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 . SCHUH R AND SUNNEY CHAN I.
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 . K . HUMPHRIES D HARDEN . MCCONNELL M AN M
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 BUNOW R. 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 ELLIOT . ELSON AND L
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 . LATTMANN D L . MARIOAMZEL E A 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
... ..................CONTENTS xi 310 310 312 313 316 319 7.2. ....1...... 337 337 338 338 339 340 341 341 342 343 344 345 346 347 349 8.. .. 8.......... Collimation ....2..... 8...... ......... ........5.................. The Scattering Signal .............. 7.. .....6..... 7... ....... 7.......... Introduction .....8... ...... 8............7....2. Earlier Reviews .........10.5.... 8........ ....... ......4.1..... Equipment ... ... . ...3... 7... . 324 7...... .......... Flight Path ... . ....... Diffusion Coefficients ...... ... 8. .... .. Large Particles . ... Relationship between Photon Autocorrelation and Special Analysis . .... ... 333 8.......5... 8......... MOORE B 8........ ........1.... 326 330 331 7... SrnallAngle Apparatus: General Features . Determination of Electrophoretic Mobilities . 7.. ...... SmallAngle Scattering Techniques for the Study of Biological Macromolecules and Macromolecular Aggregates by PETER ........ The SmallAngle Compromise ..4....8......... ......1....... ........ ....... .....2....... Characteristics of Measured Autocorrelation Functions .......2.............2...2...............1....... 8.9...1.......4......1..6.... X Rays . Blood Flow ....... ... ...... 8..2.. Motility Measurements .......3...2......2....... Instrumentation and General Techniques .......... ............ .3.....7.. ... ......... Monochromatization of X Rays ...... 8.3... ....... . ............ Purpose ..... The Sources of Current Interest .. 7.. 8....... 8......... ...3.. ...... ........2....... Gels and Solutions of Fibrillous Proteins . ....... The Experimental Problem .... ..... 7. Isolation of the Macromolecular Signal .......3... Applications in Cell Biology .3. 8.... .. ...1.......... ..... The Nature of the Technique ... Historical Comments ..2........ .. . 8...... ........1...... ........... 7..........
...... 8..... Monochromatization of Neutrons .16....18. 8.. Absolute Intensity Measurements ...12... .. 8.. . .. . ........ . .... Quantities Related to the Radius of Gyration .. .. ...... ....... .. . .....10.8.. . ... 8..7.... .. ...... Solution Scattering Studies on Molecules of Known Structure . 8... .2...... . 8..... ..... . ..16........ . .......xii 8..15.....3...1 1 ....... ... .... .. . ... Beam Monitors . . 8.. ...... ... ..... . ..... ....... ...... ........... . .3. Molecular Size and Shape: Radius of Gyration ..... .........1 1..... . .. . ...4.2... ...2.13.....2.2.. . 8. Apparatus Designs . 8.............12...2.... .... ..2..... .......18.10.. .. . . . .... ....... Neutrons ....3. . ... . .... ..... ....... ..... .... 8...... 8.... ..... Data Correction ... .. ... .. .. .... ... ... .15......... ....1..6.... 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.. ....... Absolute Scale and Contrast Variation ... .3. ......... 8...3. . ..3..3.......... 8..... The Dependence of Radius of Gyration on Contrast ... 8.. 8.. .... . ...5. 8.... ... ........2.... . ...3. . . Molecular Shape ....... 8.....3.. . ............... 8. Spherical Objects ..9...........3..3. .... ........ Contrast Variation in Practice: Radius of Gyration .. ...... ........ ..... . Molecular Weight and Partial Specific Volume ..... .. .... .... .... .... ..... 8.. .. . Neutrons versus X Rays ... . Macromolecular Aggregates: Distance Finding ..2.2. General Design: Collimation . . Length Distributions .19. ......... ... ......... .. 8..3. ....... ... ..... ..17.... .... .. . .. ...17... .... . .. ... 8......... Available Instruments . Data Analysis ....... ... 8. 8... .2. ... . .....3.. .... 8...... ........... .. .. ... .. ..3...........2... ..... 8.........14.........13.... Macromolecular Aggregates: Synthesis Methods ..... Flight Path and Detection .... CONTENTS Detection ... 8......3. . ..9........ .... . ..... . ..3. 8.... .........3.. . Characteristic Function Parameters . .. Contrast Variation in Practice: Extended Scattering . The Hydrogen Exchange Problem . ..... . .3.... ... ............ Pulsed Reactors ... .........3.. . ... .... Dependence of the Extended Scattering Profile on Contrast .... Contrast . ......... .... . . ..14..3. 8.
......... 8... Radiation Damage ...... . .... 430 9..3................ .. Electron Microscopy as a Tool for Structure Determination.. ............4.. GLAESER M 391 395 395 397 398 401 401 403 404 406 9.....2.....1..4.. .........2...........4.... 413 9.....5.........6.............. . 9.. Concluding Remarks .......... 9.....1......2...2..................3....... .... Empirical Studies of the Radiation Damage Effect under Electron Microscope Conditions ............ DirectSpace Methods ... Difficulty of the Problem ........ 9.............. 8....4. Complications...2...................... 422 427 427 9...... 9 .... .........1.... 430 9........1. 409 9.... 9..... .....2.... Contrast Transfer Function Theory . ..2..................4....2.....2...4... Complications ...... 9.... ....3............ ..4....4........... .... 9..1...1........ ... .. Specimen Hydration within the Vacuum of the Instrument ... ....... 416 418 419 9. .. 9...C0N T E N T S xiii 388 388 389 8...... ..............3............ ShotNoise Limitation for HighResolution Electron Microscope Work .......... Image Formation in the Electron Microscope ...4.... 430 .............. .. ..... .. 9.... .5...... Fourier (Crystallographic) Methods ............ .......6......5........... Radiation Physics and Radiation Chemistry ..... .... . 409 9.2..3........1.. Critique . The Need for Maintaining the Hydrated State with Complex Biological Structures ......................3........ The Hollow Cone Problem .............. Summary . Solution of the ShotNoise Problem by Image Superposition .... 9...........3........3.. Image Restoration ...... The Envelope Function: Partial Coherence .5...... ...... 9.6......... Electron Microscopy by ROBERT . Origin of Phase Contrast in Electron Microscopy .... 9..... .... 9... A Simplified Picture: The Mass Thickness Approximation . 9............ ThreeDimensional Reconstruction .... ..... 9.5......... The “Weak Phase Object” Model ..3.
External Patch Isolation ... ... 482 482 484 486 487 497 500 501 ..... . .... . .. 10.... ...... 10.. ...... . . 10........... ...3.5 .6. ......5. DarkField Methods .4...... ... .4.... 9.... .. .. 9..... Introduction ...7..6... .. 9. . Voltage Clamp with Microelectrodes .. 10.... ..... .... .. ...... Node of Ranvier .1...................... General Principles of Voltage Clamp ... SucroseGap Methods .... by FRANCISCO AND ROBERT . ... .... 9..... .3.....1. .6..... 9... ... .. .... 473 10...4... ..... .. . 445 447 451 452 455 459 461 463 470 10.. . .. ..... Voltage Clamp of an Isolated Patch Using External Pipettes . . AxialWire Voltage Clamp .... ...4.. .2.. ...5. .... ... ...1.... ... . .3....... 10...... ... ..... .7.6....... Three Microelectrodes ... ...... ... ... .... .. ..6.. ... 10.... ..... Errors Introduced by the Finite Length of the Gap .. ...3. ..1. 10. . SeriesResistance Compensation ...6... ... 10. ..... Voltage Clamp with Gap Isolation Techniques ... . 431 433 433 438 439 441 444 10...... ....6.. ....... Pulse Generation and Data Acquisition .1. .... Technical Solutions to the Hydration Ploblem ....3... ........... . ... Measurement of Membrane Current .....7... . ....... .. Two Microelectrodes .. ..... 481 10. ......... . Internal Access ....... 9. ..7. .... ....... 10.. .. ...... ......4.......7... JULIO VERGARA... . ....2.... ...... . ..5..2. .2. 473 10.............2.. . .....4... ....3...... ... ..3. 10... .....7..... ....... 10.... Electronics for the Voltage Clamp System . .. Giant Axon Preparation . . .. ...... TAYLOR E 10. Aberration Correction .. ............ HighVoltage Electron Microscopy ...... SingleSideband Images and Holography . Image Formation with Inelastically Scattered Electrons ...... . Recent Innovations in Experimental Methods . .. Cable Theory of an Axon with Axial Wire in Voltage Clamp .3.3.. .. . ......3.... Voltage Clamping of Excitable Membranes BEZANILLA. 10.... .. 10.5... . . ............ 10.2.....3.xiv CONTENTS 9.. VaselineGap Techniques in Single Muscle Fibers .... ... .. 10.........1... .. ... .
... 11....... Single Channels in Bilayers ......... ...... 539 11......... . ...... C ...... ... Multichannel Bilayers ......9.... Molecular Channels .........9......... .. .... ... 513 515 516 519 520 521 523 524 525 528 11. ... .. .........2. 1 I .. ...... ..4... 1 1....... Direct Transport of Hydrophobic Ions 11...... Techniques for the Measurement of Ion Transport .. WALDBILLIG A 508 11 1 ......... Potential Distribution for a Fiber in a Gap Voltage Clamp ... . . 541 545 SUBJECT INDEX ..... 536 1 1....... . . 565 ..2.......... Spherical Lipid Bilayers ..... 11. . 1 1.........1 ..... ...... .. .. .............. . .......... .. ... ........... Planar Lipid Bilayers ....A... ...... 1 1......2. ..... ... . ..... .. ..... .. Pure Lipid Bilayers . Ion Transport Mechanisms .... .. .... .........2..5.......... 10.3.. ...... ...4.... .. . Circuit Equations of VaselineGap Voltage Clamp ..... .... 535 11. Appendix . ... Lipid Model Membranes by G ....... ....... ..... . 11. ...4..........1........... 11.CONTENTS xv 504 504 504 10.6. 10.. .... Lipid/Solvent Systems ............. ......10.... SZABO N D R ... . 529 530 ... AUTHOR INDEX....... 10..A... . Concluding Remarks .. ...... ........ ................. SolventFilled Bilayers ... CarrierMediated Ion Transport ..4..... 11.. .. ..............4....... . 11. .......... Lipid Monolayers ..... . ...A........ ........ .... .... SolventDepleted Bilayers ..9.... Relationship between Channel Structure and Function . ...7.... .3 . .... ........... .1. Biological Membranes . ......... ...... 11... ................ ...... ... ..... ....... ...... .............. ...... .8.....7...4........ ....
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Washington University School of Medicine. New Haven. Baltimore. Louis.National Institutes of Health.~ M.* Zthaca. Missouri 631 10. Departments of Chemistry and Molecular Biophysics and Biochemistry. Bethesda. University of California. Maryland 20205 (299) * Present address: Department of Biological Chemistry. California 91125 ( I ) ELLIOTL. Baltimore. t Present address: Institute for Medical Research. California 94305 (53) PETERB. School of Medicine. xvii . New York 14853 (197) Department of Biophysics and Medical Physics. Connecticut 06511 (337) RALPH NOSSAL. ELSON Department of Chemistry. Pasadena. Stanford. Yale University. California 94720 (391) GILLIAN K. Maryland 21205 (229) Stauffer Laboratory for Physical Chemistry. California 90024 (445) MARGARET BUNOW. Stanford University. SUNNEY CHAN. Bethesda. San Jose. National Institutes of Health. University of California. BunkyoKu. M. Stanford University. MOORE. M. Stanford. MaryR. A. Maryland 21205 (229) Department of Physiology. Department of Physics. A. Los Angeles. Tokyo 113 Japan (163) EATON . St. Department of Biophysics.CONTRl BUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin. LATTMAN. ROBERT GLAESER. California 94305 ( 5 3 ) TAKAYOSHI KOBAYASHI. California Znstitute of Technology. FRANCISCO BEZANILLA. HARDEN MCCONNELL. University of Tokyo. Johns Hopkins University School of Medicine. L. MARIO AMZEL. Noyes Laboratory of Chemical Physics. Johns Hopkins University School of Medicine. E Department of Biophysics. Cornell University. California 95128. HUMPHRIES. land 20205 (123) I. Berkeley. Stauffer Laboratory for Physical Chemistry. .
. University of Calgornia. Noyes Laboratory of Chemical Physics. A . Department of Physiology and Biophysics. University of Texas Medical Branch. The Weizmann Institute for Science. National Institutes of Health. SZABO. Galveston. California 90024 (445) R. Laboratory of Biophysics. Texas 77550 (513) . Maryland 20205 (445) JULIO VERGARA. Israel (197) JOSEPH R. Pudadena. University of Texas Medical Branch. TAYLOR. School of Medicine. Galveston. Texas 77550 (51 3 ) ROBERTE. SCHUH. Department of Physiology. Bethesda. Cal$ornia 91125 ( I ) G . Rehovot. Department of Physiology and Biophysics. Los Angeles. National Institutes of Neurological and Communicative Disorders and Stroke.xviii CONTRIBUTORS JOSEPH SCHLESSINGER. WALDBILLIG. C. Department of Chemical Immunology. A California Institute of Technology.
Claire Marton. and by Dr. Future volumes will be edited by Dr.. University of Colorado. Boulder. xix . We are fortunate to have such qualified and experienced people join us. Judah Levine of the Joint Institute for Laboratory Astrophysics.C. Celotta and Dr. Bill. Both Claire and her late husband. Robert Celotta of the Electron Physics Group. Their passing is both a personal and professional loss to us. Levine had already started to work with Claire. Colorado. National Bureau of Standards.PUBLISHER’S FOREWORD It is with sadness that we inform readers of Methods of Experimental Physics of the passing of Dr. Radiation Physics Division. Washington. Dr. were associated with Academic Press almost from its founding. D.
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in retrospect. 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. more volumes on this subject are called for. this may not appear surprising. This volume is a proud addition to the treatise. Given the subject matter. Certainly. MARTON xxi . 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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xxiii . This approach. 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. Thus ESR spectroscopy is restricted to molecules with unpaired electron spins. A major theme of Part 1 is the use of nuclear magnetic resonance to measure relaxation times. Parts 14 discuss several types of spectroscopy. it is sometimes possible to add covalently a group that contains an unpaired spin. When these conditions are met. and any sampling of methods is somewhat arbitrary. Unpaired spins are present in biological molecules containing transition metals and in free radicals created as intermediates in biochemical reactions or by radiation damage. either directly or by means of model systems.PREFACE This volume describes three types of measurements that have been of intense interest to physicists working in biologyspectroscopy of macromolecules. the absorption frequency depends on the magnetic moment of a nucleus. Almost every type of spectroscopy has been used with some success to study biological macromolecules. called spin labeling. and the first two parts are about these methods. Parts 59 are concerned with methods used to visualize the structures and motions of large molecules and cells. We have emphasized methods which can be used to study molecular motion and conformational changes of macromolecules. Magnetic resonance spectroscopies have found considerable use in studies of biological systems. and electrical measurements on cell membranes. and are primarily concerned with the determination of molecular information. In nuclear magnetic resonance. structure determination by the scattering of particles or radiation. Electron spin resonance (ESR) similar to nuclear magnetic resonance is in concept. early work centered on the proton. but utilizes the interaction of a magnetic field with an electron spin rather than a nuclear spin. is the subject of detailed discussion in Part 2. With the improved sensitivity now available. Parts 10 and I 1 discuss methods for studying membranes. Part 1 includes an example of the N use of natural abundance *T M R . Since protons have large magnetic moments and are prevalent in biological material. other nuclei with magnetic moments are often studied. For systems without unpaired electron spins.as well as other new and more sensitive methods.
or whole cells. Fluorescence spectroscopy has been very successful because of the prevalence of chromophoric groups in biological molecules. Part 6 deals with xray diffraction. nucleic acids. visible. These methods allow the determination of molecular motion within a cell membrane. Thus these parts are representative of the current emphasis on Fourier optics. and circular dichroism (CD). are based not on the particular frequency of absorption. which has been the prime source of . however. but are not described in this volume. such as molecular motions. Although similar information can be obtained from spin labeling (Part 2). and lipid membranes. the lack of interference from liquid water. The fluorescence methods presented in Part 5 . the fluorescence methods are somewhat more flexible in that they can be used to determine motions over relatively long distances. optical rotatory dispersion (ORD). Other types of spectroscopies which have been used extensively to study macromolecules. A number of IR. but on the bleaching of fluorescent molecules and the interdiffusion of bleached and unbleached molecules. Whether the objects are macromolecules. which is discussed in Part 4. and rates of reactions. and the high sensitivity inherent in fluorescence measurements. fluorescence can be a very valuable tool in the measurement of absorption spectra. 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. the possibility of incorporating fluorescent markers. where previous methods have been inadequate to resolve the rapid transitions which occur immediately after the absorption of a photon. Parts 69 describe different approaches to the problem of obtaining images of small objects. ORD and CD signals. are especially valuable spectral indicators of conformation. which are sensitive to helical content of macromolecules. cell organelles. As previously mentioned. We have chosen to include a paper on Raman spectroscopy (Part 3) because it combines several important advantages. and sensitivity to homonuclear bonds. distances between groups. include fluorescence.xxiv PREFACE the spectrum of the spin label can be used to determine a number of important parameters. Raman spectroscopy has been utilized effectively in studies of heme groups. and U V spectroscopies have been successfully used for biological studies. is a relatively new method that has considerable promise for several types of biological problems. 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. Picosecond laser spectroscopy. such as the possibility of recording from small samples.
Two examples are cytoskeletal architecture and membraneprotein complexes such as bacterial rhodopsin. However. Different types of motion produce different Dopplershift characteristics. it complements the method of neutron diffraction (Part 8) to provide a good description of the structure of ribosomes. For those macromolecules that can be crystallized. 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. The laser. and ribosomes) that cannot be grown as crystals. The concepts involved in smallangle xray and neutron scattering. scanning electron microscopy. viruses. In particular. they can be used to analyze the shapes and interactions of macromolecules in solution. Molecular structures of particular interest are the ionselective channels responsible for excitability and ion “pumps .” the . 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. Consequently. xray diffraction provides a wealth of information. These shortwavelength methods provide information complementary to that obtained by crystallography. Present membranetransport research focuses on characterizing those molecular structures in the membrane that are responsible for the various transport processes. has had a strong impact on all of optical technology. are similar to those involved in light scattering. since the wavelength of radiation is so small (of the order of l &. 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. and electron diffractiontechniques that provide new and striking images of key supramolecular structuresare considered here. 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. The last two parts describe methods for studying membrane transport. and Part 11 describes methods for making lipid model membranes and for determining their properties. the scattering is confined to “small” angles near the forward direction. discussed in Part 8. Part 9 emphasizes those aspects of electron microscopy that have grown out of physical considerations of the process of image formation. Even for structures (such as fibrous macromolecules.PREFACE xxv our knowledge of the detailed structure of biological macromolecules. Highenergy . highresolution microscopy. Part 10 describes voltage clamping of excitable cell membranes. For example.
Claire Marton and Dr. the many studies of synthetic ionophores implanted in bilayers have yielded major insights into the transport mechanisms of membrane channels and carriers. both recently deceased. support. and patience. Among the systems that have been reconstituted are NaK ATPase pumps. Finally. GERALD EHRENSTEIN HAROLD LECAR . the use of noise analysis to determine the properties of the channels as they switch on and of€at random. Dr. As discussed in Part 10. the electrical behavior is extremely nonlinear. This approach has contributed greatly to the currently accepted paradigm for membrane structurea lipid bilayer matrix resembling a twodimensional fluid. L. and photosynthetic reaction centers. in which various membrane proteins are embedded. Without this control. we thank the authors for their effort and for their willingness to accommodate their individual papers to the needs of this volume. Part 11 discusses methods for using synthetic lipid bilayers as analogs of cell membranes. We hope that the sample of methods presented in this volume shows some of the technological ingenuity that biophysical problems have generated. We should like to thank the editors of this series for their encouragement. For example. postsynaptic acetylcholineactivated channels.xxvi PREFACE membranebound enzymes that use metabolic energy to maintain concentration gradients of ions. making it virtually impossible to disentangle them. More recently. lipid bilayer studies provided the first measurements showing that membrane ionic current passes through discrete channels. membrane potential and membrane conductance would vary in an extremely complex manner. In addition. significant progress has been made in reconstituting functional entities from biological membranes into lipid bilayers or lipid vesicles. and the measurement of gating current (the displacement current caused by molecular conformational changes during channel opening or closing). the voltage clamp has proven to be the major tool for studying such highly nonlinear systems. 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. We should also like to thank the staff of Academic Press for their 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 nonlinearity is crucial to excitability. such as nerve axons. In electrically excitable cells. have taken considerable interest in furthering interactions between physicists and biophysicists. In fact. Marton.
Robinson xxvii Interactions (in . Griem and Ralph H. Classical Methods Edited by lmmanuel Estermann Volume 2. Mechanical and Thermal Properties. Magnetic. Part B: Free Atoms Edited by Vernon W. Second Edition (in two parts) Edited by Dudley Williams Volume 4. C . Atomic and Electron PhysicsAtomic two parts) Edited by Benjamin Bederson and Wade L. Electronic Methods. Structure. Lovberg Volume 10. Johnson Volume 7. Fite Volume 8. Schultz Volume 5. Marton and W. Second Edition (in two parts) Edited by E. Plasma Physics (in two parts) Edited by Hans R. F. Solid State PhysicsPart A: Preparation. 0. Marton Volume 1. L.METHODS OF EXPERIMENTAL PHYSICS EditorinChief C. Molecular Physics. LarkHorovitz and Vivian A. Yuan and ChienShiung Wu Volume 6. Haxby Volume 3. Hughes and Howard L. Bleuler and R. Nuclear Physics (in two parts) Edited by Luke C. Hornyak Volume 9. Physical Principles of FarInfrared Radiation By L. Part B: Electrical. and Optical Properties Edited by K. Atomic and Electron PhysicsPart A: Atomic Sources and Detectors. Problems and Solutions for Students Edited by L.
V. A. Tang Volume 16. Part C: Physical Properties Edited by R. Accelerators in Atomic Physics Edited by P. Carleton Part B: Radio Telescopes. J. PolymersPart A: Molecular Structure and Dynamics. Weissler and R. Fava Volume 17. Biophysics Edited by Gerald Ehrenstein and Harold Lecar . Ultrasonics Edited by Peter D. L. Part C: Radio Observations Edited by M. Coleman Volume 12. Quantum Electronics (in two parts) Edited by C. Carlson Volume 15. Meeks Volume 13. Solid State Physics Edited by R. Vacuum Physics and Technology Edited by G. Fluid Dynamics (in two parts) Edited by R. Part B: Crystal Structure and Morphology. Richard Volume 18. Spectroscopy (in two parts) Edited by Dudley Williams Volume 14. L. Edmonds Volume 20. W. Emrich Volume 19. L. AstrophysicsPart A: Optical and Infrared Edited by N.xxviii METHODS OF EXPERIMENTAL PHYSICS Volume 11.
and R. on relatively simple chemical systems. Torrey.idcmic Prc\*. 1 Ciipyright @ 1982 hy Ac. 69. Particular emphasis is placed on recently developed techniques and their usefulness in studies of biological macromolecules and systems. Phys. PHYSICS. In the next two chapters we briefly outline those aspects of magnetic resonance theory necessary to facilitate the discussions of these newer NMR techniques. We conclude Part 1 with a number of illustrative examples taken from the recent literature to highlight the achievements of the method to date.. recently developed techniques and improved instrumentation have altered this state of affairs considerably. Instead.Pound. including the concepts of magnetic shielding. In this part we review some of the advances in NMR spectroscopy that have resulted in its successful application to problems in biology. magnetic dipolar interactions. Initial studies were done. such as small organic and inorganic molecules.37 (1946) C. 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. as well as the manner in which these phenomena manifest themselves in liquidstate spectra and have proven useful for structural analysis. Hansen. 127 (1946). W. H. Since 1946. it is assumed that the reader is familiar with elementary magnetic resonance. and M . W . 20 ISBN 0124750629 . I .V. NUCLEAR MAGNETIC RESONANCE By Joseph R. etc. we emphasize nuclear magnetic relaxation phenomena.Packard. particularly those techniques and features of NMR relaxation which permit *E ‘ F. Purcell. out of necessity. Chan 1. Schuh and Sunney I. Low spectrometer sensitivity and the increased spectral widths associated with larger molecules rendered it difficult to extend the method to macromolecules of biological interest. Phys. This review will be followed by a presentation of selected techniques that have found applications in biological NMR. Rev. However. 69. Rrr. Inc All rights d r c p r o d u c t i o n 111 .”’ this method has undergone continuous development toward the realization of its full potential. when the first successful NMR experiments were reported..1. VOI. In the discussions to follow. electroncoupled spinspin interaction.iny limn r c w v e d METHODS OF EXPERIMkNTAI. chemical shifts. M .
Jamcs. Marsh. Meth0d. For a nucleus of spin I . = . Physirs 3B. the angular momentum vector of an isolated spin is characterized by 21 + 1 distinct spatial orientations. For a more comprehensive treatment of NMR in biology the books by James.* should be consulted. 1973. the angular momentum is denoted by J = hI and its magnitude is h . Rattle. and Knowles et a1. S . Press.’ Dwek. 1.” Academic Press.1.1) J . 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. E. “Magnctic Resonance of Biomolecules. New York. Amsterdam.2. 1976. F.” Wilcy. A . 465 ( 1 974).’ Oxford Univ.6 Wuthrich’.2. Biomolecular structure has received considerable attention in biological NMR in the past. New York. Memory and G . D. and H. Press (Clarendon).2 1. 197s. We concentrate on the dynamical aspects here. L. Each of these projections will assume a value m I h with m. “The Principles of Nuclear Magnetism. Abragam. The spin angular momentum of a nucleus imparts to it a magnetic moment p along an axis collinear with hI. Quantum Description A large number of atomic nuclei possess an intrinsic spin angular momentum. P.. T. London and New York.” Oxford Univ. W. NorthHolland. Parker. . Knowlcs. / m . ) I . where h is Planck’s constant divided by 27t. as a number of recently developed techniques are bringing these measurements within the realm of possibility. In quantum mechanical terms. (1. . A . NUCLEAR MAGNETIC RESONANCE one to sample the details of motional processes in molecules. 1961.” Elsevier. edited by J. especially those aspects which pertain to biological function. . ’ K.s Exp.I. Only half integral and whole integral values of 1 are allowed.I . Wiithrich. These two quantities are related by )r = $1. Dwek. Waugh.2.I + 1) . “Nuclear Magnetic Resonance (Nmr) in Biochcmistry. “ N M R in Biological Research : Peptides and Proteins. W. 1976. London and New York. . These quantized states correspond to the 21 + 1 projections of hI along an arbitrary axis in space. Biophysics deals in part with the structure and dynamics of biological molecules and assemblies. “Nuclear Magnetic Resonance in Biochemistry. ’ K. Phenomenon of Magnetic Resonance 1.1.
