich and R. Thus. Carlson. The rad dose is again easily calculated from the stopping power by first calculating the energy deposited per unit volume as above and then dividing by the mass density of the material. and the stopping power can be taken to be constant throughout the thickness of the specimen. Washington. M. 38 (1954). RLT. Res. (1964). According to stoppingpower theory the average amount of energy lost per unit path length by a charged particle traveling in matter is almost inversely proportional to the square of the velocity of the particle. 33 J4 .Empirical Studies of the Radiation Damage Effect under Electron Microscope Conditions A number of factors concerning the conditions of electron irradiation in electron microscopy are rather different from the conditions that exist in radiation chemical experiments. The average amount of energy deposited per unit volume is then given directly as the product of stopping power times thickness times electron exposure.33 For thin specimens. 93. 1133.34 The energy deposited in the specimen is often expressed in units of rads. Extensive tables of stopping power for electrons of different energy in a variety of different materials are given by Berger and Seltzer. it is necessary to calculate the energy deposited in a specimen for a given amount of electron exposure in the electron microscope. one rad corresponding to 100 ergs per gram. Berger and S.Natl.422 9. Natl. to compare the radiation damage effects observed in electron microscopy to direct radiation chemical studies carried out on similar specimens. D. Council Publ. the majority of the radiation chemical literature has dealt with the use of energetic x or gamma rays. 9. Seltzer. 20268. These G values represent the number of molecules created or destroyed per 100 eV of energy absorbed from the incident radiation. M. Sci. The statistically expected value of the deposited energy can be calculated from stoppingpower theory. and for these the radiation chemistry is virtually identical to that of highenergy incident electrons.” pp. the conditions are still sufficiently special in nature as to make their description worthwhile.2. J. ELECTRON MICROSCOPY the creation of a particular product molecule. Acad. the latter being expressed in units of electrons per unit area. and the microscopist should always be conscious of these F.C. in ”Studies in Penetration of Charged Particles in Mattcr. C. which is a macroscopic representation of the inelastic scattering processes. the incident electron suffers a negligible change in velocity over the whole specimen thickness. Phys.
For specimens that are kept in the hydrated state. Thus. that it has been necessary for electron microscopists to carry out empirical studies on the damage that is produced in their specimens under normal electronmicroscopic conditions. has been to observe the changes that occur in the electron diffraction patterns of crystalline organic materials. Eventually.5. the doses received by specimens in the electron microscope are quite commonly many orders of magnitude larger than the total doses that can be realistically achieved by radiation chemists. One of the most informative methods of observing radiation damage. Put another way. In addition. A somewhat more quantitative characterization involves the measurement of the electron . and in many materials the higherresolution Bragg reflections are seen to fade more rapidly than do the lowresolution reflections. for comparison. These high dose rates open up the possibilities of introducing doseratedependent effects. A third factor is the exceedingly high dose rates that can commonly be achieved in electron microscopy. Thus. will in fact escape from the specimen without doing any damage. It is for all of these reasons. Finally. the crystalline diffraction pattern disappears completely. to introduce chemical reactions that obey second. it is unlikely that the vacuum conditions in the electron microscope will have any significant effect on the radiation damage problem. the secondary electrons that do escape cannot form negative molecular ions. For anhydrous materials one would not expect any change in the radiation chemistry of specimens in air or in vacuum. RADIATION DAMAGE 423 differences.or higherorder kinetics. under very highdoserate conditions. and all that remains is a continuous. which is highly characteristic of amorphous materials. Thus part of the energy which is lost by the incident beam. which would themselves undergo chemical reactions including bond scission. The fading of the diffraction pattern is often characterized in a semiquantitative way in terms of the electron exposure that results in a “complete loss” of the crystalline diffraction pattern. One initially sees a general fading of the diffraction spot intensities. This means that many of the secondary electrons created by primary impact may in fact escape from the specimen. a dose of lo6 rad is a rather high dose as far as the radiation chemist is concerned. from a structural point of view. First. but particularly to explore the effects at exceedingly high doses. the specimens used in electron microscopy are necessarily exceedingly thin. and which would be calculated to be deposited in the specimen according to the use of stoppingpower theory. while a dose of l o t 2 rad is not at all uncommon in electron microscopy at high magnification. A second difference regarding the electron microscope conditions of specimen irradiation has to do with the fact