00104 0. (1.28 16.98 1.4 93. so TARLE Properties of Some Biologically Relevant Nuclei I. Table I summarizes the NMR frequencies for a number of biologically important nuclei.26 26.064 75. As a result of the interaction between the magnetic moment and the applied magnetic field.05 100 0.00470 0. this degeneracy is totally lifted. In an NMR experiment one actually samples an assembly of spins.49 338. For an equal number of nuclei at constant field.037 10.0291 0.2.833 0. In the absence of electrostatic and magnetic interactions. .000508 0.000 0. However.ml. For an isolated spin.00965 0.0156 99.00268 0.04 89.22 Natural abundance 99.3) Typically. NMR frequency" (MHz) 360.2.6 kG.13 ' In a field of 84.00101 0.74 55. Since contiguous spin states under this circumstance are separated by an equivalent amount of energy.52 36.80 48. transitions will occur only at AE = y h H o .68 145. when an external magnetic field Ho is applied.11 Spin Isotope (x) Relative sensitivityh 1. I78 0.0925 0. the projections of I will be quantized along Ho. AE/h is in the radiofrequency (rf) range.1.01 95.08 0.23 24. (1.64 100 0. quantum mechanics allows only transitions between adjacent levels. the 21 + 1 spin states are degenerate.23 35.00 90. PHENOMENON OF MAGNETIC RESONANCE 3 where y is the magnetogyric ratio.81 22.2.0663 0.365 100 100 0. and each mr state will assume a different energy given by E = yhH.2) Nuclear magnetic resonance is a spectroscopic method which permits one to monitor the transitions between the various spin states in a magnetic field.01 59 0.
However.2. such as those relating to the properties of an individual nuclear spin. (1.kcal/mole of spins.n). precessing nuclear magnetic moments with I = +> in the presence of exterior field H. thus it will be necessary to turn to quantum mechanics when discussing other aspects of magnetic resonance. n .2. The nct rnagnctization M . a limitation that has hampered the application of this technique to biological problems. NUCLEAR MAGNETIC RESONANCE r.eyfiHn’kT)/(l eyfiHaikT). (a) Zeeman splitting of spin states for nuclei having I = : (b) Classical depiction of .n?) = (1 . + (1. it should be noted that the classical approach is applicable only when discussing the net magnetic properties of a system. it will be used whenever possible. la) we may write signal a (n. Inasmuch as the induced transitions between any two levels occur with equal probability. so that the energy change being measured is only about 10. ’ 1.. the Boltzmann factor deviates from unity by only 3 parts in lo5. determincd by the Boltzmann distribution.2. Thus NMR is a spectroscopic method of low sensitivity. is along the z axis aligned with the field. Classical Description Many aspects of NMR theory can be appropriately described by either classical mechanics or quantum mechanics. the signal one detects is proportional to the difference in the populations of nuclei occupying the different spin states. signal K (nt . . .2. 1.I/ 2 j AE = YhHo Y I _ 0 I 1’0 (a) (b) FIG.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. Thus for an I = system (see Fig. Since the former affords a more graphic explanation.5) For a proton NMR experiment at room temperature and 360 MHz.4 1. the measurement represents the net energy absorbed or emitted by a population of nuclei at the resonance condition.
For a given distribution of spins between these two states. each of the individual spins that make up M experiences a torque in the presence of H. the nuclear magnetic moment vector p of each spin will assume one of two orientations with respect to the field. In the presence of the external magnetic field H.1. Motion of a Spin in a Magnetic Field.2.2.10) To demonstrate this result. The frequency of this motion is given by the Larmor equation 00 = yHo.7) aligned in the direction of the external magnetic field (see Fig. we rewrite Eq. (1. (1.2. the population distributions among spin states are given by the Boltzmann factor.. . the result given by Eq..2. In the lowenergy state p will be aligned with the field. (1. lb).2.2. corresponding to the two quantum mechanically allowed energy states. (1. the sample will exhibit a net magnetization M. (1.9) in a frame of reference The result is which rotates about the z axis at angular velocity a. Under these conditions. and the magnetization associated with the N . = Y ( P ) x (Ho + dY>.2. we now consider a population of N identical nuclei having I = ).2. the effect of the torque on the magnetic moment vector is d(P)ldt = Y ( P ) x H.2. (1. 1. In order to illustrate the principles of NMR spectroscopy more fully. Since the distribution of the azimuthal angles would then be uniform. (Kp>/Wr. The equation of motion is dJ/dt = (p) x Ho.. In the absence of other external forces an assembly of noninteracting spins exhibits no preference for the azimuthal angle. The Concept of Magnetization. In view of the relation1 ships J = A and (p) = yAI.9) This equation describes the precession of (p) about an axis parallel to the imposed field.2.2.2.2. Classically.6)does hold for a system of spins at thermal equilibrium with the external degrees of freedom. (1. I = f. PHENOMENON OF MAGNETIC RESONANCE 5 1.2. while for those nuclei in the highenergy state p will oppose H.8) where (p) denotes the classical magnetic moment. spins at thermal equilibrium is a vector of magnitude M. where M = z i p i .1..11) . = t N y h tanh(yhHo/2kT) (1.6) Although experimental situations may still be found where these components are nonvanishing.
In magnetic resonance one typically applies a linear H1 field perpendicular to Ho.2. at resonance. the torque exerted on (p) by the H1 field will cause the magnetic moment to precess about the H./dt = y(M. The Bloch Equations.2. when colt = nn. oscillating sinusoidally with radio frequency orf coo./dt = y(M.1 5 ) dM.6 1. including the Zeeman .H. 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. (1. . 1. the equations become dMJdt = y(M. is the derivative computed in the rotating frame. . i.3.h i s the magnetic field. In the rotating frame.2. while the counterclockwise component produces no net effect. (1. + 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 .Ho).. 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. NUCLEAR MAGNETIC RESONANCE where (d(p)/at). sin urft M . The above results can be generalized to an assembly of noninteracting or weakly interacting spins. = i.. The component rotating in the same sense as the precessing spin will therefore appear stationary with respect to the magnetic moment when orf wo. Magnetic Resonance.2.13) where n = 1. . Through interactions of the spins among themselves and with the surroundings. cos wrft). there must be an exchange of energy between the rf field and the nuclear spin as it precesses about H I .2. These equations. ( 1. (1. Such a linearly polarized H1 field can be dex composed into two circularly polarized fields rotating in opposite directions. H . This is the classical analogy to the quantummechanical transition that occurs between two nuclear spin states when I = $.rt . . For such a system the torque equation is dM/dt = yM x H.H.e.. HI cos u. do not take into consideration relaxation processes which attempt to return the magnetization vector to its magnitude and direction at thermal equilibrium.12) 1.e.2. Since the energy of (p) depends on its orientation u i s . however.2.M.14) When the effects of Ho and H1 on the components of M are explicitly spelled out. sin urft).4.Ho + M..3.5. axis at frequency w1 = yH. dM..2.
is slowly varied through the resonance condition while a radio frequency field H I is imposed.3. component involves an energy exchange. is the time constant for the return of the M . cos o. Phys. we shall have reason to return to them in subsequent sections. both the precessional frequencies of the spins as well as their lifetimes in the spin states can be affected. magnetic resonance is observed as the decay of the induced signal. 460 (1946). between the spins and the surroundings. These component equations of motion for the magnetization vector have proven to be extremely useful in the development of advanced experimental techniques..1.2. Bloch. Two types of relaxation processes may be distinguished corresponding to the decays of the M . dM. . denoted T2./dt dMJdt dM. while the decay of the M . component to its equilibrium value M. In the second method. ( M y ) components of the magnetization. 1.. The Bloch equations are also useful for describing the effects of molecular motions on electron spin resonance signals. Taking these relaxation processes into consideration one obtains the wellknown Bloch equations.Mo)/T. sin w. . (1. The spinspin or transverse relaxation time. powerful pulse of rf energy at the resonance frequency. etc. the spins can readjust their relative phases and redistribute themselves among the Zeeman levels of the system. . H . a frequency spectrum is obtained when the magnetic field H. for example.t) ( M . consequently. and explicit solutions are given in Part 2 of this treatise. After the pulse is turned off. called the freeinduction decay (FID)./dt = = = y ( M y H o + M .y ( M ./TZ. H .. Detection of Magnetic Resonance In the introduction to his classic paper on nuclear induction. denoted T. R w . Bloch noted that magnetic resonance could be observed in two ways. vibrational/rotational/translationaldegrees of freedom. This procedure is called the continuous wave (CW) method because of the requirement for an uninterrupted rf field.is the time constant for the complete decay of the net magnetization in the x y plane. .t) y ( M . Associated with each decay is a characteristic time constant.. f t .M.2. Both the FID and the frequency spectrum contain the same F.. The spinlattice or longitudinal relaxation time.Ho)  M. PHENOMENON OF MAGNETIC RESONANCE 7 field. thus./T2. It is important to note that these two types of relaxation processes are intrinsically different. H . and M y components does not. the relaxation of the M . In this manner.M.2.' In the first method. sin q f t + M y H . 70. cos O .. all the nuclei of interest are excited simultaneously by a short. and M . the electronic cloud.16)  .
If the sweep rate is slow enough.2. (1.. 93 (1966)." Thus.17) Initially the system is far from resonance. This enhancement is due to the fact that under appropriate experimental conditions. 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 fictitious field. to obtain a frequency spectrum experimentally it was necessary that a continuous H1 field be imposed. Ernst and W. tips toward the xy axis under the influence of the continuous H1 field. Anderson. since this information is more explicit when expressed in the frequency domain (i. for the same data acquisition time. Subsequent averaging of the accumulated data produces a marked improvement in the signaltonoise ratio.210. Prior to the development of FTNMR. Under these conditions. the spectrum). The spectrum is then obtained by varying either the Ho field strength (field sweep) or the rf frequency (frequency sweep). Norberg. as the resonance condition is approached. 37. . the FID (the time domain function) is obtained in a fraction of the time (10. the signal response from a population of resonating nuclei is consistent for each FID.. its theory is included in this section.e. using a mathematical operation. RCLI. Eq. A.E. Lvwe and R. it is more convenient. However. NUCLEAR MAGNETIC RESONANCE information characterizing the system under study. As a result. However. J . the effective field in the rotating frame is ( 1. the experiment can be repeated many more times. In order to take advantage of the more efficient FID signal. ContinuousWave NMR. this is done by measuring either the energy exchange lo R. RBZI. Since this method is the basis for many of the techniques that will be discussed subsequently. Ernst and Anderson introduced Fouriertransform (FT) NMR spectroscopy in 1966.8 1. necessary to acquire a frequency spectrum by the CW method. 46 (1957). cancel each each other [cf. o/y. Insirurn.1. R.10)] and He. On the other hand. Thus by averaging the data from N freeinduction decays. so that HeRz Ho.2. This gives FTNMR a distinct advantage over the CW method in that. Sci. while the signal due to the background noise will fluctuate randomly. Typically.3. 1. 107. Phys.2. 1. one can obtain the NMR frequency spectrum of a sample from its FID." 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. thus giving M a measurable component in the xy plane. This component is the resonance signal that is detected experimentally.3.and H. the introduction of techniques utilizing FTNMR to increase sensitivity has had a particularly significant impact on biological studies. the net magnetization will follow HeR.
2.' 1. (see Fig. the individual magnetic moments will begin to dephase freely.18) where A is the frequency range spanned by the nuclei under examination. its duration is of the order of Tf. a nuclear moment is magnetically screened by the electrons in a molecule. field for a pulse experiment is usually several orders of magnitude larger than it is in the C W method. is t . Pulse NMR.2. where the asterisk indicates that this T2 value includes contributions from field inhomogeneities and may be dominated by them. In order to accomplish this. is long enough to rotate M by 90" (n/2).3. However. the strongest signal will be observed when the duration of H.2.20) In practice.21) Immediately following a 90" pulse. Similar pulse methods have also been designed .1. This results in the decay of the induced voltage in the receiver coil (the FID). and the restriction in Eq. As is well known.2.is dictated by the range of nuclear frequencies to be observed. Hi % H. must have a magnitude such that yH. A is determined by the range of chemical shifts for a given type of nucleus. + a/y. Since the dephasing occurs in the xy plane. ' In order to obtain the intrinsic T2 values a number of multiplepulse techniques have been developed which eliminate the contribution from magnetic field inhomogeneity.2. (1. H. t.. i. (1.18) requires that t . In general.Instead M precesses about H. In general.2. 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. Inasmuch as the detector only measures the components of M in the xy plane. and T % Tf. and magnetic nuclei of the same kind exhibit different resonance frequencies depending upon the chemical environment. % 1/4A. (1.19) Accordingly. since the duration of the rf pulse is usually on the order of microseconds. 2). % 2 4 (1. The pulse method differs from CWNMR in that all the nuclei of interest are excited and observed simultaneously rather than individually. = n/2yH1. In addition. T.e.2. the pulse experiment is always done very close to resonance.* is quite short and we have both T.. (1.. The H. the rf field H .2. The length of this 90" pulse. Hefi s HI for the period in which H I is turned on. the magnetization vector cannot follow He#. This dispersion of the N M R frequencies according to chemical environment has been widely exploited for structural analysis and determination. because of field inhomogeneity effects and other instrinsic relaxation processes.
rotating at the Larmor frequency.1. (a) Net magnetization Maligned along the Zeeman axis in a rotating frame of reference. thus (1. the FID.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 (provided. is to tip Mtoward theyaxis.2. which allow one to accurately determine the longitudinal relaxation time. that the Zeeman interaction dominates). The latter is a measure of the frequency components that comprise the response function f(t). 1. FourierTransform NMR.givingthemagneticvectoracomponent alongboth thez and the y axes. Mathematically. (b) Net magnetization at resonance with rf field H.2. of . one can describe a time domain function as the Fourier transform of a frequency domain function F(o).4. It is these various frequency components that interfere constructively and destructively to give the decay function in the time domain. TI.2.We discuss these techniques in more detail in Section 1. These interactions cause the transition frequencies to be different for different resonant nuclei. In keeping with the properties of Fourier integrals these two functions are merely Fourier transforms of each other .3. Classically. arises from variations in local electrostatic and magnetic interactions. The decay of an NMR signal in the x y plane.22) and .1. The effect o f H .3. NUCLEAR MAGNETIC RESONANCE 2' X' / (a 1 (b) FIG. course. following a 90" pulse.
PHENOMENON OF MAGNETIC RESONANCE 11 Naturally.2. We Lorentzian with a full width at half height Ao. these two functions contain the same information about the nuclear spin system. we consider a simple spin system which exhibits an NMR transition at frequency o = o.3.1. (b) FID of the system depicted in (a).25) X I Time ) (b) FIG. (1. The numbered points on the FID correspond to the points in (a) marking positions of the net moment in the x y plane. we obtain the response function (1.2. shall assume that the resonance line is .wO)'].equal to 2/T2. (a) Path followed by the net magnetization in the xy plane after a 90" pulse./[l + T.24) Taking the Fourier transform of this. Inward spiraling is caused by transverse relaxation of the moments in the x y plane. so that F(w) = AT.. To illustrate this.2.(w . The trace is circular because the resonant nuclei precess at a frequency different from that of the rotating frame. . The recorded signal is induced in a phasesensitive receiver coil positioned t o sample the net magnetization along y.
From James.wo1. and the FID will appear to be quite complex. Freeinduction decay signals (lefthand side) and their Fourier transforms (righthand side) for (a) singleline. NUCLEAR MAGNETIC RESONANCE (C 1 FIG. 4. This is illustrated in Fig. (b) twoline. along with their respective Fourier transforms.4. where o # wo. the precesssing magnetization will describe a spiraling arc in the xy plane.except that the amplitude decays with a time constant of T.. The return of the magnetization to its equilibrium value along the Zeeman axis is described by the spinlattice or TI relaxation time. Since this component of the . 3b). Spin Relaxation Following an rf pulse one typically creates a nonequilibrium magnetization along the Zeeman axis and in the xy plane. In practice. of course. Note that from the periodicity of this decay and a knowledge of o we can calculate the frequency of the NMR transition w o .3. This important result can be graphically illustrated in two ways. Alternatively. is merely a sinusoidal function with frequency w o . but since its magnitude is concurrently decreasing exponentially because of the T2 relaxation processes. one is usually sampling more than one nuclear species exhibiting various transition frequencies. where the FIDs for three different samples are shown. The decay of each of these two components is described by a distinct characteristic time. Each of these will contribute to the induced signal. 3a we plot the magnetization of our simple spin system as it appears in a frame of reference rotating at w. and ( c )multiline spectra.' This result indicates that the FID signal.12 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. In Fig. following a 90" pulse. 1. 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. the magnetization will be precessing with frequency I o .In this frame of reference.
where we have a spin manifold of weakly interacting nuclei of I = 5. a nonequilibrium distribution of the spin population can only return to equilibrium through transitions between the two Zeeman levels. 1.1. the phases of the spins can be modified via energy exchange among the individual spins themselves. Although in T2 relaxation there is no energy exchange between the total spin system and the surroundings. In the simplest case. 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.3.1) where g(w) describes the distribution of frequencies in the field.2) Therefore spinlattice relaxation will be most efficient when the value of the distribution function at the Larmor frequency is at a maximum. which is distinct from the spinlattice relaxation time since only the phases are involved. .1. This nonequilibrium state decays to zero with a characteristic time T. 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. Such a condition obtains only when the stochastic modulation time of the fluctuating field is approximately (ao).This characteristic time constant is the welll. 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. Thus the efficiency of any relaxation mechanism depends upon the nature of the interaction and the frequency with which it fluctuates.. known correlation time zc.3. except that only those Fourier components which match the frequencies of the quantum mechanically allowed transitions will be effective.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. (1.3.3. it follows that 1/Tl = Is(wo)12. These fields act on the nucleus in a manner analogous to the applied H I field.3. Since energy exchange between spin and lattice systems can only occur in quantized units of the spacing hwo between the two spin 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.
. caused by transient variations in the frequency of precession for individual spins. The introduction of phase coherence by an appropriate rf pulse results in the creation of a nonzero xy magnetization in the rotating frame. occurs as the individual magnetic moments lose phase coherence by precessing at different frequencies. 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. . then the nonzero M x ymust decay within the limit of TI. The magnitude of this homogeneous contribution to T2 depends upon both the time scale and nature of these fluctuations. Correlation Time In the preceding discussion of spinlattice relaxation. if the spinlattice relaxation rate for the system is sufficiently fast. decay of M.3.. it is useful to distinguish two major contributions to this process. . SpinSpin Relaxation For an assembly of noninteracting spins the equilibrium value of the net magnetization in the xy plane is exactly equal to zero. 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). only local field components collinear with H .. It follows. such as in liquids and gases. 1.3) and the M. The value of z. motional changes in the position of the nucleus bear some relation to each other. we introduce the concept of a motional correlation time z. that the energy levels will be broadened by a width of the order of AV z 1/2Ti (1.2.14 1. the positional correspondence is lost and the motion appears to be random. Thus the magnitude .3. In this case the spin vectors precess about the field at the same average frequency but exhibit a statistical oscillation in their phase coherence. under this condition. In many cases. since there can be no phase coherence of the individual magnetic moments under these conditions. Of course. The length of this period may be considered the “memory time” of a molecule.. NUCLEAR MAGNETIC RESONANCE 1.However.3. For the socalled the inhomogeneous contribution to T. along the zaxis contribute to homogeneous T2processes.. analogous to the way local fields affect TI. In discussing the relaxation mechanisms responsible for T. this contribution to the dephasing will be included as part of the homogeneous T.which will decay to zero in a characteristic time T2.3. Dephasing of an otherwise homogeneous set of spin vectors can also occur due to random fluctuations in local electric and magnetic fields. M x y . At times longer than z. This means that for the interval zc. component will decay to zero according to this time constant.
say Em and E k . (a) The spectral density function J((..5) While the function G(z) tells us something about the rate at which a randomly fluctuating field loses its positional correspondence. will be a function of the size of the molecule. AS the temperature decreases. (1.. In order to illustrate this more quantitatively. This condition obtains = w o .. and the temperature of the sample... the similarity will be lost. its Fourier transform gives the spectral density function (1." . its value at time t. (TJ' 9 w. but for t + z 9 t. described by is Gmk(r) = (mIXl(t + z)I')('I*l(t)lm). (b) Proton spinlattice relaxation times for glycerine at different temperatures and two different frequencies.4) where Gmk(z) the autocorrelation function. the viscosity 1 increases. of a transition induced by XI between these states can be shown to be related to G(z) by (1. three different values of T ~ : ( T ~ ) .as shown graphically in Fig. SPIN RELAXATION 15 of z. we now consider the energy of a local fluctuating field.. differ will from that at Xl(t + T). (.3.5. For very small values of z. Since relxation will be most efficient when the frequencies of the fluctuating fields lie close to the Larmor frequency of the resonant nuclei.. (). described by the Hamiltonian operator Zl.3. the probability W.) FIG. X l ( t ) .6) which describes the probability that a fluctuating field will occur at a particular frequency. the viscosity of the solvent. (). thus T~ becomes longer.1.+ w. 5a where J(w) is plotted for when (zJ T.3.) . A measure of the correlation between X 1values as they vary with time is the autocorrelation function G(z). From Farrar and Becker. for ' ' = w.3.(sec) (5.. If this field fluctuates in a random manner. we expect S l ( t ) x Xl(t + z). TI will be at a minimum when J ( o 0 )is maximal. For a nuclear magnetic moment which can occupy two energy states.
16 1. z. J(w) increases for low frequencies. At high temperatures. Tl is relatively long and frequency independent. reaching a maximum at wo. multiple correlation times can contribute to the spinlattice relaxation. Purcell. D.679 (1948). The dependence of TI on z. but the . C. is short.) begins to decrease. " Academic Press. Similarly.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 . 5a remains constant !). accordingly. However. For convenience we assume that all motions are sufficiently rapid to average the various interactions to first order. the transition probability will be equally low for a wide spectrum of frequencies. Reo.8MHz. E. The TI minimum is shorter at 4.8MHz are equally low. Pound. such as those found in membrane systems. for motionally restricted molecules. with a rigid molecule undergoing axially symmetric anisotropic motion (where zll << zL) two minima may occur. first at the higher frequencies then at the lower ones. However. where the ~ proton spinlattice relaxation time for glycerine is plotted at two different frequencies as a function of temperature. 'This is shown in Fig. where thermal motion is rapid. V. although TI may exhibit an additional dependence on the average orientation of the molecule relative to the magnetic field. New York. "Pulse and Fourier Transform N M R . is short and the spectral densities of the random fields at either 29 or 4. it should be noted that for large macromolecular complexes exhibiting long correlation times T . transition probability decreases at higher frequencies. 5b are greater at 29 than at 4. Beckcr. responding to temporally distinct correlation times. 5b. At lower temperatures. the spinlattice relaxation rate becomes frequency dependent. For example. Phys. the increasing Tl values depicted on the right side of the curves in Fig. As zC increases so that (z.4. Farrar and E. M .3. Thus far we have assumed that molecules undergo isotropic Brownian rotational and translational diffusion in the liquid state. a plot of TI versus z may yield a surface exhibiting multiple minima. and J(o. For this reason. l 3 N.' Thus when z. ' 1. in the case where molecular reorientation or translational diffusion is anisotropic. 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. is well demonstrated by an experiment from the classic work of Bloembergen et ~ 1 . . 1971. and R . Fig. Bloembergen.)approaches the resonant frequencies. 7 increases further. NUCLEAR MAGNETIC RESONANCE three different correlation times. including wo. cor. However. when z is long. 73.
(1.g.8f) iI. 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 . 3 . Berlin and New York. + i~.3.)(S. (1.3.iI. 1. If r makes an angle 0 with respect to the field and 4 is the azimuthal angle.3.3.8) ZZSZ(l 3 cos2 O). 13C.3. Slichter.)(S. + iZ. . “Principles of Magnetic Resonance. For a twospin system consisting of p1 = yJhI and pz = yshS separated by a distance r.i~.1.) + ( I .  (1..iS.3 cosz O).”F) is through magnetic dipoledipole interactions with other nuclear magnetic moments.)]sin’ 0 e”+.3.3.)Iz](sin 8 cos 6 23.8a) B = $C(Z.” SpringerVerlag. .3.7) This dipolar Hamiltonian can be conveniently rewritten in a polar coordinate system involving the interspin vector r and the applied magnetic field. the energy of interaction between the spins is14 Z D= (pi * p z ) / r 3 . P.3. Magnetic DipoleDipole Interactions.3(v1 * r)(c~z r)/r5. ZD ( y r y s h 2 / r 3 ) ( A B = + where A = + C + D + E + F).8b) (1.8d) (1. ) ~ ~ ] ( sOi n 8 e’”). (1. 31P.8~) + iZZ)sz (s. + cos D = &Ix .1.)Sz + ( S . . O For this Hamiltonian. + is. C = $[(I. . E = . + i ~ . SPIN RELAXATION 17 the expressions used here to describe relaxation will not necessarily be valid. F = a[(Z. 1978.)(s.$ [ ( I .iZ.  (1.3.4.)]sin~ eZi+.)(S.iS. + iZ. The predominant relaxation mechanism for nuclei having spin number I = $ (e. ‘H.8e) (1.)](l .
for example.11) For intramolecular dipolar relaxation.3.3.14) From this result we may gain some insight into those factors that influence the intramolecular dipoledipole relaxation rate. of course.6 .2)]. as. Doddrell.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.3. Chem./(l 022. If we may assume that any rotational anisotropy in the molecular motions is small. For example. where r is fixed. NUCLEAR MAGNETIC RESONANCE . woz.2)]. (1. In any case. 189 (1971) .10)can be shown to be + 02z. t. (1.. when they are covalently bonded to each other.18 1.3. Frequently. In order to illustrate how these ideas may be exploited we have selected some results from the work of Allerhand and c o . when the extreme narrowing condition obtains. This is. and R . D.I('). the spectral density functions for Eqs. and 5") are the spectral density functions associated with the J"). that is.~ o r k e r s on the Tls for '~ the I3C nuclei of cholesteryl chloride dissolved in CCl. J")(o) = +$r6[zc/(1 + J'O)(w) = $$6[z. to be expected since y is proportional to the nuclear magnetic moment p. J'"(w) = &r6[z.9) and (1. whose magnitude determines the strength of the microscopic magnetic field acting at the resonant nucleus. (1. 55.2)].12) It is generally assumed that molecular reorientation in the liquid state satisfies the StokesEinstein relationship.. Phys.h2S(S + 1)TcP. since the spectral density functions are proportional to r . Komoroski. (1.3. J . 4 1. all the l5 A . = T~ = 4nr]a3/3kT. It can also be noted that the dipolar relaxation rate will be highly dependent upon the gyromagnetic ratios of the interacting nuclei. Tl = Tz and the intramolecular dipolar relaxation rates are given by @:)intra = (Ri)htra= $y:y.3 . (1.3./(l + w2t. a situation which is not always satisfied for large biological macromolecules. This is the socalled extreme narrowing condition. Allerhand. fluctuations in F'O' = (1  3 cos2 Q r . the dipoledipole interaction will be maximal when I and S are in closest proximity.
but if the extreme narrowing condition is applicable and the spins are of the same kind. which shows that carbons with 0.6 .3. which despite the three directly bonded protons is 1.1.3.49. (From Allerhand et a1. the number and the gyromagnetic ratio of the nuclei coupled magnetically to the carbon atoms and the respective internuclear distances.0 sec for these carbons. d = 2a.The Tls for these nuclei will then be a function of structural factors only. This expectation is clearly borne out in Fig.5 sec. Because of the low natural abundance of 13C (about 1%) and the strong r . in the case of the methyl carbons attached to the terminal end of the aliphatic tail segment.0... CI 02 . (1. we predict that Tl for the ring carbons should decrease with the number of protons bonded to them. Accordingly. respectively. (1.26sec. it can be shown that @:)inter = (A.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. namely. here the increased rotational freedom of the methyl rotor results in a faster effective z than t. 6. 13C spinlattice relaxation times (sec) for cholesteryl chloride in CCI.3. The situation is therefore somewhat more complex. However.15) where N is the density of spins per cubic centimeter. for intermolecular dipoledipole interactions the interspin distance r is also time dependent. Finally. This effect of z. and D is the translational diffusion coefficient.”) . and 2 protons have average Tl values of 3.6 dependence of the relaxation rate.4. on the relaxation rate is even more pronounced . in additional to 6 and 4. Within the Stokes formulation D and d are related by D = kT/6naq.1. d is the distance of closest approach. and 0. An apparent anomaly is the TI value for the ringbonded methyl carbons..)y14h2W + 1)N/dD. the only significant dipolar contribution to the 13C Tl should come from directly bonded protons.7 FIG. the Tl values are about 2. SPIN RELAXATION 19 carbons located in the rigid ring structure will have the same correlation time 7. in which case the r values will be similar as well.
Of course.0. where we show the ' j C spectrum of the choline methyl resonance from a solution of phosphatidylcholine dissolved in a mixed solvent of chloroform. It should be evident that X j can become time dependent when either one of the spins undergoes rapid nuclear relaxation. + 1. For this mechanism only a single correlation function is involved.2. and the two nuclei are spin coupled with J / h of 3.7 Hz. ( (01 (1.3.3. Since I = 1 for 14N.J. l6 R. The spin Hamiltonian that describes this interaction between two spins is given by Xj = I * J *S. Baumann.1j 2 1 J  + (0. describing the probability that S and I are still coupled at t + T. + (xe/{l + . corresponding to the three possible orientations of the 14N spin in a magnetic field. and water (50:40: 15)./(l _ and R 2 . SpinSpin Coupling.3. Soc.~ s ) ~ t f ) > ] S+SI).16 Here. mI = .20) where re is the chemical exchange time constant. This effect is called spinspin or scalar coupling and can lead to fine structure in the resonance signals. . In addition to the direct dipolar coupling of nuclear magnetic moments.3. spectral splitting vanishes and the timedependent scalar coupling affords another mechanism for relaxation.4. 13Cis chemically bonded to 14N._:J2cT. NUCLEAR MAGNETIC RESONANCE 1. 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.19) (1. . (1. In solution. Chrm. An example of how spinspin coupling manifests itself in the frequency spectrum is illustrated in Fig. If the spin coupling interaction fluctuates in time it can also act as a mechanism for relaxation.ws)2z:>]s(s + 1) CT. 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. when this condition does not obtain. 103. Murari and W.the effect of this coupling is to split the I3C resonance into three lines.1. methanol.  (1. 1238 (1981). Assuming that I is always bound to some S spin.3. Am.17) where J is the tensor describing the bilinear coupling of nuclei I and S through interactions with their electrons.18) where J is the wellknown scalar spinspin coupling constant. there can also be indirect interactions between nuclei which are mediated via the bonding electrons. namely. one can show that the relaxation rates for I are RI1 . J.20 1. 7. or if the coupling between I and S is modulated by rapid chemical exchange for one of the spins. only the trace of J can be measured and the spin Hamiltonian simplifies to X J = J(1 S).
~s)'(Ts)~}].65 ppm). For spinlattice relaxation.3. where stationary fields also can contribute. sn1 CH. (1..P (64.0 . e. Insert is an expansion of the N(CH.(35 )S(S 2 2 + J J ~ S (+ S l)eiosr e71Tf.16 Copyright @ 1981 American Chemical Society.45 ppm).4.94 ppm). and in fact only those components of the S magnetization not aligned with the Zeeman field will be important.3.26ppm). (1. only the timedependent components of the spinspin interaction having frequency wI will contribute to T i . Therefore R'..).R. (1. Reprinted with permission from Murari and Baumann.02 ppm).g.24) . and (b) D 2 0 .3. sn3 C H 2 . = J(')(w1) ~J'"'(O). CH2N (66.1.22) (1. 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. (63.21) where J(')(w)is the Fourier spectrum of the autocorrelation function G(~)(T) The result is R I1 . 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.7. Protondecoupled 20MHz 13C NMR spectra of egg yolk phosphatidylcholine in (a) CDCl3/CD3OD/D2O(50: 50: 15).3) is predominant for the second nucleus.O. 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 / ~ ~ . and sn2 CH0R2 (70. it is necessary to include a second autocorrelation function involving the z components of S. POCH2 (59.79 ppm).3.3.Peak assignments in (a) are given for: N(CH3)3 (54.23) + l)[TS/{l + + (uI . quadrupolar (see Section 1.)3 peak.3. For the transverse relaxation rate. This may occur when a different relaxation mechanism.
(1. M.3. M. Lipids 25.15 and 0. This coupling is usually referred to as the electric quadrupole interaction. The Hamiltonian operator for this problem is given by + &?a= 1 .3.29) (1. D. 7 (1979). while in chloroform. A. depends upon the aggregation state of the phospholipids in solution.0006 sec. London. Behr and J .1)][(3I: " J. NUCLEAR MAGNETIC RESONANCE where J(O)(w)is the Fourier transform of the correlation function G(O)(~) = [&Ps(s + ~ ) ] e . (wI (1. I573 (1972). R. 49.I . E.25) (1. c ) of Q or V. Wilson. Matwiyoff.1)]V. a medium where phospholipids do not aggregate. (1. . by Q = [eQ/21(21 ..9 . The interaction of this asymmetric nuclear charge distribution with the electron cloud can partially lift the 21 1 spatial degeneracy of the nuclear spin. the corresponding values are 0. even in the absence of an external magnetic field. 1 . ) ] . we get X Q= [eQ/41(21 . methanol. b.3. the I4N splitting in the choline methyl resonance of the I3C spectrum are collapsed. 7). The spinlattice and spinspin relaxation rates for the I4N nucleus in phosphatidylcholine are controlled by the rotational rate of the CH2N which.)] P. Biochem. . in turn.4.3. V. and water (50: 50: 15). Expanding X a in the principal axis system (a.3. Lehn.06 and 0. Chem. Scalar relaxation of the second type can be illustrated from the phospholipid studies of Murari and Baumann.26) Thus the expression for the transverse relaxation rate is Ri = [+J'S(S + l)][T:/{l + . the 14N T. (1. Quadrupolar interactions. Atomic nuclei having I 2 1 possess an electric quadrupole moment because the charge on the nucleus is distributed asymmetrically.30) which may be simplified to X'.Z2) + (KO V.Zz) .' / ~ ? . respectively.3. T.3.3.27) where Q is the quadrupole energy tensor and is related to the electric field gradient tensor at the nucleus. and N. Biophys. Res.I. = [e2qQ/4Z(2Z .I6 which we cited earlier in this section (Fig.28) where e is the electron charge and Q the electric quadrupole moment of the nucleus.)(Z: . Commun. In accordance with this expectation. Concomittant with the increased relaxation rates of the I4N in the liposomes.1)][Kc(31: .22 1.177 sec. and T2 values for phosphatidylcholine liposomes in D 2 0 are 0. Walker. Phys.W ~ ) ' ( T $+ ~ } ) T:]. 1. Is + q(I. E.
33 respectively.32) where u is the anisotropic chemical shielding tensor. cause mixing of the nuclear spin states.3.31) 1. If the rotational reorientation of the molecule is sufficiently fast. the contribution of electric quadrupolar interaction to the relaxation rates can be shown to be R. For solid samples this orientational dependence gives rise to the wellknown “chemical shift anisotrop y. reorientation of the electron cloud uisuuis the magnetic field via rotational tumbling of the molecule will “confuse” the nucleus.‘H.4.4. This screening effect arises from the electronic currents induced in the molecule by H. Similarly. cos’ 8. the larger Q is.the effective magnetic shielding oz along H.4 x lO”/V m2. In biological systems. In terms of the principal values of u. in units of e x cm2. For the 35Clanion..016. Obviously. such that ooz. the most important spin relaxation pathway for quadrupolar nuclei. 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. Since the electronic environment is usually asymmetric about the nucleus. the most frequently studied quadrupolar nuclei are 14N.0. Q for ’H. in fact. the electric field gradient.. This mechanism is. however. ~ * (1. the effective field seen by a nucleus will differ from H.” With magnetic shielding.Q)27 . 14N. = eq and q = (KO &b)/l/cc* The importance of the electric quadrupole interaction is determined by the magnitudes of Q and q. and 79Br are 0.and 35Cl.H. When a quadrupolar nucleus is placed in a magnetic field. and promote nuclear spin relaxation. the greater the nuclear charge and the degree to which this charge distribution deviates from spherical symmetry. the electric field gradient has a value of 38. broaden their energy levels. exhibits the following angular dependence: oz = coo sin’ 8 cos2 4 obb sin2 8 sin’ 4 of. (1. and 0. SPIN RELAXATION 23 by taking advantage of Tr V = 0 and introducing V.3. In the presence of an external magnetic field.(l  u).=R 3 (21 ’ . by a small amount due to magnetic screening by the surrounding electron cloud. as in CH3Cl.33) + + .1. Chemical Shift Anisotropy.3. measures the deviation of the electron charge distribution from spherical symmetry at the nucleus..40 Z’(21+3)1) (l +  y) (e2. the magnitude of the shielding will depend on the orientation of the molecule relative to the external field.3. For example. Generally.3. q = 0. g 1. the Zeeman Hamiltonian is modified to XZ=p.0028. when the chlorine is covalently bound. q. (1. 23Na.
Phys. R w . that is. . where Aa = a l l . B. 25. The characterization of a molecule undergoing anisotropic motion requires at least two rotational correlation times. however. Anisotropic Motions. and 31Pnuclei in a number of c o m p o ~ n d s . Holm. J . i. the approximation of isotropic motion is often a gross oversimplification. 1289 (1956) H. M. In less rigid systems. a part of the molecule may also undergo segmental motion at rates quite distinct from those motions of the whole system. crZ is time dependent because of the rapid reorientation of the molecule. McConnell and C. More likely.e.5. Cliem. it can be seen that rotation about the long axis will be much more rapid than molecular rotations perpendicular to this axis. Although we do not treat anisotropic motion here. and Rz = (1. With the advent of superconducting solenoid NMR spectrometers capable of NMR observations at magnetic fields greater than 10T. We have assumed in our discussion of relaxation mechanisms that the molecular motions that modulate the various interactions are rapid and isotropic. Phys. "F.24 1.JH~(AB)~T. Observations of spectral width and T1dependence on H i having been reported for I3C. Gutowsky and D. 104. For example.1. In studies of biological systems. Effects of Anisotropic and Restricted Motions 1.34) (1. H. it is important to emphasize that. in the interpretation of relaxation data. S. In solution.. this modulation of the shielding field can contribute to spinlattice and transverse relaxation..35) &.uL. motions which take the molecule over all directions in space with equal probability.3. anisotropic shielding effects are likely to become more prevalent. and the condition of extreme narrowing holds. 1. the relaxation rates for this mechanism are R1 = & y 2 H i ( A ~ ) 2 ~ . NUCLEAR MAGNETIC RESONANCE where 0 and 4 define the polar and azimuthal angles of Ho relative to the principal axes of u. Woessner. ' indicating that anisotropic shielding can indeed contribute ~*~~ to nuclear relaxation in some systems..3.3. if we consider the motions of a rodshaped molecule in solution. treatments based on single correlation times are l9 2o H. but with quite different rates of reorientation about the various molecular axes.3.. E. If we assume for sake of simplicity that u is axially symmetric. = a l land aaa= obb= al.5. The inclusion of such motional details can have a profound effect on the NMR relaxation times. we expect that molecules will undergo anisotropic motions. 843 (1956).
Pulse Techniques 1. where motional restrictions prevent the averaging of certain spin interactions. EXPERIMENTAL METHODS 25 not applicable for systems where the molecules are undergoing anisotropic motions. T. calls for an initial pulse which rotates the magnetization vector M 180" from its position along the Zeeman axis (see Fig. Measurement of T. TI can be determined by . Some of these methods were chosen because they are of general utility. it cannot be directly measured from the FID. rotating M by 90" and thereby placing it in the xy plane. TI. Since TI characterizes the time required for the M . component to return to equilibrium..5. Such motional restrictions lead to firstorder dispersions in the NMR spectrum due to chemical shift anisotropy and incompletely averaged dipolar and quadrupolar interactions. will begin to relax back toward equilibrium along +z. While these effects are not difficult to treat. If.4. we present in Section 1. These techniques hold great promise for studies of complex biological phenomena...4. However.3 an example to outline some features of the NMR of motionally restricted systems.1.1. The initial height of this signal is a measure of the magnetization along the z axis at time z after the first pulse. after time T a second pulse is applied. One such procedure.5.4. utilizing a twopulse sequence. M . 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.1. Following this pulse..2. By repeating this sequence for different values of . and T. 1. macromolecular assemblies and arrays and exhibit a high degree of order. often referred to as the 180"t90" method. 8a). In studying complex biological systems one is often interested in molecules that are found in large.1. 1. 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. a signal will be induced in the receiver coil. 1. which reflects only the decay of M X yHowever.4. such as those involving cell membranes. In addition some recent advances in solidstate NMR have been included. Experimental Methods In this section a number of experimental techniques are described. however. while others have proven especially useful in studies of biological systems. space limitations preclude the discussion of these details in the present chapter. Restricted Motions.3.
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.. . 94. M is rotated onto the x y plane. Following a 90" pulse. rotating M . returns to equilibrium.).M&TI (1. Rrz. may be determined by plotting In(Ab. This procedure assumes that the line shape of the resonance is Lorentzian. to M . 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." This method employs a 90".( M . = M. If the height of the ith signal in the partially relaxed spectrum is A:. Measurement of spinlattice relaxation by the 180°~90" method: (a) H I is applied along x'. (1. Purcell.4.3) and its spinlattice relaxation rate l/Ti. at t = 0 gives the following expression for the magnetization recovery: M .nT. t. a condition not normally attained in highresolution NMR. = .'' t.2) For a heterogeneous spin system containing a number of distinct populations of resonant nuclei. . Integrating d M .180" pulse sequence and is illustrated in Fig. After a period t there will be a loss in the phase coherence of the magnetic moments..1) with the initial condition M .4. NUCLEAR MAGNETIC RESONANCE FIG.M . wherein T2 is related to the linewidth at half height by Av = l/(.26 1. one obtains a measure of the rate at which M . H I is applied long enough to rotate the net magnetization along y'. but if at this time a 180" pulse is applied. . (1.8a. 630 (1954). then ln(AL  Af) = ln(2AL) . 8b. inhomogeneous T.(1  2er'"'1). T. / d t = .Z I T .A:) versus t. Y . Phys.4. Carr and E.z . processes will '' H. The transverse relaxation time T2 is often measured directly from the linewidth of a resonance signal. M . and that the linewidth contribution from magnetic field inhomogeneity is not significant. The intrinsic T2 is best determined by the CarrPurcell spinecho techniques. . (b) after a time T.
. However.. However. It is evident that the amplitude of the induced signal decays exponentially with a time constant equal to half the homogeneous T2. the peak heights in the frequency spectrum may be measured after Fourier transformation of the FID at 22.y ' . Although T2 measurements do permit one to sample slower motions. He. Thus by appropriate choice of HI. inhomogeneity across the sample volume. the maximum signal induced in the receiver coil at this time will have decayed by e . H.8b. the efficiency of relaxation will depend upon the amplitude of local fields fluctuating at y H . Spinecho method in the rotating frame: (a) 90" pulse H I along x' rotates M . at resonance and the spins become locked to H.12 bring the moments back in phase after another period 2. this is rarely done because of the difficulties in obtaining a properly phased spectrum. rather than yH. Tl. (b) magnetic field inhomogeneity causes nuclei to lose phase for a time T.4..y'. the sensitivity of the method does not extend beyond times slower than about sec. Measurement of Diffusion Coefficients..y ' half of the x'y' plane. measurements provide a convenient parameter to sample motion in biological systems. When the spins are locked by H.1.4. Although T. The diffusion of molecules can introduce significant errors in the measurements of T2 because of H. However. there exists a third relaxation time TIP (referred to as T. and applying a second 180" pulse to refocus the spins and obtain nuclear induction at the receiver coil. in the laboratory frame.1. very much like the alignment of M along H. However. EXPERIMENTAL METHODS 27 FIG.relatively slow molecular motions can be sampled. TlPis measured by switching the phase of H I following a 90" pulse so that it becomes aligned with M x . The cycle can be repeated by allowing the spins to dephase for another time 2. processes.2 r i T z because of homogeneous T. occurring near 1081010 sec. to the y' axis. in the rotating frame) which allows one to sample motions as slow as lo4 per second. .2. (c) 180" pulse along x' rotates dephasing magnetization into the . 1.For systems having more than one spin species. under the spinlocked condition yields TI. they are limited in that spinlattice relaxation is only effected by relatively rapid motions. The decay of M. (f) dephasing of spin echo. (e) maximum magnetization along . in the rotating frame. under appropriate .. particularly from complex systems with coupled spins. (d) nuclei rephasing towards ...
58 (1973) . Decay of the proton signals from a dimcthyl sulfoxideH.= M o e ( . 9a).A + ~ Data Acquisition FIG.3.. James and G .4. NUCLEAR MAGNETIC RESONANCE Field Gradient . MultiplePulse Methods.22 1. Mot. b . 9b).O ( 1 : 1) solution as a function of the echo development time T shown in (a). 9b. Fourier transformation of the resulting FID allows one to calculate the diffusion coefficient from any spectral peak that can be resolved (see Fig.4) The selfdiffusion D can be measured by comparing M(2r) determined in the presence and absence of G at various values of 2.1." conditions one can take advantage of this effect and use it to measure the molecular diffusion coefficient D (cm2/sec). the spinecho amplitude at 2 2 ." 0 10 0 200 T 0 100 200 (rnsec) Fic.2 r / T 2 ) k 2 / 3 7 2 G 2 D r 3 (1. M q n . The angular dependence of dipolar interactions is of no consequence for molecules undergoing rapid. McDonald. is given by M ( 2 r .4. L. Spectra were obtained in the presence [(b) and (d)] and absence [(a)and (c)] ofa pulsed field gradient. Echo development occurs in thc prcscnce of a gradicnt which is then turned off to sample the echo decay envelope.28 1. R m m . 11. J .. G .9a. A significant improvement in this method can be realized by utilizing a pulsed gradient imposed on the sample following each rf pulse (see Fig. For a sample in the presence of magnetic field gradient G . random reorientation since the dipolar interactions are averaged to zero by such '' T. Pulse scqucncc and data acquisition pcriod for a pulsed gradient experiment.
it is evident that XD be averaged to zero both in real and can spin space. 16. Lrtr. W.7) where the subscripts denote the axes along which the pulses are applied. Phys. 20. Phys. . Phys. However.^^. However. and 20.900.90Yx . Waugh. Lett. Prog. EXPERIMENTAL METHODS 29 motions.z6 and Vaughan.22 .5) From this result.S. The purpose of the WHH fourpulse cycle is to averageout XD through an appropriate choice of pulses so that (31. y . we get (1. 180(1968). U. z for durations of 22 each. Waugh. D. N M R : Basic Princ. Suppl. Inasmuch as this pulse sequence over the period 62 of the cycle aligns the magnetization along all three axes x. 1096 (1966). Haeberlen. Reson. M q n .4. Chem. L. 23 24 *’ ’’ .. R w . this averaging does not occur and the signal is broadened. this effect masks many of the weaker spin interactions. Rewriting the simplified Hamiltonian. these sequences permit unmasking of the weaker interaction^. S.^^ To illustrate this. 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 . 22. then P. Annu.4. 391 (1978).Unfortunately. Huber.2 . Mehring. (1.2. M. Mansfield and D. D. and Haeberlen (WHH).E .6) This averaging in spin space may be accomplished by imposing the rf sequence 90: . Vaughan. this led Waugh and coworkers to formulate a number of multiplepulse sequences that allow for the coherent averaging of dipolar effects in solids.90. Reti. we can disregard the terms C . E. R E D .4.I ’ S) = 0.1. Unfortunately. there ~~*~~ was a concomitant loss in the chemical shift and spin coupling effects. Huber.8) since they serve only to induce relatively weak absorptions at frequencies O. Lett.3.and sixteenpulse sequences. and F in Eq. in systems where this condition does not obtain. S. 11 (1976). Haeberlen.22 . it is of great interest to have a method which allows them to be observed.4. we will briefly discuss the initial fourpulse sequence developed by Waugh.27 although better averaging is now possible by utilizing eight. (1. J. ** R. Ware. Ado. and U. 26 M. (1. 29. such as chemical shifts and spinspin couplings. I (1976). . Since these parameters hold important information concerning molecular structure. which also contribute to the spectrum. Ostroff and J. Phys. . Details about these further developments may be found in HaeberleqZ5 Mehring. such as solids and membranes.28 For dipolar broadening in a twospin system consisting of I = S = 3. 133 (1966).
10. (1.4. Resolution of the ”F rnultiplet from polycrystalline C. In this section we describe the technique of magicangle spinning. Clearly. the magnetization is sampled once every cycle at a 6t interval. to minimize dephasing effects t must be chosen to be much smaller than T 2 .30 1.6) is satisfied. for the timedependent .the timeaveraged dipolar contribution to this system is (1. In a typical multiplepulse experiment. 1.8) where 2 is the timeaveraged value and %(t) is the timedependent Hamiltonian having a mean value of zero.2.4.4. We illustrate in Fig.4. it was shown how dipolar broadening could be removed by coherent averaging of the spin operators in spin space. Similarly. (1.9) 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. 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). MagicAngle Spinning In the preceding discussion. NUCLEAR MAGNETIC RESONANCE ~ 0 .F. and the averaged spectrum is subsequently obtained by Fourier transformation of the accumulated decay signals. which averages the lattice operators in real space..FI2 by application of a WHH fourpulse sequence.z6 I. . . 10 the striking effect of coherent averaging in spin space on the I9F spectrum of polycrystalline C.& = +I * S and Eq. If the axis of sample rotation forms angle p with H. GAUSS5 C652 200K 2000 1000 0 1000 2000 3000 400C dHz) FIG..
1 0.10) the value will average out to zero when the frequency of sample rotation o. Magn. 203. EXPERIMENTAL METHODS 1/r 31 (kHZ) 10 0 01 . in “ N M R and Biochemistry” (S. I 1 0 1000 10. the condition or l/zc > A > E. then it can be shown3’ that. (1.01 FIG. Prog. Dekker. resulting in the loss of information about these tensors. New York. pij is the angle between internuclear vector rij and the axis of sample rotation. and lo’ sec. J . p. If the frequency of such motions is on the order of l/zc. In both expressions.1 . Effect of magicangle spinning rate v . Reprinted from W a ~ g h . Inc.~ 4ij) + + sin’ fl sin’ pij cos 2(o. Thus. Waugh. Reson. The major reason is probably that biological systems always display some motion. 1 (1971). R. 1 1 . under appropriate conditions of sample spinning. 4 .t+ 4ij)]. one can effectively remove dipolar (as well as quadrupolar) interactions from the spectrum of a solid. S.000 v I N 73 3 0. magicangle spinning experiments result in highresolution spectra such as those observed in liquids and solution^. J. Opella and P. Nucl. 1979. is greater than the linewidth A of the rigid lattice. eds. dipolar term x [sin 2p sin 2pij COS(O. by courtesy of Marcel p. 8. in order to obtain further narrowing by sample spinning. Lu. Dekker. on linewidths from samples having correlation sec. Rapid spinning also removes the chemical shift anisotropy.'^ At the present time. Andrew. 2y 30 .4. magicangle spinning has not received the attention it deserves in NMR studies of biological systems. ~ ’ 207. times of co. Accordingly.).
32 1. Double Resonance Numerous methods have been devised to take advantage of the coupling interactions (dipolar and scalar) that exist between nuclei. 1. Doubleresonance techniques employ a second rf field which acts on one of the coupled nuclei while the other is being sampled.87 HOD L . This effect is illustrated in Fig. 25. Proton N M R spectrum of a sample containing 33 wt. change the spinstate distribution for one type of spin by 3' D. Doskocilova. respectively. 12). a significant reduction in linewidths may be observed when samples are spun at the magic angle (see Fig. z A. E. "/. 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. for systems displaying l/z. Schneider.4.3. 11.3 HMD5 MDS (a 1 (b 1 6. 6 ~ assigned to the fatty acid and are terminal methyl group protons and the choline methyl group protons. Chapman. . I I I I J I I I 600 400 200 0 0 200 400 600 I I c/s c/s FIG. Under appropriate conditions these methods can remove the spinspin splitting between scalar coupled nuclei (spin decoupling). D. Of course.O at 25" and rotating at 500 Hz about an axis perpendicular (a) or at the magic angle (b) to the external field H . . 261 (1972). egg yolk lecithin in D. 3 1 The resolved peaks in (b) at 9 .12. NUCLEAR MAGNETIC RESONANCE must be met. HMDS is an cxtcrnal reference. FEBS Lett. Oldfield. such as a phospholipid multilayer. and B. l ~ 6 .
However the linewidths Av of these carbons are all slightly broadened due to the strong J coupling ( 2 120 Hz). at the resonance frequency of the coupled nucleus such that y H . however.4. off resonance from the proton frequency. Spin Decoupling. 1. R. Saturation Transfer. The multiplet is observed only because the rate at which transitions occur in the coupled nucleus causing the splitting is slow. The usefulness of spin decoupling is apparent. 13b. The effect of this incomplete proton decoupling is shown in Fig. In addition. If. . 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. Childers.3. which experience only weak longrange coupling (I Hz). and the spinspin splitting is collapsed. Here the I3C spectrum for the aromatic region of tryptophan is complicated by interference between signals from protonbonded and nonprotonated carbon atoms. and simultaneously sample the species of interest.. we now observe only a single peak at the center of the multiplet of the undecoupled spectrum. we impose a second rf field H.4. The technique of double resonance can be taken to great advantage when two spins are dipole coupled. 15 are easily resolved and unambiguously assigned. and E. Oldfield. the magnetization transfer experiment employs a second field which is strong enough to equalize the spin populations (saturate) of the irradiated species. Unlike spin decoupling. F. facilitating spectral assignments. 13. spin decoupling may be used to simplify complex spectra. 1. 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.1.3. Since Av oc J 2 / H i .2. and increase the signal intensity of a dilute spin species by transfer of polarization from an abundant species (cross polarization). An interesting example is presented in Fig. for example by setting H . Since the area under the spectral peaks is proportional to the number of nuclei. This is because the dipolar interaction provides a mechanism for transferring magnetization from one spin species to the other.1. In this section we briefly describe these techniques and illustrate their usefulness. Since the 32 A .32 The peaks from the protonbonded carbons all appear as singlets due to proton decoupling. $ nJ. where a relatively weak rf field is used to excite one of the scalarcoupled nuclei. Under these conditions the peaks arising from nonprotonated carbon atoms. EXPERIMENTAL METHODS 33 equalizing the spinstate populations of a coupled species (saturation transfer or nuclear Overhauser effects. Allerhand. Biochemistry 12.4. 1335 (1973). the collapse of spinspin splitting can result in considerable signal enhancement. any protonbonded carbon resonance can be effectively broadened by reducing H.
brought about by the equalized populations.and lowenergy spin orientations.3683 (1972). will cause a redistribution in the Zeeman populations of the nonirradiated species.wo)(<sz) SO). Chem. as modulated by the dominant spin interactions. Glushko.( z. V. called the nuclear Overhauser effect (NOE). Also shown are the transition probabilities K.4. which couple the spin states to the lattice degrees of freedom. "The Nuclear Overhauser Effect. (b) Offresonance protondecoupled spectrum of tryptophan. 1971. . Under these conditions. and A.34 1. 3 4 D. Allerhand. Phys. the equation of motion for the observed I spin magnetization ( I . respectively. Noggle and R.'^ For the sake of discussion we shall consider a simple twospin system consisting of I = S = +. Doddrell. This effect. where a and /?denote the high. w. H. NUCLEAR MAGNETIC RESONANCE I I I I I I 55 I 60 65 70 75 00 05 COOH J 90 PPM FROM CS2 . Schirmer. 56. w (1. the resulting increase in the relaxation rate of the saturated spins.13. Zeeman states of the two nuclei are coupled by dipolar interactions. The coupled Zeeman levels are shown in Fig." Academic Press. 'I' I ' FIG. New York.11) 3 3 J. (a) Fully protondecoupled "C N M R spectrum of the aromatic resonances of tryptophan in aqueous solution. is manifested by a change in the intensity of the NMR signal from the unsaturated spins when the latter are amp led. 14. J . Reprinted with permission from Allerhandet Copyright @ 1973 American Chemical Society. ) is described by34 d(J. E.)/dt = (wo + 2 1 + wz)((Iz) 10).
14. and So are the equilibrium values for ( I . because of the dependence of f r ( S ) on the relaxation mechanism. (1.Zo(1 + fr(S))].wo)/(wo+ 2Wir + wz)l~s/~r.4. so that ( S . Overhauser effects have been used to sort out the relative relaxation contributions in several welldefined ~ysterns. (1. In fact. 52. However. EXPERIMENTAL METHODS 35 FIG.3.1. with subscripts 0. D. Thus for dipolar relaxation of small molecules undergoing rapid motions.) . M. K .4.12) The effect of saturation is therefore to change the steadystate value of ( I . the NOE is also influenced by the onset of nuclear spin relaxation mechanisms other than dipoledipole interactions. the zero quantum transition probability will also be important and the NuE modified accordingly.3439 (1970) . % Wo and the fractional enhancement reduces to ys/2y. Chem. For nuclei exhibiting a significant scalar contribution to the relaxation rate. J . ) and (&).~ 35 K.1. ) = 0. Energy level diagram for coupled spins I = S = i. F. W. ) from its value at thermal equilibrium by the factor 1 + fr(S).Transition probabilities between fi (high and low) energy levels are denoted W. a and I .4. If we now impose a saturating rf field at the S spin frequency. From these relationships.13) For I = S = f the transition probabilities are where K = hysy. Eq. and J("(o) are the spectral density functions discussed in Section 1. where fr(S) = C(wz .. and R. or 2 representing the quantum of energy absorbed or emitted by I or S or both. Phys.4. it can be seen that the net NOE is determined not only by the relative signs and magnitudes of the nuclear gyromagnetic ratios. + W2)[(I.4. Harris. 1. (1. Grant. but also by the relative probabilities of the various relaxation pathways.11) becomes d(Iz)/dt = (wo + 2w1. Kuhlmann.
Rrv. 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. 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 . where (w. Cross Polarization of Dilute Spins. Their data. BothnerBy. Chmi. Dadok. Hahn. For this reason. J . is given by fi(S) = ( 5 + w'TZ.4015 (1972). 128.1. 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. Am.4017 (1972) . obtained by monitoring the aromatic protons of the peptides SMeCysPheIleNH. but for long correlation times. 1. 94. The usefulness of this approach is illustrated by the work of Balaram et a1. f.'*P. (1. A.36 1. BothnerBy.3. and E.(S) = 0. Balaram.. Hartmann and E. These spins are then brought into contact with the rare S spin system by imposing a second field H at the S frequency.3. Breslow. A. Phys. Soc. Balaram. 94.4w42:)/(10 + 2 3 ~ ' ~+. .4w42:). A. For a system of two dipolarcoupled protons.(S) = . R .w. L. A . Bioclzemistry 12.4. Chern. A m . A. BotherBy. J .4695 (1973). 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. and J.. Based on preliminary studies showing a differential broadening of certain aromatic protons in position 2 or a tripeptide hormone analog.)'zf < 1.4. 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.G 1) results in f. Under these conditions both spin magnetizations may T P. .5. Rreslow. and E.15) which for rapid motions ( w ." . 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. over W. P. ~ . these studies usually take advantage of the fact that spin interactions are distance dependent. Balaram. Soc.3638 who used proton NMR to characterize the hormone binding site on the protein neurophysin 11. A. 3h 3' .show a decrease for the resonance intensities of the 2 . '"S. any measurable NOE can then be taken to infer that two nuclei are in reasonably close proximity. the dependence of the fractional enhancement on z. 3 . The physical basis for the negative Overhauser effect associated with slow motions is the predominance of W.2042 (1962). In this experiment the abundant spins are irradiated with an rf field of amplitude H i at the Larmor frequency of I .
569 ( 1 973). the observation of a dilute spin signal still presents difficulties for solid samples. Waugh. S. the z components. of course. the interactions of the xy components of the precessing spin magnetizations are negligible.fields so that one can effectively match the Zeeman splittings of the two nuclei along their respective HI fields. for studies of labile systems or transient effects. will have the same frequency. since yIH. In an effort to mitigate this problem a technique based on the aforementioned HartmannHahn experiment was introduced in 1973 by Pines. (1. Although the advent of FTNMR and signal averaging techniques have alleviated many of the difficulties associated with investigations of rare spins. such as 13C(1. 4n A . Chem. as described in Eq. and W a ~ g h . This allows energy exchange to occur through the z components of the dipolar interaction. precessing about the imposed HI fields with frequencies Qr = Y~H: (1. the abundant I spins are In polarized and brought into contact with the S spin system. # ysH. This exchange. where dipolar interactions are not averaged out.98 %) to enhance the signal from relatively rare spins. as previously described.16) and sZs = ys H s . . in this case. the use of lengthy signal averaging is impractical.1. only results in a small decrease in the I magnetization. it is the rare S spins that are sampled directly. However. J . Early applications of the technique utilized isotopically abundant protons (99. EXPERIMENTAL METHODS 37 be considered as vectors in the xy planes of their respective rotating frames. and J. (1.1%). 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. there will be a net transfer of magnetic polarization from the abundant I spins to the rare S spin system. Similarly.4. Phys. 59.4. Gibby. fields in the rotating frame.3. Pines. In the laboratory frame. However. M . however. As the two spin systems reach internal equilibrium.8a). thus allowing one to sample the rare S spins indirectly. Repetition of this cycle ultimately results in a measurable decrease in the I magnetization.4. which are generated as the spins precess about their respective H. Gibby.17) By adjusting the amplitudes of the H. ~ ' this experiment.
as in the HartmannHahn experiment. S.. " As a result. the energy conserved from the longitudinal relaxation (in the rotating frame) of the I spins causes an increase in the S spin magnetizaY tion along H in the rotating frame. this method is commonly known as protonenhanced nuclear induction spectroscopy.1 I *I T 2 i " i '72' 3 i ~ i 1 N. The two systems are next brought into contact so that energy can be exchanged via dipolar cross relaxation.. rN 1 I * time t I .~' 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... in Fig. Grifin. 17... The rare spins are then sampled by turning H off and recording the FID while continuously irradiating the Y abundant I spins in order to decouple the remaining dipolar interaction. Pulse sequence for a cross polarization experiment in which the magnetization from the abundant spins (M.I S Spins .. Biochmiistrj. G. This is done. 41 R. The FID of Msis sampled during the periods denoled by T ~ . denoted t . During this period. FIG. L. A recent application of this technique included measurement of the angular dependence of the * P chemical shift for oriented phospholipid bilayer~. the accumulated FIDs are averaged and transformed to yield a highresolution spectrum of the rare S spins. Only in this way was it possible to define the chemical shift tensor in relation to the plane of the membrane. (1. One version of the protonenhanced nuclear induction spectroscopy experiment is shown in Fig.4. 15. and P. Finally.. 15. 15. NUCLEAR MAGNETIC RESONANCE I 1 . This cycle is then repeated until the rotating frame magnetization of the I spin system becomes depleted. the I magnetization is locked along Hi so that it decays in a time Ti. Following a 90" pulse.18) are satisfied.2718 (1978) . Powers. by imposing Hf such that the conditions of Eq. Pcrshan.38 1.) is transferred to the rare spins (M\).
64. we consider the case in which a complex spectrum is resolved by effectively spreading it in two dimensions. R. Ernst.4. 88. Ernst.4. Jresolved spectrum. W. J. C % m . Jresolved spectroscopy43344). as we have noted. For the sake of illustration. based on the spinspin coupling and chemical shift (a technique referred to as 2D. Phys. In its simplest form. Jresolved spectrum. Bartholdi. twodimensional (2D) NMR. Most of the techniques that have been developed to simplify complex spectra do so by suppressing some of these effects.1. P. 31. 16.4. the basis for it can be outlined. E. each direction displaying the effect of a single variable (e. Wuthrich.4226 (1976). Chern. P. 90" 180" I FIG. J . E. Phys. R. P. L. 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.4' a new method which holds great promise for unraveling the complex spectra of biological macromolecules. 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. If the coupling constants J are small relative to the chemical shifts between coupled nuclei.g. Rev. shown in Fig. Aue. Aue. Rrson. However.2229 (1976).43 is simply the spinecho method repeated at different values of t l . 42).133 (1978). K.. and R. Pulse sequence for obtaining a 2D.45This is due to the fact that dephasing effects from 42 43 44 4s W. . Another approach is to disperse the signal along different dimensions in frequency space. Karhan. 64. thus much important information is lost. then the spinecho amplitude will be a function of J. where t l is the echo development time. Hahn and D. Maxwell. The experimental scheme for obtaining a 2D. Ernst. R . Phys. Mugn. K. spectra are obtained by Fourier transformation of a signal possessing more than one independent time variable. Bachman. 16. E. and R. and R. 1070 (1952). EXPERIMENTAL METHODS 39 1. J . either time or frequency. Although the mathematical development of this method is beyond the scope of this chapter (see Ref. J . spinspin coupling). This is the rationale for the development of multidimensional NMR spectroscopy. chemical shift. Nagdyama.
4. The decaying echo signal is then sampled for time t .O / 0.8 0. Jresolved spectrum of the same sample with the individual multiplets displayed along the J axis and the chemically shifted.19) This data matrix can then be Fourier transformed in two dimensioils to give a corresponding matrix in frequency space.8 / / 0.20) where 0jk = nj + v j k and = 2n 1 Jj. (a) Standard proton NMR spectrum of bovine pancreatic trypsin inhibitor in the highfield region. displaying the unresolved multiplets arising from 19 of the 20 methyl groups present. Repetition of this experiment using different values for the parameter t . (1. 0) cos(vjkt1 .Ojk)21112 vjk (1. results in a data matrix of the transverse magnetization components as a function of t l and t 2 . projected in trace ( c ) .1 j 2 2 > + (O2 .4. (1. m{k.4. . NUCLEAR MAGNETIC RESONANCE / 1.0 2 ) = lMjk(O.v j k ) 1. The contribution each resonance line k in the multiplet of nucleus j makes to the matrix is given by Mjk(t1.2 1A 1.40 1. Thus by changing the spinecho development time one can obtain a measure of the spinspin couplings in the absence of chemical shifts.17.6 / 1.which is a running variable of constant length. ~ ~ chemical shifts and magnetic field inhomogeneity are reversed following the 180" pulse.19)can be shown to be IS(jk(W1. (b) 2D. 0)[1/T.Wjkt2)er1'T2Jk12'TtJk. .4 / / 6( p p w FIG.jk C1ITT?k + (01 . The contribution arising from the resonance described in Eq. t z ) = Mjk(0. decoupled peaks along 6.
425 (1979). Biochem. 86. Nagayama. 47 D. Bachman. J .1. Res. For many years biological chemists have worked at unraveling the molecular mechanisms responsible for enzyme catalysis.5. Proteins: Probing the Active Site of an Enzyme Practically every chemical reaction occurring within a cell is catalyzed by an enzyme. Hoult and P. P. where the proton spectrum of bovine pancreatic trypsin inhibitor is shown. 1. Cornmun. Lauterbur. I. It is hoped. R. R. Thus by displaying S ( o . ~ ~ This type of information can be used for the unambiguous assignment of resonances from similar groups located in different parts of the macromolecule. The effectiveness of this technique in resolving complex spectra is illustrated in Fig.5. Reson.5. 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. 218 ( 1 979). . 17. SELECTED STUDIES ON BIOLOGICAL SYSTEMS 41 ojis the Larmor frequency of j . 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. In this case the onedimensional spectrum contains overlapping spin multiplets from nineteen chemically shifted methyl resonances. and K. many excellent and important examples must necessarily be omitted in this limited treatise. Ernst. J j . Mayn. r~ 1. Wiithrich. allowing investigators to directly observe the effects of spin decoupling on each r e ~ o n a n c e . 34. However. In these efforts much information has been derived from the investigation of enzyme reaction rates and substrate binding constants. We have also decided to forgo any discussion of NMR zeugmatography since NMR imaging studies are highly technical and require specialized equipment. and mlk are the magnetic quantum numbers of the coupled nucleus 1. Unfortunately. C . is the spin coupling constant. in two dimensions the data are well resolved. while the spectrum projected along o1shows the spin coupling multiplets. More recent studies employing both xray crystallography and NMR have also allowed the definition 46 K. Biophys. 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~the literature cited therein. . w 2 ) along the o2axis one obtains the chemical shift information.1. however.
Smallcombe. Chem. value. of the aspartyl residue is unusually high (6. the NMR approach has an additional advantage in that it can be done under physiologicalconditions. Isotopic Labeling. Two schemes have been proposed for the involvement of this triad in the proteolytic cleavage (Fig. In addition to their similar functions. W. the pK. containing the same triad of amino acid residues (aspartic acid. and serine) in close proximity. ~ ' ~ ~ ~ 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. NUCLEAR MAGNETIC RESONANCE of the activesite residues of enzymes and the orientation of the bound substrates. Biochemistry W. H. Of course. Hunkapiller. In addition. 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. the reader should consult the biological NMR texts cited in Chapter 1. In some cases. specific enrichment allows for greater sensitivity. because this enzyme has only one histidine residue. H.8041 (1978). D.1. the chargerelay mechanism depicted in Scheme I is possible only if the pK. Am. D.42 1. 18a). a host of other NMR techniques have been successfullyapplied to the study of proteins. For discussions of these methods. if Scheme I1 is operative. histidine. 100.4~ Since 13C and "N are normally present in low isotopic abundance (1.1.9. W. The first technique allows one to characterize the protein residues at the active site. the enrichment provides an unambiguous assignment of the reasonances arising from this residue. 4732 (1973). 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. and J . Richards. 12. This approach has been used to investigate alytic protease isolated from cultures of M y x o b ~ c t e r . for the histidyl group should be normal. Roberts. 6. these proteases share a common type of active site.365 %. On the other hand. respectively). R. Whitaker. The mechanism of action of Elytic protease is of interest because it is representative of a ubiquitous group of enzymes called serine proteases.11 % and 0. S.5. 1. 49 . both of these problems can be obviated by incorporating labeled precursor molecules into a biosynthetic system which produces the macromolecule to be studied. While both xray crystallography and NMR provide detailed information at the molecular level. Soc.9) and then only if the histidyl residue has an unusually low pK. J . Bachovchin and J. In the elegant work of Bachovchin and these investigators exploited the sensitivity of the I5N chemical shift of the imidazole 15N 48 M. with enzymes and substrates remaining in solution.1.9). In the following section we describe two approaches for using NMR to study enzyme mechanisms. Based on the pH dependence of the enzymatic activity (maximum at 6.
99. SOC. be present at the active site where the substrate binds. J . Paramagnetic Probes.1. Chem.__ *1=Ir) 1 6 8 I 1 0 I PH ( 0 ) fb) FIG. Chem. Z. Blomberg.5. 37. (a) Proposed mechanisms for the catalytic activity of the serine protease family of enzymes.for' paramagnetic ~ systems. SELECTED STUDIES ON BIOLOGICAL SYSTEMS 43 SCHEME I FI k t \ \ \ \ \ \ SCHEME Ii \ \ \ \ tetrohedral \ \ '\ mlermediale 140 4 I .2.1. Connick. Maurer. 40.2686 (1964). See text for details.5. thus disproving the popular chargerelay mechanism. 18b. 1. 7. 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. The longitudinal relaxation rate is primarily determined by five parameters: (i) the ratio of the concentration of paramagnetic species to ligand ( f ) . as well as the extensive application of NMR relaxation t h e ~ r y ~ ' . Ruterjans. Phys. . The results of these 15N experiments.(ii) the binding stoichiometry or coordination number of the electronnuclear complex (4). indicate that the histidyl residue does have a pK. from either a paramagnetic ion or a spin label. . (b) Effect o f p H on the I5Nchemical shifts of "Nenriched histidine nitrogens in alytic protease: 0 .8149 (1977). (iii) the  50 51 52 F. Luz and S . and (b) isotropic nuclear hyperfine coupling of the unpaired electron at the nucleus under consideration. Am. Two interactions can contribute to the nuclear spin relaxation : (a) throughspace magnetic dipolar interaction between the nuclear spin and electron spin . J. respectively. and A. 307 (1962). J. Chem.18. The approach outlined in this section requires that an unpaired electron. Phys. and H . W. I5Nenriched at N3. Reprinted with permission from Bachovchin and Roberts. J.enriched at N I and N3. shown in Fig. E. Meiboom. Swift and R. T.49 Copyright @ 1978 American Chemical Society. resonances to their state of p r o t ~ n a t i o nto ~ ~ examine the pH titration behavior of this residue.
(iv) the correlation time modulating the dipolar interaction between the nuclear and electron spins (T. 19a.Note that the ratio of TIP at two frequencies is a function of z. provided that the dipolar mechanism dominates the relaxation. This procedure may be repeated for several substrate nuclei. In a typical experiment. or the experiments can be done at temperatures where this condition is fulfilled. 322 (1978). Nucleic Acids: The Dynamic Structure of tRNA Transfer RNAs (tRNAs). K.5. and z.1) Here S is the electron spin.2) Of course. frequently TIM9 z M . The general structure of tRNA is illustrated in Fig. is usually large enough so that ~ f z : 9 1 and the contribution from modulation of scalar nuclear hyperfine interaction in Eq. however. unlike proteins. 1.). and in this manner the orientation of the substrate relative to the paramagnetic probe at the active site of the enzyme can be ascertained.5.2.5. Mrthuds Enzyrnul. z. . Mildvan and R. g is the electronic “g” factor. assuming that t. one determines l/Tlp. NUCLEAR MAGNETIC RESONANCE lifetime of binding at the active site (z. is the effective correlation time for the scalar interaction. ’’ A. Gupta. all display a similar structure since they all perform an identical function in the process of translating messenger RNA. can be obtained.5. A is the nuclear hyperfine coupling constant. (1. is probably best determined by ~~ measuring T IPat two or more f r e q ~ e n c i e s . only. In this case the longitudinal relaxation rate has been shown to be (1. The value of z. Instead.1) may be neglected. For an enzymebound paramagnetic center. z M must still be known.44 1. p is the Bohr magneton.). The nuclear spin relaxation is then dominated by magnetic dipolar interaction between the nuclear spin and the paramagnetic center. the paramagnetic contribution to the longitudinal relaxation rate. 44G. (1. S. and (v) the distance between the paramagnetic center and the nucleus (r). the distance between the nuclear spin and the paramagnetic center. one does not measure TIM directly.. 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). The dipolar contribution to TIMprovides a means of determining r.
. 19.. I I I .) The numbered assignments for the cloverleaf (secondary) base pairs correspond from Reid et to the diagram (inset). Kim. Am. 100.(a) i LCIOP h i m (b) I SlEhl I _ > CND ACCEPTOR STEM so5. Inc. Copyright @ 1978 by Scientific American. . . while the resonances from the tertiary base pairs are marked with asterisks. Inc. by courtesy of Marcel Dekker. (b) 360MHz proton NMR spectrum of yeast tRNAPhe (Reprinted p. A11 rights reserved.I3 12 I1 10 9 PPM FIG. ." C. (a) Schematic representation of the threedimensional structure of tRNA. .D &*a A I) " LOOP 1 7 M ANTICODON STEM . 52 (1978). " em \ 9RIABLE I . ! 15 14 . Sci. H. . Reprinted from A. 238. . . 511 . Rich and S. .
the rate may be determined from deuterium exchange studies. D.46 1. 3599 (1977). 1979. and R. Initial studies have utilized a technique called saturation recovery to measure these hydrogenbond proton exchange rates. Woodward and B. Dekker. Rev. For hydrogenbonded protons which exchange very slowly compared with TI. Rcid.55In this method. the ‘H NMR spectrum provides a fingerprint for the secondary structure of the tRNA. Biophys. that the rate of exchange is greater than the intrinsic longitudinal relaxation rate of the resonance monitored. Lu. The rate of this recovery measures the rate of replacement of the hydrogenbonded proton by the unsaturated water proton. provided. In addition. G. p. C. R. ’‘ ‘’ . the hydrogenbonded protons of a particular kind are spectrally dispersed according to base proximity relationships determined by the sequence. s 5 P. Accordingly. Azhderisn. New York. These hydrogen bonds resonate at very low fields in the ‘H NMR spectrum (. The signal intensity is then measured from the frequency spectrum taken after the saturating field has been turned off for various time intervals z. Johnston and A. Bincng. K . 9 1 .15 ppm from DSS). D. Hilton. E. although features characteristic of the tertiary folding are often discerned54 (see Fig.99 (1979).” (S. Redfield. Generally these studies have confirmed the cloverleaf model for the secondary structure of tRNAs. 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. a saturating rf field is imposed at a specific frequency corresponding to the resonance of the specific hydrogen bond to be sampled. 19b). The cross rungs represent hydrogen bonds between complementary base pairs. Annu. E. N u c l ~ i cAcids Rcs. 4. in “ N M R and Biochemistry. However. Hurd. . The assignment of the individual hydrogenbond resonances in the lowfield spectrum has received considerable attention in recent years. since the nucleic acid bases consist of unsaturated ring molecules which can induce ringcurrent shifts in adjacent spins. 8. following the decrease in signal intensity when D 2 0 is substituted for H 2 0 in the medium. J. of course. eds. 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. Opella and P.11 to .). Some important insights into the tertiary folding of these molecules in solution have also emerged. there have been attempts to measure the rates of proton exchange between tRNA hydrogen bonds at specific sites and the solvent.56 B. the expotential recovery of the saturated signal can be followed. By varying z.
720 (1972).~ define the orientation of the major and minor axes of the electric field gradient tensor relative to the external field. The ’H NMR spectrum of a specifically deuterated ” S.I) = yhHorn. and q is near zero. )175. are given by E(. particularly the effects which lipids might exert on the proteins and vice versa. The results for the case of 2H ( I = 1) are shown in Fig. that is. D . The current dogma of membrane structure is the fluid mosaic model. The resultant NMR spectrum can be seen to be a doublet displaced symmetrically about the Zeeman frequency by v . (1. They serve as highly specialized barriers.2 x [(3 cos2 e . 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.” This model raises many questions about the role of lipids in cell structure. when the Zeeman interaction dominates over the quadrupolar interaction. e2qQ/his typically 170 kHz. L. Of these. not only between the cytoplasm and the extracellular medium.vo = k$(e2qQ/h)[(3 cos2 8 .5. 1  ZZ ( + l)] (1.5.4) For a CD bond in an alkyl chain. but also in compartmentalizing many activities within the cell (e.g. J.1) + ‘ sin2 e cos 241. Singer and G . + [e2qQ/8Z(2Z . there has been considerable interest in the structure and motional state of phospholipids in bilayer membranes. in order to provide benchmark information on this system.3. 20.1. the electric field gradient is essentially axially symmetric about the CD bond. C .5. To answer some of these questions.1) + q sin2 0 cos 241. . with proteins intercalated into a lipid bilayer matrix. Nicholson. Membranes: The Restricted Motions of Lipid Chains The important role played by cell membranes in controlling biological functions is firmly established. The energy levels of the combined Zeemanquadrupolar Hamiltonian. the most informative has been deuterium NMR studies on phospholipids with perdeuterated or specifically deuterated methyl and methylene groups. Science (Washington. A number of NMR techniques have been applied to probe the orientational order and the molecular mobility of the phospholipid molecules in bilayers.3) where the angles 8 and r#.1)][3rn.. SELECTED STUDIES ON BIOLOGICAL SYSTEMS 47 1. nuclear and lysosomal membranes) while providing a highly specialized surface to promote certain biological reactions.5.
longchain alcohol (1. . (1. SpringerVerlag.+) \___= El= yhHo+$ e2qQx(B. Seelig. Biophys. and the ’H quadrupolar splitting may be rewritten as A V Q = $((e’qQ/h)sc~(3 COS2 8’ . __c I_____ . but it is significantly smaller than the value of 170 kHz expected for motionless acyl chains. p.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.20.I).3). Niederberger. Biochem. 96. 5 9 Note that one frequently deals with unoriented multilayers in membrane studies and only a powder spectrum is observed. 21b.l= yhHo + $e2qQX(B.58A quadrupolar splitting is indeed observed.2069 (1974). (1. SOC.$) . The ’ NMR spectrum of a powder sample of dipalmitoyl lecithin H specifically deuterated at carbon 5 in both acyl chains is shown in Fig. 57. the chain motion takes a molecular axis symmetrically about a director perpendicular to the bilayer surface. From “Membrane Spectroscopy” (E. ed. 21a. The origin of the reduced splitting is.406 (1974). . of course. .. Chem. In a phospholipid bilayer.5.ldideuterooctanol) intercalated into hydrated sodium octanoate and oriented at various angles with respect to the external magnetic field is shown in Fig. E.). (a) Nuclear energy levels of an I = 1 nucleus in a strong magnetic field. +I E~=  $&ox(e.I.5.+) Zeeman + Quadrupolor YO FIG. 59 J. 12.\ Q. function ~ ( 04) is defined in Eq.48 1. The angular . Res. Grell. Seelig and W. The effect of motional averaging on the electric quadrupole interaction is usually expressed in terms of an order parameter. Heidelberg. Commun. J . Am. Seelig and A . (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. The relative contributions to the splitting from the nuclear Zeeman effect and the nuclear quadrupolar interactions are not drawn to scale. motional averaging. SCDshave been measured for methylene segments located at different ’* J. NUCLEAR MAGNETIC RESONANCE ml (a) .
1. as depicted in Fig. Stockton. P.8 MHz) of specifically deuterated fatty alcohol intercalated into hydrated sodium octanoate and oriented at various angles with respect to the magnetic field. C. 954 (1976).5.5. Q. 1. (b) 'H NMR spectrum (13. Reu. FIG.8 MHz) of a powder sample of dipalmitoyllecithin specifically deuterated at carbon atom 5 in both chains. J . Biochemistry 15. From these data the motional state of the phospholipid bilayer membrane can be quantified and expressed in terms of the flexibility profile. Smith. 96. investigators have G. (a) 'H NMR spectra (13. 6 1 J. C. Reprinted with permission from Seelig and Niederberger. Temperature 60°C.21. The interested reader is referred to a review by Seelig6' and to the Specialist Periodical Reports on NMR published by the Chemical Society. 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. Soc. The angle 0' is between the magnetic field and the bilayer normal. 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. 353 (1977). 2069 (1974). P.59) positions along the hydrocarbon chains of a phospholipid bilayer. SELECTED STUDIES ON BIOLOGICAL SYSTEMS 49 . 10KHz . and I.4. Biophys. Chern. Seelig. 22. Tullock. W. .60 'H NMR is now widely used in biophysical studies of biological membranes. F. F. Am. (From Seelig and Seelig. 10. Polnaszek. Hasan. A. Copyright 1974 American Chemical Society.
Data taken from Stockton et a1. even when the data are successfully gathered. Both of these studies have used NMR to characterize the metabolism of living cells.50 1. Such work involves not only the immense technical challenge of maintaining the sample under conditions compatible with life. 31PNMR. 31Pis an abundant nucleus (natural abundance of loo%). many problems. However. has . but they illustrate the diverse range of problems the technique is capable of tackling. 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.4. We view the outlook for exciting breakthroughs and giant strides in this field with some degree of optimism. Quadrupolar splittings from specifically deuterated steatic acid in egg yolk lecithin dispersions plotted as a function of the hydrocarbon chain position. but also presents interpretational hurdles. and tissues and organs not of interest."" persevered to the point where today living tissues and whole animals are finding their way into the spectroscopists' magnetic field. if only slowly. interference from resonant nuclei located in organelles. being overcome.5. such as those involving magnetic field inhomogeneity caused by air bubbles.22. are gradually. 1.1. NUCLEAR MAGNETIC RESONANCE 35 r 30 t 0 0 250 20 QVQ (kHz1 1 5 0 0 0 0 0 0 Carbon Number FIG. In this section we briefly review two recently published studies in this area.
and D. R. Richards. we cite the recent studies of Dawson and c o . (d) Decline in the isometric force development of the stimulated muscle during the time period of the experiment. J . a spin of ). SELECTED STUDIES ON BIOLOGICAL SYSTEMS 51 FIG. Gadian.703 (1977) h4 R . G.These investigators ~~*~~ were interested in delineating the chemical changes occurring in frog muscle as it fatigues due to repeated contractions under anaerobic conditions. (London) 267. R. Moon and J. together with the fact that metabolically important molecules bearing 31P are usually small. H. stimulated for 1 sec every 60 sec and recorded at the time intervals indicated. To illustrate the utility of 31P NMR for monitoring metabolic changes. Wilkie. p. D. In addition. Chrm. Dawson. (a)(c) "P NMR spectra from anaerobic frog gastrocnemius muscles. These properties. B i d . Gadian. 862. . G . J . with short correlation times and concomitantly long relaxation times.1. render 3 1 P NMR an ideal tool for the study of cell metabolism. Nature (London) 274. Copyright @ 1978 Macmillan Journals Limited. Wilkie. From Dawson e t a / . and exhibits a large chemical shift range. Dawson. D. J.~ o r k e r s . 861 (1978). 7276 (1973). Physiol. In b2 '' M . 6 4Reprinted by permission from Nature 274. 8.23. and D. J. 248.5. inorganic phosphate displays a characteristic pHdependent "P chemical shift62which can be used to simultaneously determined pH changes within the cell medium during metabolic studies. M.
6096 (1979). 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. U. T. 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. phosphocreatine (PCr). Because of the isotopic enrichment they were able to obtain wellresolved spectra at oneminute intervals. S. using [l”C] glucose and [6’3C] glucose as substrates. K . Acknowledgment This work was supported by USPHS NIH Grant GM22432 and American Cancer Society Grant PF1628.1. based on their quantitative relationships to the directly measured metabolite^. Brown. and the production of the fermentation end products ethanol and glycerol. NUCLEAR MAGNETIC RESONANCE their pioneering experiment. free ADP. . Although the NMR experiments permitted only the direct measurement of ATP. 3CLabeled Substrates. this method can be useful in following the fate of a labeled substrate molecule as it is metabolized within a cell. 1. they suspended the muscles in a specially designed chamber which allowed them to perfuse. Some of their data are reproduced in Fig. 6 5 J.2. stimulate. and determine the strength of muscle contraction. A . and R.5. Thus by exploiting NMR as a noninvasive probe one can investigate reaction kinetics and the stereospecificity of enzymes in their natural environment.52 1.1 we noted the great advantages of enriching an isotopically rare nucleus at a welldefined position in a protein.5.4. while simultaneously using 31PNMR to record the level of various metabolities as well as the cellular pH. Proc. In addition. R. 23. 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. and lactic acid. Similarly.^^ Taken together these results established an unambiguous correlation between muscle fatigue and the energy state of the cells.6bisphosphate. G . Shulman. This approach may also prove fruitful in unraveling the origins of various metabolic diseases. den Hollander. Ugurbil. Natl. Acad. the production and consumption of the metabolic intermediate fructose 1. In Section 1. Sci. A. it was also possible to infer indirectly the concentration of creatine. 76. This approach has recently been utilized by den Hollander et aL6’ to investigate anaerobic glycolysis in yeast cells.
Boundaries are no longer convenient if they are allowed to become barriers to creative science. there is no room for esoteric science. specialization and generalization . it is vital that information be communicated in a manner which is palatable to biologists. The recent course of science has been remarkable not only for its tremendous growth.the former being concerned with how things differ from one another and the latter with how they are the same.2 spinlabel theory is presented in a simplified form with the aid of copious diagrams rather than mathematical equations. They aspire to the eclectic and are especially dependent upon the lines of communication between themselves and the specialists in areas of their interest. is essential in 53 METHODS OF EXPERIMENTAL PHYSICS.2. as do spin labels. with a view to discovering selection rules which control immune responses to membranes. at two levels. we have taken the unusual step of presenting a physical method having great realized and unrealized potential in biophysical applications. which is a review of recent work in the McConnell laboratory concerned with using spin labels as antigenic determinants. We hope that Chapter 2. In Chapter 2. This latter phenomenon should not be surprising because many such boundaries are not natural but exist as a convenience to scientists and librarians. and chemists alike. 20 Copyright 0 1982 by Academic Press. McConnell 2. Those who engage in highly interdisciplinary science are usually searching for unifying principles rather than specifics. Inc All rights o f reproduction in any form rrsrrved. This method has been used many times by Humphries to present spinlabel theory to medical and biological scientists.1. physicists. ISBN 0124759629 . 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.4. Humphries and Harden M. VOL.2 will enable any scientist to follow Chapter 2. If biophysical chemistry is to be effective and intellectually stimulating. but also for the increasing difficulty with which we construct boundaries between “different ” scientific disciplines. NITROXIDE SPIN LABELS By Gillian M. Introduction Systematic natural science has advanced by means of two interdependent processes. but has not been published previously. K. Guided by this belief.
Examples are shown below. Seelig.2. J.” Oxford Univ.’’’ However. The choice of this topic allows us to illustrate the main features of electron paramagnetic resonance (EPR) spectra of nitroxide spin labels. A. p. EPR enables us to reach conclusions regarding the distribution. Shelnitz. R. G. Waggoner. 2.1. SpinLabel Theory: A Descriptive Treatment 2. eds. ed. R. A. 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.(3. either with respect to work from this laboratory or from others. New York. Dwek. Theory and Applications. M.6. “Spin Labeling Methods in Molecular Biology. Gaffney. P..” Academic Press.4dimethyl2. orientation. Cliem. B. Grifith. 267 (1972). Jost. We also hope that Chapter 2. In brief. “Biological Applications of Electron Spin Resonance. H3CQH3 H3C 0 2.” Wiley. 161 (1974).. 1. 1976.Carboxypropyl)4. New York. H.Mol.). 1976. and refer the reader to other recent monographs or reviews which accomplish that purpose. A.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. Rothfield. Bolton. S. Reuben. C. Cohn and J . trans. 32B. and dielectric environment of spinlabel antigenic determinants.).2. Such a molecule is host to an unpaired electron: it is a stable free radical and therefore paramagnetic. rate of motion.54 2. M. J . “Nuclear Magnetic Resonance (N. 418 (1977). our discussion will touch upon certain of the most common uses of spin labels. 214 (1971). J . C. Berliner. Swartz. Borg. and D. ’ ’ .) in Biochemistry. H. M.2. I (1972). Dwek. ed.tridecyl3 oxazolidinyloxyl L. 1973. Methods Enzymol.” New York. Res. Berliner. L. R. Academic Press. Press. Biomembranes 3.6Tetramethylpiperidine. 4. in “Structure and Function of Biological Membranes”(L. We do not endeavor to provide an encyclopedic review of the use of spin labels. 83. and 0. 1971. Likhtenstein (P. 49G. J. J . R. NITROXIDE SPIN LABELS any study designed to test this proposition. F. 1. Arc. 1972. Adz$.1 oxyl I CH3 (TEMPO) 2. New York. Methods Enrymol.3. “Spin Labeling. London and New York. Relaxation Processes 4.
which is predominantly associated with the nitrogen atom. occupying a 2p n orbital. The conventional assignment of z and x axes is given. If the carbon atoms adjacent to the nitrogen atom are not tertiary.OL . disproportionation takes place as shown below. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 55 Such molecules are stable in the p H range 3.?. t h e y axis is normal to the page.1. . 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. 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. it is partly governed by the y 3 CN?.2. also indicated in the diagram. Diagrammatic representation of the nitroxide group in a typical spinlabel molecule. This is an oversimplification. in fact. This leaves a single unpaired electron.to )( 0 I FIG. Similarly. 1 and legend). with the oxygen atom. This sharing has a concomitant effect on the extent of charge separation (also indicated by the structural representation). it is also significantly associated. chemical reduction by appropriate agents such as ascorbate or glutathione destroys the paramagnetic properties of spin labels as shown below. but to a lesser extent.10. so generating the charge separation indicated in the diagram.2. 0 OH I The products are no longer spin labels because they do not contain unpaired electrons. The figure is drawn to indicate that the unpaired electron is solely associated with the nitrogen atom.
2. they can be thought of as little bar magnets. for a sample of spinlabel molecules at equilibrium. (So long as the EPR spectrometer is adjusted correctly. 2).2. The unpaired electrons exist in one of two possible “spin states. the rate at which +fstate electrons revert to 4 electrons by “spinlattice + 4 4 ++ FIG. 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. This is the condition of resonance and is the essential step for generation of EPR spectra of the type which we consider here. there will be a population difference. on the energy level E of the permitted orbitals of unpaired electrons having spin states S of +f and +.3. (b) Diagrammatic representation of resonance: absorption A of microwaves of frequency v as a function of field strength H .4. NITROXIDE SPIN LABELS dielectric properties of the environment and is an important factor leading to certain spectral changes which we discuss in Section 2. The energy levels of these two spin states are not identical in the presence of an applied magnetic field ( 4 being higher than 4). it may absorb it and assume the state. unpaired electrons in the ++state being slightly in the minority with respect to those in the state. If an electron is not paired to another of equal and opposite spin in the same orbital. This is covered in detail in Section 2.) In a manner analogous to the induction of a magnetic field by an electrical current passing through a coiled wire.” designated +$ and 9. with the microwave power set low enough. spinning electrons are each associated with their own magnetic moment. 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. (a) Effect of external magnetic field strength H .56 2. (The population difference at equilibrium is controlled by the temperature and the difference between the energy levels ofthe two spin states. The energy of a quantum of electromagnetic radiation is given by its frequency v multiplied by Planck’s constant h. .2. it demonstrates appreciable interaction with any magnetic field to which it is subjected. As a result. by unpaired electrons not influenced by nuclear hyperfine splitting.
9.2.2.2.) The principal features of an EPR spectrometer. If the spacially fixed magnetic field is sufficientlystrong. a machine which is designed to detect the resonance condition.” This is covered in detail in Section 2. are illustrated in Fig. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 57 relaxation” is fast enough that the system does not “saturate. 3.3. .3. The axes are the same as in Fig. 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.
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.. 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 .I ) on the energy level E of permitted orbitals of unpdired electrons (spin states S = ++. the strength of the spacially fixed magnetic field is varied at around 3000 G. Typically. This is experimentally convenient because the radiation is supplied as standing waves in the “cavity. 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.e. 0. The reason why nitroxide spinlabel spectra are interesting is that the unpaired electrons are strongly influenced by the nitrogen nuclei. +) (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 . and the wavelength of electromagnetic radiation (supplied by a klystron) is kept constant at approximately 3 cm (i. which would vary little in shape or position with changes in motion. for the spinlabel work. .58 2. orientation. In summary. If the only relevant facts were those which have already been presented. 2b). distribution. NITROXIDE SPIN LABELS electromagnetic radiation of a convenient wavelength. in the “microwave” range). or dielectric environment of the spin labels (see Fig. 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. we would expect a field versus absorption spectrum showing a single narrow absorption peak or “line”: a very boring bump.” a carefully engineered structure suspended between the poles of the magnet with dimensions appropriate to the wavelength.
4b). 4. versus H . (as shown in Fig. 0. Therefore. absorption spectra are easier to understand theoretically.. and the minimum ' .e. is parallel to x and when it is parallel to y.2. Effect of Orientation Figure 1 illustrates the disposition of the 2p n orbital of the nitrogen atom. is parallel to the z axis of the nitroxide group and minimal when it is perpendicular to the z axis.2. 2. (as shown in Fig. 4c).2.0. the N. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 59 spin states (+ 1. Because of the anisotropic disposition of the unpaired electron (i. This phenomenon is an example of nuclear hyperfine splitting. corresponding to the + 1. and we therefore continue to use them as models in the following discussion. EPR spectra are usually not recorded as microwave absorbtion A versus magnetic field strength H . the maximum value for the hyperfine splitting between adjacent peaks is known as IT. there is a small difference between the splittings when H . with which the unpaired electron is predominantly associated.l). The effect on the permitted energy levels of the electrons is illustrated by Fig.. in the p orbital) its interaction with the large external applied magnetic field H .0. onethird of all the unpaired electrons in a spinlabel sample are associated with nitrogen nuclei in each of the three spin states. If the orientation of such a crystal in the cavity of the EPR spectrometer is rearranged in many different ways. but usually it is sufficient to consider an average of the . 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. and z (the long axis of the 2p n orbital) are parallel. the observed hyperfine splitting (the distance between the absorption peaks) changes such that it is maximal when the external field H . because the electron is partially shared by the oxygen atom. 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. Because the populations of nitrogen nuclei in each spin state are approximately equal under normal experimental conditions. this is maximal when H . but as the first derivative of the absorption with respect to field strength d A / d H . . . as 7 [In fact.1 spin states ofthe nitrogen nuclei.2.and two tertiary C atoms are placed in a single plane in the diagram although this is not usually strictly correct. the CNO angle being a function of the particular nitroxide molecule considered. and therefore exhibit magnetic properties themselves. However.. is governed by the orientation of the spinlabel group with respect to the direction of H. As a simplification. This is convenient for the instrument's design and because firstderivative spectra provide information in a more measurable form than absorption spectra do. This shows that resonance conditions are obtained at three different positions of the field sweep. In actual practice. and the conventional manner of assigning Cartesian coordinates to the molecule.
( 2 ) The curve associated with the outer lines has been removcd. 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). Figure 5b is a simplified version which approximates to Fig. . For orientations of spinlabeled crystals other than H . (a)Truechangein nuclear liyperfincsplittingsand linepositionsasacryslalcontaining oricnted nitroxide spin labels is rotated about a spatially fixed external magnetic field of variable strcngth If. 5b are all occupied by spin labels but. with concomitant shifts of the outer lines.. the signal generated by such a sample would be a composite of contributions from the randomly oriented labels. Note that the center line is equidistant from the two outer lines at all values of 0. when 0 = 90” or 90” the z axis of thc labels is parallel to the magnetic field.3. 5. Two simplifications have been madc.60 2. is parallel to any line drawn in the xy plane. the hyperfine splitting values are intermediate.” is shown to be constant (see Section 2. 5a and allows us to present a version of spinlabel theory without recourse to mathematics. i. NITROXIDE SPIN LABELS values obtained when H . If we assume that the various positions indicated by the lines in Fig.3 but need not be of concern for the purpose of the present discussion. for further simplification.e.. This is due to a change in the “g value. and when 0 = 0”it is perpendicular. If we were to take the carefully prepared crystal that we have been discussing and crush it to a powder. 11 z or H .5).” which is discussed in Chapter 2. 1 ) and H . 0 is the angle between the xy plane of the nitroxide groups (see Fig. Iz .] 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. we consider labels with similar positions as groups or “packets” k‘rc. (1) The “ y fiictor.. (b) Simplified version of(a).. 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. .
(Fig. 6c) has the same shape as the absorption spectrum of the packet of labels with z /jH. See Fig.. 6b).2. 6c) we see that the packet of labels that includes those with z 1) H . the EPR spectrometer records spectra not as field versus A . is responsible for the field positions at which C A commences to (i) increase increasingly rapidly and (ii) increase less rapidly.3.).and TL.6. 6. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 61 which generate absorption spectra of fairly narrow width. etc. Simplified version of the generation of a powder spectrum. However. The firstderivative spectrum (Fig. 6d. This fact is also derived mathematically in Section 2. therefore. 6b). versus d AIdH..8. both commence at the same lowfield position and achieve their maximum value at the same position. This shows that a powder spectrum is related in a very interesting way to spectra derived from single crystals rotated relative to H o . the spectrum may. obtained by adding the individual spectra shown in (b). be used to determine both T. but as field versus the first derivative of A (i.. (b) Superimposed absorption spectra of all packets of labels shown in (a). (d) Firstderivative spectrum taken from (c). . (a) As Fig. 5b but indicating anoncontinuous distribution of labels as an aid to interpretation. (c) Composite absorption spectrum of all packets of labels.  "0 FIG.2. The composite firstderivative spectrum may similarly be used to detect contributions from the packet of labels having their z axes perpendicular to H. 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. 6d) of the lowfield shoulder shown in the composite absorption spectrum (Fig.e. If we consider the lefthand (lowfield) side of the composite absorption spectrum (Fig. that is to say. we arrive at the prediction illustrated by Fig. H . 4 for use of symbols.
labeled 0 = +90 to 90. a “powder spectrum” will be obtained if the motion is very slow relative to the instrument’s data gathering interval Ho HO * FIG..). Even if labels are moving. We then say that the rotational sec. at any time. shown in (a) as a function of H .7. [It should be noted that for an isotropic discorrclation timc of the molecule is I tribution there are. . Diagrammatic representation of the effect of isotropic motion on the spectra of isotropicdispersions of spin labels. not TI^ + TJ as suggested by this diagram which does not take this “weighting” into account. Because the instrument has a fixed. (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. 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. effectively twice as many labels oriented with z l H . . A comparison of Figs. at some time during the data gathering interval.2. Thc “strings of beads” represent packets of labels that absorb at approximately equal average values of H . 2. (a) The tilted Vshaped solid lines to either side are the nuclear hyperfine splittings appropriate to zero or very slow motion (see Figs.) and may be compared with q. at the orientation 8.]   . 6d is derived using several simplifications which are describedin the legends to Figs.62 2. .3. relates to these. 5 and 6. The EPR spectrometer has a characteristic time interval for receiving data which can be likened to the shutter speed on a camera. labels which alter their orientation appreciably during this time interval are detectcd via their absorption at an average H . NITROXIDE SPIN LABELS It should be borne in mind that the spectrum shown in Fig.Effect of Motion The individual spin labels in the powder sample discussed above are essentially motionless. as there are labels oriented with zJiH. and TL for calculations regarding the motion of spinlabeled molecules. 5 and 6). and were. (b) The firstderivative spectrum of the microwave absorption of the packets of labels. and the ordinate. characteristic data gathering interval (sec). Therefore the value for the observed splitting in the true case of rapid motion is f(T11+ 2T. 6d and 13 should be made. 0 being the angle between the x y plane of nitroxide groups and H . to observe similarities and differences between the true and synthetic spectra. appropriate to their orientation excursion during sec.
. [i. but yet it is impossible to observe measurable maximum and minimum splittings. 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.4.. Soc.e. Chem. with decreasing motion the amplitudes of the high. For intermediate cases. This phenomenon is responsible for the effect discussed in Section 2.” Frequently.321 (1972). Commun. for example. McConnell. motion is not sufficiently fast to give rise to a spectrum in which the splittings are completely averaged. have been used to obtain absolute or relative values for motion. Hubbcll and H.2. M. 18. Fig. l3 I‘ E. 7 and 11 and also Section 2. and the nitrogen nucleus’s share of the unpaired electron increases relative to that when the spin label finds itself in an apolar. J . Soc. this being the weighted average of splittings appropriate to the various orientations of z with respect to H .2.)]. 93.3.). 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. Biochem. J . Broomfield. W.7. J. J . Morrisett and C. Am. 93.e. If. It is sufficient for the present discussion simply to say that the higher the electron density on the nitrogen nucleus. “hydrophobic” environment. M. sec. polar or “hydrophilic” environments) the polar character of the NO bond is encouraged. isotropic spectrum the amplitudes of all three peaks are almost equal (see Fig. when spinlabel lipid haptens report the “melting” of lipid bilayers.2. the bond lengthens. Similarly. at the beginning of a data gathering interval. 46.4. or shorter than.7297 (1971). McConnell. A. $(TI1 + 2T. 11).and lowfield peaks decrease relative to the center peak. In such cases one can take advantage of the relative amplitudes of the various peaks in order to obtain information regarding motion13. a single value for hyperfine splitting is observed. a packet of labels might be oriented with z [I H . for a true fast motion. however. SPINLABEL THEORY : A DESCRIPTIVE TREATMENT 63 (. charge separation is favored. This phenomenon has a marked effect on EPR spectra. Effect of the Electrostatic Environment In environments having high dielectric constants (i. spin labels are moving so fast that their rotational correlation time is equal to. 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. various degrees of mixing of splittings become apparent. Am. *’ L. Biophys.. D. 2. Such spectra are termed isotropic or “fast tumbling” (see Figs. Changes in observed maximum and minimum splittings. 314(1971). relative to those obtained from powder spectra. Chem. Shimshick and H .l o p 9 sec). Res.4.
8. of all packets of labels shown in (a) as a function of H. J. S r i . ' ~illustrated by Fig. H. (For further discussion see Chapter 2. F. In both environments. 17. Nor/. In other words. polar environments increase splittings. Acad.3) This is the basis of a phenomenon described in Section 2. Linden. 6 and 7. Biochrrnistry 12. Wright. phase. or hydrophobic. apolar environments decrease splittings. the spin labels are in rapid isotropic motion and the amplitude of the firstderivative signals (2h and 2 p ) are approximately proportional to concentration. NITROXIDE SPIN LABELS "0 FIG. Proc. (a) The timeaveraged nuclear hyperfine splitting (see Fig.. S. 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. 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.6 and illustrated by Fig..4.64 2. K. 2271 (1973).(c) First derivative spectrum taken from (b). M . of a phenomenon which forms the basis of a wellknown method for determining lipid phase transitions. and C. Diagrammatic representation. phase and the aqueous. Shimshick and H. (b) Total absorption. 70. l4 l5 . It is also the basis of a wellknown method for detecting temperatureinduced phase transitions in lipid bilayers or cell memb r a n e ~ . C. The compound TEMPO partitions ~'~ between aqueous and lipid phases with a coefficient which is a function of the fluidity of the lipid phase. 2351 (1973). M. A. The highfield lines of these two spectra are sufficiently well resolved that they can be used to calculate the relative amounts of TEMPO E.A . derived in a manner analogous to that shown in Figs. Ll. 7c) of TEMPO in the lipid. Fox. or polar. McConnell. McConnell. 8. the greater is the influence of the nucleus and therefore the larger the hyperfine splittings.
22.2. The g shift is down field for apolar environments. again broadening the peaks of the spectrum and decreasing their amplitude because an element of inhomogeneity has been introduced into the magnetic environment. the second label is moving slowly. as it will be in the case of lowmolecularweight spin labels in lowviscosity solvents at concentrations 2 1 mM.5.6. thus averaging the effects of the two nuclei.5. If.3. the direction of its magnetic field is totally averaged and cannot affect the first spin label. (See also section 2. If the second label is tumbling very rapidly and isotropically around the first.3. An example of this effect is illustrated by Fig.2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 65 in the two phases.1. If the collision frequency is sufficiently high. 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. SpinLabel Theory: A Mathematical Treatment 2. broadening by spin exchange also results in a decrease in amplitude of the firstderivative spectrum.3. the orbitals containing unpaired electrons overlap sufficiently that there is a high probability that the electrons will exchange orientations.12. (The resolution is aided by the fact that there is a “g value” change affecting the position of the set of three lines. 2. or nonisotropically.) 2. 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. it may still be affected by this second label in another way.) 2. we consider spinexchange broadening first.3.2. so that the lowfield lines of the two spectra overlap extensively but the highfield lines are better resolved than they would otherwise be.3. The Resonance Condition The term “magnetic resonance spectroscopy” has usually been applied to those forms of spectroscopy i which the frequency of radiation that is n absorbed or emitted is nearly or exactly proportional to the strength of a .Exchange Broadening There are two ways in which EPR spectra are affected by the distance between individual spin labels. this spin exchange has a marked effect on the spectra. Effect of Distribution or Concentration of Spin Labels: Spin. its magnetic field will affect that of the first spin label. The ratio may change dramatically at temperatureinduced phase transitions. As is the case when the lines are broadened by a decrease in motion. If labels collide. This is covered in more detail in Section 2. however.
9.000 MHz range.10. (2. (2. E.”) An electron is a negatively charged particle. (2. Fizrol. .0023 when the orbital contribution to the electronic magnetic moment is zero (complete orbital quenching).S. A circulating motion of the electron gives rise to a magnetic moment. This magnetic moment is parallel to the vector spin angular momentum Sh.4) ” E. and c is the velocity of light. Fizrol.1) where h is Planck’s constant. The quantity g is the “spectroscopic splitting factor”.. Zb. where 8 = eh/2mc.8 x 10. (Electron paramagnetic resonance was discovered by Zavoisky in the U.R. Equation (2. m is the mass of the electron. 2a). Electron magnetic resonance spectroscopy is also called paramagnetic resonance spectroscopy. Zavoisky. p = g(e/2mc)Sh. 9.3. In organic free radicalst the orbital magnetic moment is largely quenched and makes only a small contribution to the total magnetic moment. NITROXIDE SPIN LABELS laboratory magnetic field acting on the sample. The absorption of radiation by a sample placed in a magnetic field can be discussed in terms of the equation hv = BE. one giving an orbital magnetic moment. An electron can have two kinds of circulating motion.l o em).3) We shall write Eq. and the other giving a spin magnetic moment. and the radiation frequency is in the 900035. 245 (1945).000 G range. The electronic magnetic moment p and the spin angular momentum Sh are parallel to one another. at least for large fields (see Fig. The electron spin magnetic moment is a vector and is denoted by p. in 1945. Here h is Planck’s constant divided by 2n and S is the spin angular momentum in units of h. in order to avoid any uncertainty in algebraic sign: p If’ = 9l8lS.3.16.66 2.2) is often written in terms of the Bohr magneton 8. 181. AE is exactly or nearly proportional to the strength of the applied laboratory magnetic field H. (2. In electron magnetic resonance spectroscopy. v is the frequency of the radiation that is absorbed (or emitted). 211 (1945).2) The charge on the electron is e (e = 4. Zavoisky. the applied fields are usually in the 3000. and AE is the separation of two energy levels.3. g = 2.3. Zh.3.3. t Except for diatomic CH.S.2)in terms of the absolute value of 8. (2.
3. = 4) and E(S. When a sample containing unpaired electrons is at thermal equilibrium there are more electrons in the lower energy state ( S . (2.3.8) is also often expressed in terms of the magnetogyric ratio y : 2nv or (3 = = yHo (2. = $) than in the upper energy state (S.7) (2. 2. (2. From Eq. = +$.3. (2.5 kMHz. and thus there are two energy levels.1. = ++3 S. 2. = ++)than downward ( S .10) yHo.E(S. The numerical value of is 0. where Y = slPl/h.. (2.3. = = 3). paramagnetic resonance absorption is observed at an applied field H.. is negative. = 1) 2 transitions. then the energy E can be expressed in terms of S.3. resulting in a net absorption of radiation.11) .3. This frequency region is often termed “ X band” by microwave spectroscopists. as sketched in Fig.3.9) (2..92731 x erg/G. According to Eq. Most commercial paramagnetic resonance spectrometers operate in this frequency region.3. = 3 + S .3.2.8) it follows that for radiation of frequency v = 9. where E thus. E = p*Ho.3. Incident radiation of the proper polarization and frequency then stimulates more upward ( S . (2. Most organic free radicals have g factors that are approximately equal to 2. with an energy E.5) = gl/?lS * H. microwaves) of the proper frequency and polarization. = +$) is hv = E(S.8) hv 9lPIHo. The conditions for resonance given in Eq.6) If the applied field is in the z direction. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 67 The negative sign arises because the charge on the electron e. the frequency of the radiation v that can produce transitions between two energy levels E(S. This is electron magnetic resonance. = +$). A magnetic dipole moment p interacts with an applied field vector H.3. = +$) . Transitions between these levels can be induced by radiation (eg. S . (2. of approximately 3370 G.
and into the cavity.3. In the presence of a uniform magnetic field of strength H . 3. The resonant frequency v can be regarded as a classical Larmor frequency of the electron magnetic moment. the number of photons taken from the microwave field by the paramagnetic sample when the fieldfrequency ratio corresponds to thc resonance condition.68 2. through an iris. The principal features of an EPR spectrometer are shown in Fig. some of the electrons (nl) will be in the lower (“spindown”) energy level. the frequency of the radiation is normally kept fixed and the resonance absorption detected by changing the strength of the applied field. Paramagnetic resonance absorption is observed when the field of the electromagnet is changed until the resonance condition [Eq. radiation is absorbed by the sample. 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. .3. In the case of the commonly used reflection cavity. This particular type of resonance cavity has a length which is equal to the wavelength of the microwave radiation. where the electron spins interact with each other only very weakly compared to the thermal energy kT. 2.3.3.12) .3.16 cm.5 GHz has a wavelength i = c/v = 3. (2. Radiation of this wavelength can be conveniently l handled by means of microwave tubing and resonance cavities. microwave energy from a source passes down thc waveguide. this absorption is detected by a decrease in power leaving the cavity. At resonance.). the resonance cavity can be thought of as functioning in the following way. as discussed below in terms of the Bloch equations (Section 2. (2. Let us now discuss the intensity of the resonance absorption. or being lost by going out through the iris and back up the waveguide. The radiation can thus be thought of as being “stored” in the cavity. In free space. radiation having a frequency v = 9. 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). 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. In the simplest possible terms. Absorption Probabilities and Paramagnetic Relaxation In the above discussion we have considered briefly the fieldfrequency condition for paramagnetic resonance absorption. Note that in an experiment of this type. Consider a system of N electrons. that is. whose internal dimensions are usually of the order of magnitude of this wavelength.8)] is met. and the remaining electrons ( t i ? ) will be in the upper energy level: N = t1J + t~?.2.
(2. Let n = nL . so .3.2.14) 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. That is.3.‘)that an electron in the spindown state absorbs a photon.13) where hv is the transition energy. roomtemperature thermal energy is 200 cm’. (2. This relaxation is often a simple firstorder process.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. Thus n? and n l differ only by a few tenths of a percent. The population difference then follows the equation dnldt = 2nP.3.3. The probabilities for absorption and stimulated emission are the same.n t be the population difference. so that the transition energy measured in wave numbers is 0. The wavelength of Xband microwave radiation is 3 cm..17) According to this result the spin system saturates with a time constant of ) P and the absorption of radiation decreases at this rate. The time rate of change of the population of the upper spin state n t is thus dnfldt = .15) is the rate of loss of electrons in the upper spin state due to stimulated emission. (2. In the same units. (The probabilities of spontaneous emission of radiation can usually be neglected in magnetic resonance experiments. Under these conditions a good approximation to the difference in population n is then  nq N N(hv/kT).15) The first term in Eq. equal to g 1 p I H. and the second term is the gain of electrons in the upper spin state due to absorption.) We shall see later that P depends on the photon density o r energy density in the radiation field.33 cm’.3. 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”).3. if P is the probability (in sec. then P is also the probability that an electron in the upper spin state will be stimulated to emit.n f P + n / P . this is not usually the case because of paramagnetic relaxation. (2. (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. Fortunately.
3. and the resonance absorption of radiation does not appreciably disturb the difference in population ofthe two spin states. the region of the microwave cavity where the aqueous samples are normally placed.3. (I) is 2n times the microwave frequency. in fact.70 2. This can also be written in the following form: power absorption = [NP(hv)’/kT]/( 1 + 2PT. As already mentioned. the net time dependence of the population difference is governed by the differential equation dn/dt = 2Pn  (l/TJ(n . is parallel to the cavity axis z. Near the center of the cavity this field is linearly polarized and uniform. we have dn/dt = (l/T.nq). 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. the rate of radiationinduced transitions is small compared to the rate of paramagnetic relaxation. H . is the amplitude of the oscillatory field.). The power absorbed by the sample under steadystate conditions is nssPhv. but in the presence of a thermal bath with which the spins can exchange energy. is the electron paramagnetic relaxation time. most paramagnetic resonance experiments are carried out using a resonant microwave cavity. NITROXIDE SPIN LABELS that in the absence of an applied radiation field.22) Here i is a unit vector in the x direction. 3 and 9 are the same.3. (2. (2.3.)(n .18) Here T. The lines of force corresponding to one phase of the microwave magnetic field are shown in Fig. Let the strength and direction of this linearly polarized field be represented by HI = H C O S ( U ~6). and 6 is a phase factor that can .2 1) We now discuss the absorption probability P. (2. (2.20) From this equation one draws a physically obvious conclusion: as long as the incident power (radiation energy density) is low enough.19) Under steadystate conditions where dn/dt = 0 we find for the steadystate difference in population nss nss = nq/(l + 2PT).3. I + (2. The axis systems in Figs. 9. The shaded region is. The oscillatory magnetic component of the microwave radiation field is then in the x direction.nq). The electromagnetic radiation that is stored in this cavity has both electric and magnetic components that oscillate at the resonant frequency of the cavity. The resonance cavity is placed between the pole faces of the electromagnet so that the applied field H.
(2. Lines of force of the oscillatory magnetic field associated with the microwave radiation within thecavity. where typical linewidths are never much less than 1 MHz. 21. For convenience we write the amplitude of this field as 2 H . be set equal to zero. Harper & Row.24) An elementary quantummechanical calculation yields the following result for the transition probability for an electron’ *: p = $(?JHJ2g(v). McLachlan.26) We do not give a quantummechanical derivation of Eq. Carrington and A. (2. (2.25) 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.25) here since essentially the same result is obtained later from the Bloch equations. 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. described by g(v). SPINLABEL THEORY: A MATHEMATICAL TREATMENT 71 z Y FIG.” p. (2. and the microwave radiation has a frequency stability of better than one part in lo’. 1967. Thesampleis placed in thecentral shaded area.3. This condition is always valid for the paramagnetic resonance experiments of interest here. which is normalized as Jomg(v) dv = 1. The axissystem is the same as that used in Fig. D. (2.25) Here g(v) is the absorption lineshape function.3. New York. If the energy levels between which A.9.3. “Introduction to Magnetic Resonance. instead of Hi : 2H. 3. (2. The lineshape function g(v) can be thought of as describing the distribution of energy levels between which transitions can take place.3.3. The derivation of Eq.2.23) Hi = i(2H.3.) cos(ot + 6). E Hi. .3.
3. steady applied field.27) d (2. dM.3.29) where M is the total electron magnetic moment M = Cpi..3.33) d M J d t = . dt d dt (2. If we take the z axis to be in the direction of the large.3. According to Eq.3./dt = (Mz  Mo)/T./dt = . (2. Equations (2.3. with . If we multiply through by the magnetogyric ratio y.3. we obtain pi = ypi x H.( B J = ~i x H. then this can also contribute to the width of the distribution of frequencies given by g(v).34) Here N is the number of electrons in the sample.3. (2. 2. NITROXIDE SPIN LABELS transitions take place have a sufficiently short lifetime.29) gives the time rate change of the electron magnetization due to the externally applied fields. relax to their equilibrium value. (2. The equilibrium value of the electron magnetization in the field direction is easily shown to be M = Nh21(I + 1)/3kT. then the contributions of relaxation to the time rate of change of M are assumed to have the following form: dM.3.33) assume that the components of electron magnetization perpendicular to the steady large field H.31) (2.yM x H..3.M. zero.32) (2.3.32) and and (2. The Bloch Equations Consider an electron i with spin magnetic moment pi and spin angular momentum Mi.31).3./Tl.28) Then we may sum over all the electrons i in the sample and obtain dM _ .72 2. dt (2.MJT2. (2. with a consequent change in the direction of the angular momentum : . A magnetic field H exerts a torque of pi x H on the spin magnetic moment.3.3. but does not take into account changes in the magnetization due to internal fields in the sample that produce electron relaxation. the electron magnetization relaxes towards its equilibrium value M o with the relaxation time T.30) Equation (2.
is the steady large field applied in the z direction.. When the frequency w is close to the resonance frequency w. one of these components (H: or H.3.3. (2.. l9) The Bloch equations can be written in the form of a single vector equation Here i.35).38) (2. = yH. the applied oscillatory field (with angular frequency w ) is usually linearly polarized.3. The magnetogyric ratio y is negative for electrons.j(sin wt)]. (2.) will be nearly “in phase” with the precessing electron magnetization. (2.39) (2. (2. when viewed from below the xy plane in the positive z direction. As can be seen from Eq.3.29)] and due to relaxation [Eqs. respectively.37) This linearly polarized field can be decomposed into two circularly polarized counterrotating components: Hi = = H: + H.3.3.. 70. Reo. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 73 time constant T.29) and Fig. y..3. H: = H I[i(cos o t ) + j(sin of)]. The outofphase component of H l has a negligible effect on the motion of the electron magnetization and can be neglected from Eq. 10. .3. Therefore we include in Eq. (2. and the other component (H. + Hi. the magnetization (in the absence of any HI field) precesses clockwise about z.460(1946).3.3. Thus when y is negative. Phys.3 1)(2. Hi = 2H1i(cos wt). and H i is a (usually weak) oscillatory field perpendicular to H . (2.33)] we obtain the Bloch equations. H: also rotates clockwise and is the inphase component.3.35) only the cirularly polarized component that is in phase with the electron magnetization at resonance. (These were originally devised to describe nuclear magnetic resonance. j. Bloch.40) H. . and H = H.3.36) H.2. k are unit vectors in the x. Let this linearly polarized field be in the x direction and of amplitude 2H.z directions. ” ) F. If we add together the rate of change of the electron magnetization due to the externally applied fields H [Eq.[i(cos o r ) . or H:) will be “out of phase” with the precessing magnetization. H. (2. (2. In both microwave electron paramagnetic resonance instruments and radiofrequency nuclear magnetic resonance instruments.
3.3.3. ( W . T2/[l + T:(o  0 0 ) ~ + y2H:T1 T’]. T ~ T ~ ](2. v .3. .3.(W./dt . .42)(2. dvldt = = (wO .W ) V .3.( M .3. . 10.T:<COO w)M/[l yH.  (2. (2.42) (2.u/Tz. u.41) where u is the inphase electron magnetization.~ H .W)U (2. and M .49) . The Bloch equations are readily solved by a transformation to the rotating coordinate system i’.43) (2.46) + T. as before.w)’ + Y ’ H . u is the outofphase electron magnetization. Illustration of the axis system used in the Bloch equations.3. and M. Let M = i’u + j’v + k’M. the Bloch equations are duldt = (00 .3.47) ~ .74 2. j’ is a unit vector perpendicular to i’ which lies in the xy plane. (2.3.. is. or “slow passage” conditions where duldt = dv/dt = dM. ( = H : when y is negative). j’. (2. (2. NITROXIDE SPIN LABELS L H . the electron magnetization in the external field direction. and M .44) + ? H I M .48) The average power absorbed by the sample is then P(w) = Q ~ H : M . Under steadystate.w)’ + y2H:TlT2]. and k’ = k.  The instantaneous power absorbed by the sample is H dM/dt.45) = = M. and the average power absorbed (averaged over one cycle of the microwave field) is P(0) =   dM dt.U/T2.3. dM. [l (2. Eqs.T. k’. u. Here i’ is a unit vector in the direction of H./dt = 0. + T:(co~ ~)’]/[1 + T .3. In terms of u. Section 2. FIG. : u u = ~H.3.44) are readily solved for u..Mo)/TI.M/[l + T : ( w ~ 0)’+ y2H:T1T2].
Nuclear Hyperfine Structure The most important feature of the paramagnetic resonance spectra of organic free radicals is the rich nuclear hyperfine structure. However. . This hyperfine structure arises from the magnetic interaction between the electron spin magnetic moment and nuclear spin magnetic moments.3. y 2 ~ : ~ .” 2. (2.50) The quantity y2H:TlT2 is termed the saturation factor. (2. these proton hyperfine interactions often contribute to the observed resonance linewidths. The component of the nuclear spin angular momentum in the applied field direction is designated m.. H I ) when the microwave power is low enough. SPINLABEL THEORY: A MATHEMATICAL TREATMENT 75 This absorption curve has a Lorentzian line shape that is independent of the microwave power (e. Fig. The nuclear hyperfine interaction splits the paramagnetic resonance into a number of components. ~ 1. 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.3. (Strictly speaking. in fieldsweep spectra.1 (see Fig.) We shall refer to the amplitude of the outofphase component u as the “absorption signal.2.3. These three lines correspond to the three possible orientations of the 14N nucleus in the applied magnetic field.g. 0 (TEMPONE) The resonance spectrum consists of three sharp lines separated by 16.5 1) This result is the same as that obtained by direct quantummechanical calculation of the transition probability.4. The 14N nucleus has a nuclear spin 1 which is equal to one. du/dH. Proton nuclear hyperfine splittings are not resolved in the paramagnetic resonance spectra of many aliphatic nitroxide radicals.2.6.49) and (2. the quantitized values of rn are m = 1. If we consider the condition for resonance.3.6tetramethyl 14oxopiperidine (TEMPONE) in water. For example.21) we can obtain an expression for the absorption probability P : P = $y2H:T2. .0. P(w). .05 G. 4).. When I = 1. 11 shows the paramagnetic resonance spectrum of 2.3. (2.0 0. and compare Eqs. w = w.. the signal usually recorded is the derivative of u.3.
(2. The natural abundances of 3C and 5Nare 1. respectively.3.76 2. 11 are due to a small fraction of free radicals that contain 13Cor ”N nuclei instead of I4N and ”C. NITROXIDE SPIN LABELS FIG. 11 is g = 2.00562 & 0.1 % and 0.11. there is an electronspininduced electron orbital motion that creates a magnetic field which acts back on the electron spin. EPR spectrum of a dilute aqueous solution of TEMPONE. which is nearly but not exactly equal to the freespin g factor go = 2.52) Paramagnetic resonance transitions between these levels then occur at the transition frequency v.00002. Another important feature of the paramagnetic resonance spectra of organic free radicals is the spectroscopic splitting factor g.0023. 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.37 %. This deviation of the g factor from the freespin value is due to a (secondorder) spinorbit interaction.53) The local magnetic field acting on an electron in a nitroxide free radical has two components.3. Thus hv = 90IBIIHo + HlocI. 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. . and the separation of these energy levels is ’ ’ hv = 90IPIlHl. The additional weak signals in Fig. 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. plus local fields Hloc originating within the molecule. the hyperfine field due to the 14N nucleus. the spectroscopic splitting factor g for the resonance spectrum of TEMPONE in Fig. For example. A free electron with spin ++has two energy levels in an applied field of strength IHI. See text for discussion. (2. In other words. and the spinorbit field due to the (spininduced) orbital motion of the electron itself.
3. and for nitroxide free radicals in particular. the only effective component of Hhfis the component in the direction of H. to a good approximation. centered at the field H ..: H. 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. As we shall discuss later (Section 2. we see that the paramagnetic resonance spectrum of a nitroxide free radical is a threeline spectrum.54) becomes (2. the effective field H. and this isotropic hyperfine field is HAf isotr. due to spinorbit interaction that acts on the “free electron” spin is.54) hv = gOlbllHO f Hhf + Hgl.0. The three possible values of HLf.9).3. .55) Here g = go + 6g. for organic free radicals in general. Here Hhfis the local field acting at the electron due to the magnetic moment of the I4N nucleus and H.56) hv = slBlHo + g0lPIH. to a good approximation. (2.2. On the other hand. In general. 11. namely HLf. with two hyperfine lines centered about this resonance position.O.f. in the same direction as H.3. The anisotropic part of the hyperfine interaction makes a large contribution to the hyperfine splittings in the paramagnetic resonance spectra of oriented nitroxide radicals. such as water. IHLf 1. Thus SPINLABEL THEORY : A MATHEMATICAL TREATMENT 77 (2.1 HAf I. correspond to rn = .3. the three hyperfine lines have essentially the same intensity. at room temperature. Hhf.57) In the theory of the effect of molecular motion on the magnetic resonance spectra. the local hyperfine field acting at the odd electron is +16(f0. as discussed in Section 2. (2. Thus Eq. Since the second term in this equation is small compared to the first. That is.1.3.3. is the local field acting at the electron due to the orbital motion of the electron. and proportional to H.lHifl. = @. . Since 14N has a nuclear spin I = 1 which can take on three orientations in a magnetic field. In the paramagnetic resonance spectrum exhibited in Fig.7.3.Since these values of rn are very nearly equal in probability. the anisotropic part of the hyperfine interaction contributes to the widths of the resonance lines even when the molecular motion is very fast. corresponding to applied fields H. the local hyperfine field )Ihf has three possible values.1. (2.namely . the hyperfine field acting at the electron due to the nitrogen nucleus. IHLfl and H.3. . under these conditions only the isotropic interaction contributes to the splitting./gO)aO.05)G.has two components: + + f&f = Hhfisotr + Hhfanisotr. Also. where hv = g1B1H0.
k =YNbNk (2.IflIgNPNr3(U . As discussed above [cf. NITROXIDE SPIN LABELS The theory of the origin of the nuclear hyperfine interaction in organic free radicals has been developed extensively. For an alternative discussion.53)]. = YolPIS. ‘I ’’ .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. see Abragam and Bleaney.” p. (2. H. the freeelectron spin magnetic moment pe is P.3.60) where gNis the nuclear y factor. “Classical Mechanics.63) For a discussion of dyads and dyadics. This spin Hamiltonian provides a general and convenient quantitative starting point for the calculation and interpretation of magnetic resonance spectra. Abragarn and B. 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.58) Here r is the distance between the electron with spin magnetic moment p . The energy of this point dipoledipole hyperfine interaction can then be written x d = hS ’ T 1. Reading. Massachusetts. (2. Electron Paramagnetic Resonance of Transition Ions. . and I is the nuclear spin angular momentum in units of ti. An understanding of this theory is not necessary for most of the biophysical applications of spin labels. one obtains hyperfine A. 1 A in these equations. (2. The energy of the magnetic dipoledipole interaction is (2. see Goldstein21 The reader can show for himself that by inserting an electronnuclear distance of the order of.3. 1950.3.78 2. and the nucleus with spin magnetic moment A . London and New York.62) Here U is a unit dyadic: U = ii + jj + kk. (2. Bleaney. jjNis the nuclear magneton.3.3rr/r2). Goldstein. AddisonWesley.3. Eq.3.61) where T. = h’g. say.” Oxford Univ. 1970.3. Press (Clarendon). 147.59) The corresponding expression for the nuclear spin magnetic moment is (2.
Strathder. Since the odd electron is described by a quantummechanical distribution function. Theory and Applications” (L. Berliner. but rather is distributed in space according to the molecular electronic wave function. (2.Sect.3.2. (2. Kruger.3. 2.64) 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. BB26. 1976. nitrogen. and (ii) the electron spin magnetic moment is not concentrated at a fixed point in space.66) One can readily generalize the point dipole interaction in Eq. The spin distribution doubtless also has some norbital amplitude on the oxygen atom as well. (2. We assume that the oxygen. Academic Press.68) J . it is clear that the net dipoledipole interaction must take into account this distribution of the spin. New York. 22 23 2s H . Ac/a Crysrallo. 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. This is precisely true in one nitroxide free radical whose structure has been determined. L. Mol. Let m(r) dV be the contribution to the total electron spin magnetic moment arising from the element of volume dV. Boeyens and G. For quantitative work.~~ “odd” electron is localized The in a 2p n atomic orbital centered on the nitrogen atom. in “Spin Labeling.60) to a distributed dipole interaction as follows. LajzerowiczBonneteau. ed. I. C. . Phys.22 For determinations of the structure of other nitroxides.3.67) T.yr. = J: T. pe = Im(r) dV. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 79 interaction energies of the same order of magnitude as the observed hyperfine splittings. J . Acra Crysfallogr.3.668 (1970).3. 2.Sect. Berliner.). BB26. and two bonded carbon atoms lie in a plane. 24 J . = hs * T. 1. M. The spin distribution in nitroxide free radicals can be discussed by reference to Fig. A. p.3. J . .p(r) du. McConnelland J . see Berliner23 and LajzerowiczBonnetea~. J . (2. I198 (1970). 129(1959). (2. 239.
3 ) derived from ~ selfconsistent field Hartree functions for atomic nitrogen.3. C. Dousmanis.3. NITROXIDE SPIN LABELS The c in Eq. nuclearspin dyadic need not be symmetric. the effective electronspin. then the dipolar tensor is axially symmetric. ~ ’ ’’ A.3.72) One can make semiquantitative estimates of these dipolar terms using 2p = Slater atomic orbitals.3.69) (2. The spin density distribution function p(r) can be readily evaluated from an approximate molecular electronic wave function. This difficulty will be considered later in this section. is the average value of ( r P 3 ) . ’For an estimate of ( r . If z is taken to be the symmetry axis. then (T.447 (1962). (2.3. (T&? = (T&. Irrespective of the detailed form of this spin density distribution function. (2. is symmetric in the sense that the coefficient of ij is equal to the coefficient of ji. From the above discussion it follows that (Td)xx = (Td)yy = !dTd)zz. In molecules where there is significant contribution of orbital magnetic moment to the nuclear hyperfine interaction.)xx= (Td)?).62) in Cartesian coordinates. P\IW R w .An explicit calculation of the dipolar tensor T. and so forth.80 2. Mol. Phys.3. Tr{T. 27 G. for a 2p norbital spin distribution yields the following result: (2. LonguetHiggins.68) denotes a small region surrounding the nucleus which is excluded from the integration because of the fact that the dipolar interaction formula T. 967 (1955). . as in the case of paramagnetic transitionmetal ions. A second point to note regarding the dipolar tensor T. O.3. 1.71) Here ( r . Estimated values for nitrogen are (TJxx = (q)zz 38 MHz. see D o ~ s m a n i s . where the average is taken over the radial probability distribution for the odd electron. and in their effects on the observed resonance spectra. ’ ~ .70) + (Td)yv + (Td)zz This result follows from the fact that the point dipolar interaction itself obeys the same trace equations. (T& = +76 M H z . (2.. The point dipole dyadic T. (2. Thus the distributed dipole dyadic has this symmetry as well. 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. C . the dipolar hyperfine tensor T. 5. conforms to the following trace equation. Carrington and H. 77. becomes infinite near the origin. as one can easily show by expanding the vector r in Eq.} (Td)xx = = 0. In the case of nitroxide radicals the antisymmetric components in the hyperfine coupling between the electron spin and nuclear spins are negligible in magnitude.
1732 (1930). If the spin distribution p(r) is due exclusively to spin distributions that can be described in terms of p . relative to their intrinsic spatial extent.68). d . z are a set of principal axes. then it is necessary to consider this small region about the nucleus in detail. The physical problem concerning integration near the nucleus is that the simple dipoleedipole interaction formula. 1956. “Molecular Beams. this dyadic can be written in diagonal form (all nondiagonal elements equal to zero). Phys. so that the sphere is small enough to have a uniform spin density due to the electron. we shall use the notation (7’Jx. 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. Z .. F. of the order of interaction between the nucleus and the magnetization inside the sphere with radius R. ( T d ) y y r and (Td)zz.3. must be large compared to the “size” of the nucleus. 320 (1930). Rer.3. Eq. Let R. or size. Fermi. The axes x.. if the oddelectron spin distribution has contributions from s orbitals. and (T& in place of(G)xx. E. then the small region E need not be excluded from the integration because the divergence of T.62).3. On the other hand.2. London and New York. (2. Let us assume that the effective A. about the I4N nucleus as center. G. since for these atomic functions the spin density goes to zero at the origin. We now return to the expression for the dipolar interaction given in Eq. (2.3. y. Doerman. Ramsey. The magnetic interaction of the nucleus with the electron spin magnetization inside the sphere is s i = FN = B (2.59) or (2. W. .” Oxford Univ. Press (Clarendon). This means that R.3. . is only adequate when the two magnetic dipoles are far apart. 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). ~ ’ Draw a hypothetical sphere of radius R. (T&. 36. Breit and F. does not produce any problem in the integration.68) and consider the small region E that is excluded from the integration. (2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 81 Since the elements of the dyadic T. which is the case for nitroxide radicals. z for which Tdis diagonal are then a set of principal axes. . Phys.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 . This corresponds to the small volume excluded in the distributed dipole integration in Eq. .3. 60. N .74) YNPNISB. atomic wave functions. 29 *’ ’’ . Now consider the magnetic size of the nucleus is R. When x. f . are symmetric.73) (2.3. We assume that the spin density p(r) is uniform across the radius R. y.
E. Hazzard. that is. Rozantzev. nitroxide radicals are examples of ‘‘zelectron radicals”.81) (2.3.76) = %%3 I B ISP(0). New York. 1970.3. the anisotropic dipolar interaction is averaged to zero by rapid isotropic tumbling motions.3. 39).3. L. 301 (1963).78) where the isotropic hyperfine splitting constant a is a = h’gOIBlgNBN$XdO). As we have stated earlier in this section. The observed spin density at the nucleus is due to electron spin correlation effects (configuration interaction). The theoretical calculations yield isotropic I4N hyperfine coupling constants from Eq.77) = haS * I.75) (2. These calculations should be applicable at least approximately to aliphatic nitroxide free radicals. This leads to the following expression for the isotropic hyperfine interaction : xi = 90 1 P 1 gN PNfXP(op ’ I (2. To a good approximation.” respectively. transl.79) that are equal to +58.82 2. J. which have isotropic hyperfine interactions equal to 45 MHz. 32 p. 6 . 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. is large compared to the anisotropy of the dipolar terms.3.f = hs * T * I. (2. 39.). 31 32 . Giaconnetti and P. NITKOXIDE SPIN LABELS where B is the (uniform) magnetization inside the sphere. Nordio. This is the case for lowmolecularweight nitroxide free radicals in solutions of low viscosity. G. and also in diatomic NO give a spin density p(0) that is positive. or inverse correlation time. Phys.3.3. T =Td 4.80) (2. Plenum. This occurs when the tumbling rate.5 MHz3’ and +40 MHz.82) A?. p.UU. (2. From classical magnetostatics we know that B = $nm(O) (2. (2.79) Theoretical calculations of sorbital spin density at the 14N nucleus in planar NH.. for a single electron the spin density at the 14N nucleus is zero. “Free Nitroxyl Radicals” (B. such as TEMPONE in G . Ma/. which have been discussed at length in the literature for various nuclei (see Ref.3.3.
3.0) = (.” 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. it follows that i T r { T ) = a.3. the electron orbital motion is “quenched. we obtain T. Nonlinear polyatomic free radicals have no firstorder electronic orbital angular momentum.90) . + 45 = 7MHz..69)].84) 44.88) The energy of interaction of this magnetic moment with the externally applied field H .86) are based on the theoretical evidence that the spin density p(0) at the I5N nucleus is positive. but the induced moment need not be in the same direction as S.3.3.3.85) (2. Here the hyperfine splittings are 16. The Spectroscopic Splitting Factor g (2. Additional evidence for the validity of this conclusion will be evident from subsequent discussions.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. that is.85) and (2. corresponding to an isotropic hyperfine coupling constant a = (2.89) (2.3.} = 0 [cf.) (2.:.0 0. These theoretical estimates are compared with experimental values of T in the next section.3. 38 (2. is (2.. In such cases the observed (highfield) splittings are equal to the isotropic hyperfine interactions. and thus a is positive.3.  76 N + 45 = 121 MHz.14 MHz. 2. 11. with the experimental value for the isotropic hyperfine coupling constant.8 0.3.5.2.05 G. as is the case for the spectrum in Fig.(16.3. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 83 water. (2..3.83) (2.3.8025 MHz/G) . The magnitude of this induced moment is expected to be proportional to the magnitude and sign of S. Note that since Tr{T. Note that Eqs. If we combine our previous theoretical estimates for the elements of the dipolar tensor T. (2. Eq.86) T.3. (2. = T.
(2. McConnell and R. see McConnell and Robertson. J .PNgNI H. and g. Proc.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. M.n orbital.3. 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.6. 61.34 2.PNgN1. From this discussion it follows that gz must be close to the freeelectron g factor. For an early note discussing these ideas. Sci. including nitroxide free radicals.3. The lowestenergy transitions contribute most significantly to the gfactor deviation..3. 1018 (1957). S. Chern.. terms are expected to differ much more from the freeelectron g factor. (2.92) 34 H. (This is the mixing of lowerenergy occupied molecular orbitals with higherenergy unoccupied orbitals). virtual transitions. This contribution is expected to be the order of magnitude of Ag/g smaller than electronspin interaction itself. since the only contribution to gz comes from highenergy pxe p. + p. McConnell. shown in Fig. In this discussion of the nuclear hyperfine interaction. where Ag is of the order of magnitude of the deviation of the g factor from the isotropic g factor. NITROXIDE SPIN LABELS The term X Zis often referred to as the electron Zeeman Hamiltonian. p.766(1958).9 1) we recall first that rapid tumbling averages out the anisotropic terms. H .. . Robertson. p. The g.H. For nitroxide free radicals. Acad. E. 44. A . which is a pz orbital in our axis system. these transitions involve the halfempty 2p . To understand this spectrum in terms of the spin Hamiltonian equation (2. Natl. so we are left with an isotropic. g can be assumed to be a symmetric tensor.. p.3. we arrive at the following expression for the spin Hamiltonian : &? = 1P 1 * s g ’ H IhS ’ T * I . Phys. + pz.91) We have included the nuclear Zeeman term PNgNI* H. U. These qualitative expectations have been verified for a number of organic free radicals.1 33 . 11. For molecules such as nitroxide radicals. M. the magnetic interaction between the spinorbitinduced orbital magnetic moment and the nucleus has been neglected. Transitions between localized atomic 2p orbitals such as the following are “allowed”: p.84 2.The Spin Hamiltonian From the above discussion. timeaverage Hamiltonian = I P l g S * H IhaS. .
3. The precession frequency v is h l p l g H o 21 9. ~ ~ and h(v 2. (2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 85 The resonance spectrum can be obtained from the spin Hamiltonian equation (2. 1. hv/g 1 B I. Chem. 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.)/glPI. (2.92) by quantummechanical calculation.5 MHz when H o N 3300 G. This precession frequency is orders of magnitude larger than the precession frequency of the nucleus in the external field.3. These energy levels are only accurate for strong fields.3. Thus the secular or timeaverage interaction between the electron and nuclear spin is = IPlgHOSh + + jNgNHO1h. Priborostr. 12. The corresponding energy levels and resonance transitions are sh. this spectrum can also be obtained by means of elementary physical arguments. 43. 0. D. Ryzhkov and A. see R o ~ a n t z e v ~ ~Ryzhkov and S t e p a n o ~ . McConnell..36The free radical often takes 35 76 V. Thus H o = h(v + uI. then in a fieldswept spectrum resonance signals are observed at values of the applied field H .= +1. However. Pby. if h is the direction of the external field.g. and H.y.7. 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.own schematically in Fig. For calculations of energy levels in low applied fields. ) (2.94) I. M.2. P.a)/g 1 p I. ++ + where v is the fixed microwave frequency. or in the nuclear hyperfine field (haI)/(g [ p). J.3. Stepanov.35(1962). Thus. H. Cornell.3. that satisfy the equation (2. Griffith.93) In strong applied fields (e.3.3. 3000 G and above) the components s h and of the electron and nuclear spins in the applied field direction are good quantum numbers. W.0. 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. G~Ofiz. (2.97) A fieldsweep spectrum thus consists of three signals at applied fields equal to + a)/g I PI. M.. s h = k. 4.95) When we apply the selection rules Ash= (an allowed magnetic resonance dipole transition) and Al.2909 (1965) .96) hv = IpIgH. and h(v . ha]. = 0.3.
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. the magnitudes of the principal hyperfine interactions. As one example of this type of experiment.1 G when the applied field is perpendicular t o b. The angle 0 = 90" represents a field direction parallel to b. 12 shows paramagnetic resonance spectra of the Noxyl4'. fixed orientations relative to the axes of the host crystal. 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. The angle 0 = 0" represents a field direction perpendicular to the twofold monoclinic symmetry axis b. 5a.02 G when the applied field is parallel to the crystallographic axis b. . 12. 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. Plots such as this may be used to determine the directions of the principal axes of the nuclear hyperfine interaction.0 k 0. The maximum hyperfine splitting is 31. Fig.86 2.91 k 0. the directions of the principal axes of the g tensor. the minimum hyperfine splitting is 6. and to the z axis of the spin label (indicated in Fig. and the magnitudes of the ' 0 IOG FIG.'* 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. NITROXIDE SPIN LABELS on one or more welldefined. I).
5a can be used to determine the elements of the spin Hamiltonian discussed in Section 2. 12 and 5a from an empirical. That is. one observes a maximum splitting of 6.is the maximum field strength. The principal axis z is easiest to locate experimentally.0024 f 0. because the hyperfine splitting is not only an extremum. Many biophysical applications of anisotropic paramagnetic resonance spectra do not require a detailed quantitative analysis in terms of a spin Hamiltonian. the principal axis direction that is simplest to find experimentally is the principal axis corresponding to the largest hyperfine splitting. When the applied field H. We denote this principal axis direction z. the largest hyperfine splitting. In the case of label (I). yx and g. and a minimum splitting of 5. as well as the corresponding principal axis directions.91 _+ 0. If one plots the resonance position for the central hyperfine line. and to one another.02 G. and the direction perpendicular to this gives the third principal axis direction with intermediate nuclear hyperfine splitting. but is an absolute maximum and is much larger than the other hyperfine splittings. plots such as those given in Fig. the highest field position of this resonance defines the minimum principal value of the g tensor. strongly immobilized orientation in a single crystal. When the applied field H. This maximum field position is an absolute maximum for this direction. This principal value of g. SPINLABEL THEORY A MATHEMATICAL TREATMENT 87 elements of this tensor. utilitarian point of view. can be determined from the maximum and minimum . The other two principal axes are necessarily perpendicular to z .3. In other words. the nuclear hyperfine splitting is an extremum. Therefore we first discuss the significance of data such as are given in Figs.where H. In the case of the free radical (I) dissolved in cholesteryl chloride. and then subsequently justify this discussion in terms of the spin Hamiltonian.. x and y . so these principal axes of the g tensor and T tensor coincide. from the principal axis direction. 31. The principal axis directions of the g tensor and the components of the g tensor can be found in much the same way. can be calculated from the equation gz = hv/ I p I N. It is found that gz = 2. g.6.1 G. is observed where the applied field H.83 f 0.2.0001. 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. is parallel to the crystallographic axis b. when the applied field is rotated in the ac plane (percendicular to b and to z). These principal axis directions are denoted x and y. is parallel to a principal axis of the nuclear hyperfine interaction. The corresponding field direction gives the direction of this principal axis.1 G.3. For nitroxide free radicals with a fixed. The other two values of g. there is no firstorder change in the hyperfine splitting for any small deviation in the direction of H.31 & 0. 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.
U . It is found that g. 708 (1966).88 2.37 Griffith et permitted determination of T and g values for several simple nitroxides. 43.651 (1971). Anisotropic paramagnetic resonance spectra can also be observed when nitroxide spin labels are attached to protein molecules in single crystals. Chrm.0001 and gy = 2. J. Sci. Biol. 1359 (1970). Berliner and H . Res. The directions of these principal axes deviate from those of the nuclear hyperfine interaction by a rotation around z of about 10". 53. S. Bauer and L. J . The earliest study of nitroxide free radicals in single crystals was made by This study and others. Griffith. Mol. For example. Phys. 1 (1979). Biochem. and also in single crystals. and the OH refers to the hydroxyl group of the activesite serine. Berliner. L. 128.0060 +_ 0. S. Null. R. M. McConnell. Mol. Biol. = 2. Acud. 165 (1979). Commun. Bauer. A . J.0090 +_ 0. J. ~ ~ '' L. . J. and paramagnetic resonance spectra can be observed from these crystals. McConnell. J. Berliner and M c C ~ n n e l have shown that the active site of l~~ the enzyme crchymotrypsin can be acylated in solution. such as that of Libertini and Griffith. 129. Lihertini and 0. 55. ~ " . L. there is a similar uncertainty in the directions of the x and y principal axes of the g tensor. J . H . Berliner and H.39 according to the following reaction : 0 EOH + 0 __jc t 0 E0C II 0 t Here E0 I designates the enzyme achymotrypsin. S. Under the experimental conditions employed by Berliner and McConnellJ9 the enzyme deacylates at a negligible rate in the single crystals. Biophys. M. 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. Proc.001. More extensive studies of this reaction in solution and in single crystals have been carried o ~ t . 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 " . 3R 3y 4" 4' L. Berliner and R. J .
2.2 x lo4 G acting on an 14N nucleus.the electron spin is quantized in the external field direction. then the spin Hamiltonian becomes 8 = IpIShHh* g * h 8= lPIHsh(gz(‘z  + hShm(h.3.99) where the first term arises from the hyperfine field due to the unpaired electron and the second term arises from the externally applied field.) 2 2 l/2 . we give simple physical arguments using (2.3.3.98)that lead to a good approximation to the observed energy levels and spectra.103) . 2 = [PIS g * H  + h S .3. and is small compared to the first term (9500 MHz when H 3200 G). at least as a first approximation. + (2. The smallest value of IT I is .C. and that the direction of the hyperfine field depends on the direction of the electron spin angular momentum S. Instead. Thus the nuclear spin magnetic moment &gN I “sees” a magnetic field equal to hS * T/pNgN + H. * (2. T T h)”’.91)]. + + gyCz) + hShm(T:c: * * (2.3. The last two terms in the spin Hamiltonian are linear in the nuclear spin. (2.3. T .16 MHz. in terms of the spin Hamiltonian derived in Section 2. Eq.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.I + PNgNI H.100) The second term is of the order of the nuclear hyperfine interaction. Note that in general the hyperfine field is not in the same direction as the externally applied field.102) T:C: YxC: + T.3.3. If rn is the quantized component of the nuclear spin in the local 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 .the secular comS ponent of this field is hShh * T. (2.T 1 (2.101) The 14N nuclear spin “sees” a local field parallel to h T.4 [cf. SPINLABEL THEORY : A MATHEMATICALTREATMENT 89 We now return to a brief discussion of the resonance spectra of oriented spin labels in single crystals.T.3. Applied fields H of the order to 3300 G can then be neglected compared to this hyperfine field. Under these conditions the simplified approximation spin Hamiltonian is simply  % = lfllS*g*H hS. (2.3. defined by the unit vector h:  2@ = I ShHh* g * h + hS . which corresponds to a field of the order of 5. 7 100 MHz.I.
c.109) The applied fields giving resonance transitions are therefore HYm) = ( where m = . y principal axis system of the spin Hamiltonian.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. from labels attached to proteins in powdered crystals.3.3. The paramagnetic resonance spectrum of an isotropic distribution of strongly immobilized nitroxide free radicals is illustrated in Fig. sin 8 sin (2. sin 9 cos (6. and q. These radicals will then exhibit hyperfine splittings that range between T.c. c.8. 13. In terms of the polar and azimuthal angles 8 and (6.106) c. 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 . NITROXIDE SPIN LABELS Here c z ... + 1. for example. In isotropic distributions of nitroxide radicals. find that observed = we resonance transitions are observed at applied fields H for which hv = l/?lgH + hTm. = = = cos 8.108) Again using the selection rule for allowed electron spin magnetic dipolar transitions in strong applied fields.3. Certain qualitative features of this spectrum are easily understood and have been covered by Section 2. Z C+ T:c~ + ~ (2.1.Tm)/gIPI ) (2. or from labels rigidly attached to large. 6. (2. ex. these direction cosines are c. + 9. M v .2 and illustrated by Fig.3. or nearly perpendicular. +_$. (2.Z + g.110) 0.3. some radicals have orientations for which the principal axis z is perpendicular.3. Such spectra can arise. let 4. For brevity.105) (2. to the applied field direction.A m = 0.107) and T = ( T . are the direction cosines of the field in the z.c. AS. x. 9 = g.3.c.90 2. 2.2.3.104) (2. slowly tumbling proteins in solution.
and z when 8 is near 90°. SPINLABEL THEORY: A MATHEMATICAL TREATMENT 91 FIG. As we show below. These six resonance signals are then overlapped with the rn = 0 component of the hyperfine triplet corresponding to the principal z direction. the separation of the peaks of these two outer “absorption curves” is to a very good approximation exactly equal to T. An interesting and very useful feature of these outer hyperfine extrema is their shape. The paramagnetic resonance spectrum seen in Fig. As we see below. inner hyperfine structure is well resolved when there is axial symmetry. is exactly parallel to 2. . It is therefore not surprising that the “perpendicular” hyperfine structure is not well resolved. to a good approximation the outer signals of this derivativecurve spectrum have the shape of the ubsorption curve itself. 13 reflects the fact that these signals correspond to the H. 11 z extremum in the hyperfine splitting pattern shown in Fig. whose formula is shown. and q. 13 is the derivative of the absorption spectrum of an isotropic distribution of radical orientations. for the special case that the applied field H. We shall therefore discuss these outer signals in some detail.({).. 13. Paramagnetic resonance spectrum of a fatty acid spin label. where  5 =H = Hr(8.. however. = T. and when the resonance linewidths are narrow. Centered about each of these field positions are hyperfine splitting of T.11) . The apparent relatively high intensity of the outer hyperfine signals in Fig. 5a. to the y direction being determined by g y . g = g y . strongly Immobilized in phosphatidylcholine bilayers. Since the resonance position does not change rapidly with the angle 8 between the applied field H.2. At X band these two fields are separated by 3 G.’’ Moreover. T. The outer hyperfine signals in isotropic distributions have been very useful in many biophysical application of spin labels.3.3. #). relatively large number of molecules contribute a resonance absorption at positions of extrema. Let the paramagnetic resonance absorption spectrum due to the hyperfine state rn be a. (2.
Two approximations are in fact involved.1 13) This derivativecurve lineshape expression can now be greatly simplified by using the following approximation to the resonance line position H.3.O" ? 8 70".. This range is due to Ho FIG 14.O) + &(I .3. 5a. 4) N.1).H. 14 are the same as the data in Fig.(o. (2. The quantitative aspects of these approximations are illustrated in Fig. which shows the resonance line positions HY(0. 4): H.cos) for m = k 1.114) This approximation is very important and needs to be considered in detail.(e.1 12) The corresponding contribution to the derivativecurve spectrum is (2.8 for further discussion.92 2. 8=n/2 a m ( < ) sin 6 do. 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. 0) + ~. The data in Fig. First. 14. 4) for the cholesterol spin label (2.4.(O.cos 6). Hyperfine splittings as a function of position of orientcd spin labels.(i .3. NITROXIDE SPIN LABELS and HT(6. (2. the $ dependence of the line position is neglected."(O. except that in the upper part of Fig.. and the 6 dependence is taken to vary as cos 8.3. 4) for any possible value of 4. The contribution of hyperfine component m to the total absorption spectrum is Am(H) = S. In the lower half of this figure is given a rough plot of the total range of Hr(8. . 14 a plot is given of H. See Section 2.3._.
and that the highfield ' signal is negative since 1.3.7 we saw that the paramagnetic resonance spectrum of a nitroxide free radical depends on its orientation in space (relative to the 42 43 D. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 93 deviations of the spin Hamiltonian from axial symmetry. (2..3. D. .(H . The function a. M.Hr(0.1 G (highfield line). ' (2. 0. with the result dAm/BH = (/Z. 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.(H .3. 11 exhibits three sharp hyperfine components. 1 (1974). .3. 4 3 2.114). 14.6 G (lowfield line). Since the probabilities for the three 14N nuclear spin quantum states rn = 1. 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. as discussed in Section 2. M . Gaffney and H. Further quantitative description of the strongly immobilized spectra may be obtained using computer calculations. . Reson. using the approximation in Eq. . The observed separation of the outer hyperfine signals is equal to 2T.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). The g factor gz can be determined from the center point of these outer extrema. J . But the derivativecurve peak heights are evidently not equal. .113). McConnell.115) Here + .3.114) can now be inserted in Eq. J . . and 1.3. (2. In Section 2. (2. the integrated intensities for the three hyperfine components are expected to be equal.9.is negative. Lett.O)) + .Hr(0. (2.3. M a p . Chem. The approximation in Eq.3./m)a. = 47.2. 16. (2.113) is readily carried out.114) is invalid in this region. which is centered at the position it would have in a perfectly oriented crystal for which the applied field H. In the plot in the upper half of Fig.Effects of Isotropic Motion on Spectra The paramagnetic resonance spectrum of TEMPO shown in Fig.3. From these plots it will be seen that the approximation in Eq.470 (1974) B. Note in Fig. is parallel to the principal axis z. The indicated integration in Eq. McConnell. 25. (2.O)) is of course just the absorption curve. Phys. Thomas and H. . indicates the term arising from the 6' = 0 lower limit of the integration.1 are very nearly equal. which is dropped because Eq.' .3. 4 2 . 13 that the lowfield signal is positive since 1 is positive. with the understanding that only the derivativecurve spectrum arising from radicals for which 6 7 45" will be accurate. = 18.3..4.
when the rapid. 563 (1956).3. T I (2. Phvs.” We now turn to a quantitative treatment of paramagnetic resonance line shapes of nitroxide spin labels for the case of slow isotropic rotation. rather than the instantaneous spacing. 4. 104.44 The simplest approach is to use the Bloch equations.Mo)k + DV’M.94 2. of S and I in the field direction are “good quantum numbers. the largest residual linewidths even with very fast isotropic motion are found for the highfield line (rn = .yM x H . See Berliner’ and additional references contained therein. Here M ( 0 . there ensues the phenomenon of motional averaging. Torrey. That is. modified to include the effect of diffusion : dM 1 _ . Shimshick. Lett. 11 rather than the orientationdependent spectra.l). Pkyv. M . . This gives rise to the sharp threeline spectra seen in Fig. On the other hand. The theory of hyperfine multiplet line shapes has been developed extensively. or strongly immobilized spectra. Understandably. and the position of the highfield (m = . Cp. this rotational averaging is never perfect. 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.. t ) sin 0 de d 4 44 45 R . The basic idea is that in the presence of molecular motion the spin Hamiltonian (2. Chrm. E.3. This is because the axes of the hyperfine and gfactor dyadics T and g are molecule fixed. 115 (1972). McCalley. That is.7. and H. M = M(8. H. NITROXIDE SPIN LABELS direction of an applied magnetic field).(ui d c T 2 1 + uj) .1) line changes most with angle (see Fig. In the presence of the rapid motion considered here. Of the three resonance lines.91) becomes time dependent. Here M is the spin magnetization per unit volume (or per unit area on a sphere. then.( M . t).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.3. in the presence of rapid motion the components Sh and I . considered in Section 2. Rev.. J . C . 5a). whereas the spin vectors S and I are effectively fixed in the laboratory frame. the position of the central line (rn = 0) changes least with angle of rotation. 13. and the smallest linewidth is usually found for the central hyperfine line (m = 0). the microwaves are absorbed at the average spacing of the hyperfine energy levels. For rotational diffusion the magnetization is represented as a function of 8 and Cp. . C . see below) and D is the diffusion constant. isotropic diffusion rotation of the spin label becomes fast enough.
N 27t sin 8. M. corresponding to the center of each zone is 8. . For the simple case of a spin Hamiltonian with axial symmetry.3.3.3. (2.119) (When the direction 8 = 0 is taken to be the direction of the applied strong magnetic field H. M = M(8.117) From differential calculus the quantity V2M(8) can be written as a limit.) The polar angle 8.118) 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. The total electron magnetization in each zone is M.3.122) . t). 2. .so that the angular width A of each of the zones is the same. n.120) and the zones are numbered n = 1. For this special case V2 in Eq. we need only consider the dependence of M on 8..*)A. r = I).iA)M(O .3.M(8.116) is (2. .).3. (2. . This approximation is described below. = ( n . (2.3. V2M(8) = lim {(1/A2 sin 8)[2 sin 8 cos iAM(8) A0 + sin(8 + $A)M(8 + A) + sin($ . 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. = / On i A12 2nr sin BM(8) do. A = n/2N.A12 For small A (and a unit sphere.121) 8. 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.3.2. Divide a unit sphere into N zones between O = 0 and 8 = in.A)]} (2.
+~ (. the Bloch equations are 0= k2'3 +  .124) Here n(n k 1 + n ) = (D/A2)sin(Q.M:)k  M.]M. (2. k +A)/sin(0.3.123) include the effect of the anisotropic hyperfine interaction by regarding the effective field acting on the free electron as depending on d. [1/(27c sin O.123) (2..116) and replace M(8. Equation (2.(u. j) .+. in the limit of no saturation. 1 I ii 5 N .u. n = N. + n(n + 1 + n)u. the angle between H. x H . and the quantities n(n _+ 1 .n ) give the cor. 1 for 1 In. responding expressions for the rates of magnetization entering the nth zone from the adjacent zones. as assumed for the present calculation. the principal axis corresponding to the largest 14N hyperfine splitting.3. Under steadystate conditions.1 n + n)M.125) (2..1. yeH:.: __ = m l dt yM.1 + +. + n(rz . (2. and in a coordinate system rotating at the microwave frequency. (2. the angle describing the zone in which the principal axis of the radical lies. where M. 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. + n(n + 1 + n)M.+.1 + n)u.) by .128) .. and on the orientation of the strong applied field H. we need only consider the angle 8.TI ( M n Z.+ 1 + n(n .3.' gives the rate of spin magnetization leaving the nth zone due to rotational diffusion. t. When this spin Hamiltonian has axial symmetry. n + 1<N.+. 1..126) = (D/A2) cos A/cos $A. and k.3..96 2. The quantity t. The modified Bloch equations (2. When this substitution is made.3. relative to the principal hyperfine axis of the nitroxide spin Hamiltonian..3. k A).123) is a suitable form for computer program calculations.3.3. (2.i T 2  1 1 + u.u.. these equations have the same general form as do the Bloch equations when modified to include chemical exchange effects on nuclear resonance spectra. is the magnetization in the nth zone. one obtains the following differential equation for M.127) + n(n + 1 + n)u. = (D/A2)cos+A. NITROXIDE SPIN LABELS We may now substitute 0 = 8 in Eq. (2. f 1 .
k 2H.110): HreS(n.44 using a computer and employing a 3” angular mesh ( N = 30 zones) and a onegauss field mesh. ~ ~ transitions are induced. has been developed by H ~ d eThomas.g L . Methods Enzymol. 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 . 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.129) where Ag = gl. Hh = H .3. This time is of the order O f 5 x l o p 7sec. so this approximation introduces no significant error for approximating rotational correlation times in the range 2 x 3x sec. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 97 In these equations H is separated into its two components. Hyde. 439 (1978).3. . is the resonance m). p. B ~ o ~ ~ J24. subject to a nuclear hyperfine field due to a nitrogen nucleus with spin component m. 49. A general theoretical treatment of the effects of slow motion on spinlabel paramagnetic resonance spectra has been given by Freed (see Ref. Thomas. 53). can be obtained directly rn) from Eq. D. ~ ~ J. 480 (1977).130) that the derived correlation times should depend linearly on the viscosity. The above set of 2N simultaneous equations was solved by McCalley et a1. A h .. J. + T: sin’ + (2. D.I. termed saturation transfer spectroscopy. Rimn. In the case of globular proteins we expect from the Debye equation z2 = 4nqa3/3kT (2. 1. Shimshick and McConnell’ have used a particular extrapolation method in which the field shifts are plotted versus (T/v)”~. A convenient approximate expression for Wes(n.2.0. = + (2nv/I Y e I )C 1 + (Ag/g 11) sin’ On] rn(Ti cos’ 8.3. . Dalton. With a knowledge of the strongly immobilized hypcrfine separation. x 48 L. 149 (1976) 46 ” . . the plot of correlation time versus q/T was obtained.47and Dalton48to determine very slow . i cos(2nvJ. A novel technique. In cases where the same proteins have been studied using fluorescence spectroscopy the results of the two methods are in excellent agreement. and m = 1. 8. where Hres(n. S.Hres(n. McCalley et ~ 1 estimate that this approximation 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. The data follow the Debye equation to within experimental accuracy. (2. H = H.3. Mrryn. rn) field for an electron in zone n. R.
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. If the molecular motion is sufficiently rapid. 2. since T. we simple isotropic hyperfine spin Hamiltonian discussed above in connection with Eq. Since the components of S and I perpendicular to h precess at very different frequencies. y& = yi. (2. Ty. the spin Hamiltonian Z [cf. = 3a.98 2... (S and I may sometimes be fixed to the laboratory reference frame simply because of conservation of angular momentum. their interaction averages to zero.3. 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. Eq. 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.lh(Y. for example. = yiz = 4. Y h y = yh. are good quantum numbers. Thomas and M ~ C o n n e l have given a comparal~~ tively elementary treatment of this problem using the Bloch equations. NITROXIDE SPIN LABELS molecular motions. (2. = yhz(t) are the timedependent direction cosines of the field direction h in the principal axis system x. This is because (i) the dyadics g and T are anisotropic. (ii) the principal axes of these dyadics are fixed in the freeradical molecule.131) Imagine that a magnetic field is applied in the direction h. hh.93). S (2.10. + T.y.If the rapid molecular motion is isotropic. and I .131).3. with correlation times as long as a millisecond. where a is the isotropic hyperfine coupling constant. namely x. the hyperfine energy is Here yhx = yhx(t). z of the hyperfine Hamiltonian Z ' h f . Both the experimental and theoretical aspects of this method are too involved to permit discussion here. (2. In the presence of molecular motion. %hf(t) = h ' T ' I.f hS. + K= z)..3. the observed interaction energy is the time average of Zhf.(t). Line Shapes with Fast Anisotropic Motion and Partial Orientation.. Eq.133) .3.Frequency Amplitude Order Parameters Highfrequency anisotropic motion can arise. obtain the . to a good approximation in high fields.3. + T. If the rapid molecular motion is not isotropic in the z .3.91)] becomes time dependent. ' (2. T x x + Yi.) Consider the timedependent hyperfine interaction Zhr(f).f : z. Thus. the field being so strong that both S.
z principal axis system of T. y‘.1). y‘) such as (2.yXfz 2   + y. The residual or effective hyperfine interaction can be represented by the hyperfine dyadic T :   X f = hs * T’ * 1. and The nine quantities $.137) where yf.3. Then from Eq.139) The effective Hamiltonian is The elements of g’ are related to the elements of g by equations similar to those relating the elements of T’ and T [e. k’. Thus there are three equations (one each for z’. Instead of using the direction cosines %to describe the motional averaging of one axis system relative to the other. and let the strong applied field be in the direction of k’.  (2.g. i = x‘.3. Since the principal axes of g and T nearly coincide for nitroxide free radicals...141) . defined by the equations sij= +(3y$ . + yf. (2..134). the order parameters S. Let the principal axes of T’ be i’. it is conventional to use a set of equivalent numbers.. z‘..3. z. = 1.  SPINLABEL THEORY : A MATHEMATICAL TREATMENT 99 sense that yix = y t y = yiz = $. y. are not all independent of one another.3.137)]. j = x. are time averages of the direction cosines of z’ in the x.3.. . then there is a residual hyperfine interaction Xhf which is necessarily anisotropic. so do the principal axes of g‘ and T’. and yfCy and yf. Tk S y.. We obtain simply (2. y ..138) and also three equations (one each for z . y ) such as . (2. x’..134) A quantitative relation between the elements of T’ and T is easily obtained.3. j’. h (2. (2.2. Eq.3. x. Similar equations hold for the elements Tbfy.3.
S. Tyy). 39) have the equivalent forms c Sij i j = 0. and particularly 1 T. and g one can..SZ>=. The other order are parameters Syfx... (2.j) for phospholipid spin labels incorporated into phospholipid bilayers.. a determination In of TL. observed values of T i . . ~ = S.and S. and SyCZ obtained from the above order parameters and Eqs.144) and (2.143) 1sij= 0..138)and (2. motional averaging very often leads to effective Hamiltonians X ' which account for observed spectra to within the experimental accuracy. the second member of the righthand side of Eq.I are small for iiitroxide free radicals... a similar way. + i(S...145) for SZCz and SzCx Szry.S. In such cases. two independent order parameters can be determined..3. In view of the fact that T. (2.100 2..3. .(gyx .) .: The principal elements of T' and g' can often be determined from experimental spectra using methods analogous to those described in Section 2. where the effective Hamiltonian has axial symmetry and the symmetry axis is k'. (2. In the frequently studied liquidcrystallike systems (phospholipid bilayers and biological membranes)...Sz. (2...3. and Szfysince S z j .. 2 gzz . 1 + SzfzT.142) and (2...T..J(TX. 42) (2.&IXA . and g. From observed values of T'. Let Ti1and T . From the foregoing equations one can obtain the following convenient set of equations: ~ ~ Yzz s...143)..3. (g.3.3. determine all the elements of the orderparameter matrix..dgxx + gyy) + gyy) si 1 ~~ (2... enable one to solve Eqs. In this work it was found 1 : experimentally that Skfi: Sk!j 1 +SkCk.gpy)(2Sk'i + Sk'k) .. g'..3. The relevant equations for this case are given below T i s z = ~ ( ..3.144) A similar equation holds for g:. and g:. For example.. and g:. yields the values of S. .. NITROXIDE SPIN LABELS In terms of these order parameters the sum rules in Eqs. Tyy.These two quantities then permit the calculation of S.8.3. + SzcY= 1 ..) be the elements of T' (and g').147) These equations have been used by Gaffney and M ~ C o n n e lfor determinal~~ tions of order parameters Sk*kand Sksl(and hence S.3. T.  (2. Sr!y.3. in principle. + .
146) can often be neglected for fatty acid and phospholipid labels in bilayers and membranes. 314 (1971)..). leading to the convenient expression sk*k = (T.CH.. SPINLABEL THEORY A MATHEMATICAL TREATMENT 101 A $n FIG. is obtained .’ /NYo 0 0 ‘(cH.NMe o Me I 0 A I Me Valucs of T. (2. (b) spectra calculated using Gaussian line shapes. Am. (Reprinted with permission from Hubbell and McConnell. where k is the axially symmetric symmetry axis.3. SOC. are obtained from the observed spectra as indicated. they generally signify the order parameter S. (a) Observed spectrum. given in Eq. Copyright by the American Chemical Society. (c) spectra calculated using Lorentzian line shapes.3. 0P0CH..2. (center point of the inner hyperfine extrema).%Txx + Tyy)l* (2.).. First approximations to Ti are obtained as indicated.(CH. anisotropic molecular motion in more detail. J .’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).0CR I II I ii CH.) A  (2.3. The value of g.93.l  Ti)/[Kz . Before discussing rapid. and z is the principal axis of the largest hyperfine splitting (norbital axis). Chem. Applied field is 3300 G.. let us illustrate the development up to this point with some early spectra and calculations by Hubbell and McConnell. i t C CH. Paramagnetic resonance spectra of a phospholipid spin label in an aqueous dispersion of egg lecithincholesterol (2: 1 mole ratio).cocH I1 CH. at room temperature.148).3.15. as in the first approximation to g.148) When “order parameters” for nitroxide spin labels are referred to in the literature without further definition.
3. W.” Smith. J . The physical origins are entirely different : the isotropic hyperfine interaction is a magnetic interaction. Cun. J . J.J. 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. Mugn. The exchange interaction is analogous to the contact hyperfine interaction only in that both are isotropic. 83. Lin and J . The first is the coulombexchange (or simply “exchange”) interaction. P. The second is the (electron spin)(electron spin) magnetic dipolar interaction. P. An extreme case of a strong dipolar interaction is found in the case of a nitroxide biradical prepared by Keana and Dinerstein’ : 4y J. This process was then repeated until the best overall agreement between observed and calculated spectra was obtained. Phys. 5 1 I.2808 (1971). Chem.460(1974) J . Sac. 379 (1979). and 0. C. C. Dinerstein. I5 that the comparison between observed and calculated spectra is good. J . C. Reson. F.49 See also Lin and Freed. but not perfect. 4183 (1977). 57. 93. W. The origin of this interaction is discussed in textbooks on quantum mechanics and is not discussed further here. L. H. Keana and R. Freed. Burke. ’* ’’ . 15. H . There are two interactions that affect the resonance spectra. l(1979). Smith.” and Libertini er 2.102 2.H. see Freed. It will be seen in Fig. Phys. Am. These parameters were then used as the first step in the computation of spectra.11. Freed. A. Griffith.4. For a guide to more general treatments of relaxation and resonance spectra. using either isotropic Lorentzian or isotropic Gaussian line shapes. Chem.3. and the isotropic electron spin exchange is coulombic. Biochem. Jost. Libertini. 66. J . J . J. Chrm. NITROXIDE SPIN LABELS from the center point of the outer hyperfine extrema. analogous to the (electron spin)(nuclear spin) magnetic dipolar interaction responsible for the anisotropic hyperfine interaction discussed in Section 2.
Dipolar interactions between the electron spins on different spin labels are often manifest as broadenings of the paramagnetic resonance lines..3. Molecular models suggest that the electrons are 3.57) and (2.3.149) (2. Keana and Dinerstein have analyzed the observed spectra under the assumption that the colombexchange interaction is large compared to the nuclear hyperfine interaction.’).6 x IOR ’) is R = 4.’[ve P: . in rigid glasses.151) (2. 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. For the case of two electrons.3.152) P e = solPls.3.3. where s = s + s’. 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. 55) where D = ST.61) are x d = r . in which case the total electron spin S is a good quantum number. The paramagnetic resonance spectrum has been observed in solution.7 A.3. 54) (2.S.85. The corresponding distance estimated for two point dipoles ( D = 80. the dipolar or “fine structure” contribution spin Hamiltonian can be written + E(S: . (2.3.4. the equations analogous to the dipolar equations (2.  q). E = +(T.156) The observed spectra can be accounted for with D = 777 MHz and E N 0. (2.153) In this case. Xd= DS: (2. (2. the uncertainty reflecting various plausible lowenergy conformations of the piperdine ring.3. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 103 The two unpaired electrons in this molecule are quite close to one another.3(pe * r)(&  . and in oriented multilayers.2 A apart. P L &d = solPls’? = hS * T ’ S’.3.3. large splittings due to the electronelectron dipolar interaction are evident in the observed spectra.3.3. A particularly interesting example can be found in the work of Marsh ~ .2. Although the nuclear hyperfine structure in these resonance spectra is not well resolved.W2].150) (2.
fluctuating magnetic field. as first described by Taylor et and by Leigh. Broplzys. S.Marsh and I . S. A .5h For many enzymes. S. the resonance signal from the nitroxide group is essentially obliterated.104 2. S. C‘hem. A c ~ u ) 298 (1973). but where they occasionally diffuse together and undergo strong spinexchange and/or dipolar interactions. Marsh and Smith have used such data to study the “condensing effect ”ofthe cholesterol bilayer structure. In the presence of an intense. where paramagnetic spin labels are normally far apart and noninteracting. Another important situation arises in liquids. Spinspin dipolar interactions between spin labels and paramagnetic metal ions have often been observed in studies of metalcontaining enzymes in solution. J.3 cos2 8.” powdertype spectrum since the rotational correlation times of the enzymes are large compared to the reciprocal of the hyperfine splittings. who have observed dipolar splittings due to neighboring pairs of cholestane spin labels of (I) when incorporated into phosphatidylcholinecholesterol bilayers. this interaction may manifest itself in a most unusual way. 54”. bound spin labels can exhibit a “strongly immobilized. 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). Biochim. J . Phys. with the dipolar interaction only producing a relatively small broadening of the lines with increasing radical 54 I . 2.1 2. 52. Leigh. Leigh. Such effects have often bcen observed for various free radicals in solutions. For such molecules the dominant part of the dipolar interaction. 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. 2608 (1970). except for those (nitroxide spin)(metal ion spin) pairs whose orientational vectors make angles close to the magic angle. vanishes. 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. Sci.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. to the external magnetic field. or liquidlike systems. C. J.3. and M. 64. Sometinies the major spectral effects are due to the spinexchange interaction. Cohn. in frequency units.Taylor. Nurl. Smith. U. proportional to I . Proc. and have in fact been studied in somc detail for nitroxide free radicals. P.219 (1969) 5 b J . with comparatively little change in the characteristic shape of the strongly immobilized spectrum. 55 . Acud. NITKOXIDE SPIN LAHELS and Smith.
. whereas u(0) = u(0) = u( . one on each radical. 0. = 0 and I. because the overlap of electron wave functions is required for the effect. 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. . that is. those for which the nitrogen nuclear spins are I . Thus. We can now understand the effect of spin exchange on the paramagnetic resonance spectra of nitroxide free radicals. and that the spin wave function for the odd electron on the second radical is Q2(2) = a.3. = 1. is an operator that permutes the labels of the electrons. This can be demonstrated in a formal quantummechanical way through the use of Dirac spinexchange identity: (2. 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). the spinexchange interaction between nitroxide radicals depends strongly on the distance between the NO groups. In an applied field of strength H.3.u. 22.2. say to within the van der Waals radius. it is more accurate to say that the spinexchange interaction produces an exchange of the electron spin momentum (or magnetization) on the two radicals. and in the presence of rapid exchange (compared to a in + .h' . a solution containing nitroxide free radicals contains three groups of electron spins. g I P I H . In the presence of the exchange interaction. the spinexchange interaction becomes very strong . 2 10 A) the spinexchange interaction can be assumed to be negligible.I) = u( . The corresponding precession frequencies are v = gIPIH. = . Consider that the highfrequency (or lowfield) resonance signal is being observed. For example. The spinexchange interaction between nitroxide free radicals can be thought of as equivalent to a physical exchange of two electrons. = 0 and I. 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. Since efectrons are. This then corresponds to an interchange of spin angular momentum between the two radicals.1. identical to one another. and in the absence of spin exchange.. = . when the radicals in solution are far apart (i. In the presence of slow exchange this then leads to a simple lifetime broadening of the resonance lines. the II( I) and u(1) magnetization is transferred to the I . strong relative to the nuclear hyperfine interaction and electronelectron dipolar energies. Just as is the case for chemical bonds.157) In this equation P. of course.e.' . When the radicals come closer to one another. 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). k . SPINLABEL THEORY : A MATHEMATICAL TREATMENT 105 concentration.1) = 0.(t)cc(2) b2(t)P(2). gI/3IH0h' + a.. and this loss of magnetization is replaced by the incoming zero magnetization of the I .1 states.1 states with no loss of magnitude or phase.
If the liquid is “ideal” the lifetimes T and T . (2.106 2. 6 I the collision is weak. For our present purposes it is sufficient to imagine that the liquid has a quasicrystalline structure. be the duration of the collision. In a strong collision the spin magnetization has.3.’ is its hopping frequency.be the probability per unit time that a nitroxide radical has a “collision” with a second nitroxide radical. Let T .’) to a narrowing of the resonance to a single line. of course depend on the detailed molecular properties of the liquid. or greater. let T . and if J T . 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. are the concentrations of solvent and radical in the solution. In considering the effect of collisions on the paramagnetic resonance spectra. (2. and T. Of the collisions that take place at a rate of T . Let T . be the broadening of any given resonance line due to spinexchange collisions. we must decide first whether these collisions are strong or weak. so that the molecular environment of each radical has a relatively welldefined structure. 1/T2 = + * 1 / ~= 3. Here a “collision” means that the associated exchange interaction is of the order of magnitude of the hyperfine interaction.)P..’ . In dilute solutions of nitroxide free radicals (cf. It is assumed that T B T. That is. assumed to be strong. no recollection of which radical it was on before the collision. and thus only 3 of all collisions can be effective in line broadening due to spin exchange. only 3 of these are between radicals with different components of the nitrogen nuclear spin angular momentum.3.158) The lifetimes T and T . Of these collisions.159) where N .we see that a collision will be strong if T . Fig. we have D = :(l/z. NITROXIDE SPIN LABELS sec. Thus the linewidth enhancement due to spinexchange collisions is  ’. only result in a net spin exchange. In this quasicrystalline model T . is the average time that the radical stays in its site. If D is the diffusion constant for the free radical in the solvent of interest. and N . 22) the weak broadening of the resonance lines can be attributed to a lifetimelimited state of the radical. The number of new neighbors encountered with each step is z. Thus after a strong collision the spin magnetization has a 50: 50 chance of having the spin magnetization of the colliding partner. If J T .160) .. (2. so to speak. Let T be the “lifetime” of a nitroxide radical between collisions. are related by the equation T/Tp = NS/zN. 2 lo’ sec.5.3. Let us now consider this line broadening in more quantitative detail. B I the collision is strong.
Rer. (2. Richards.163) where wl.8 x cm2/sec.3./N.)6D/A2. N . Miller.3. and w 3 are the angular resonance frequencies of the three lines: (W] .ZN. Miller et al. for a threeline spectrum. (2. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 107 where A is the “jump distance.167) leads to an experimental diffusion constant of D 3.7 x 109(NR/N.3. A.3. in the limit of infrequent but strong spinexchange collisions. 58 T. (2. These investigators find for the enhanced linewidt h 1/T2 = 3. and P. 44. (2. M.166) and (W1 (4na)2.3.w2)2 = ( W j w3)3 =  w2)2 = (2742 (2.165) (2. (2.3.3.3. Thus l/Tz = ta%.161) Therefore. K. R . These authors found that in the slowexchange region the hyperfine linewidths are proportional to the radical concentration. J .162) and make the plausible assumptions that z 6 and A 3. Hudson and G.164) (2. Chem. the strongly narrowed residual linewidth is of the form5’ 1/T2 = &T[(Wl  W2)’ + (W2  ~ 3 ) ’ +( ~ 3 01)~]. Adams.5 x lo’. then Eq. ’’ A .2. It can be shown that. the the enhancement of the linewidth is 1/T2 = ~(.4022 (1966).167) N If we return to Eq.). Luckhurst. 69.162) In this limit the enhancement of the linewidth is seen to be proportional to the freeradical concentration. 191 (1969).50 have carried out a study of the paramagnetic resonance spectra of ditbutyl nitroxide in the solvent dimethylformamide.)(~D/A~). . Equation (2. Chem. w 2 . (2.3.3. In the above discussion. The case of rapid spin exchange between radicals corresponds to an exchange rate that is large compared to the hyperfine splitting a.3.159)can then be written T’ = (zNR/N.” the distance moved in each step. we have considered the effect on paramagnetic resonance line shapes of spinexchange collisions between spin labels for two . Phys.
NITROXIDE SPIN LABELS limiting cases.” the probability per unit time that an electron spin moment will undergo a transition. Thus we give here only a very brief discussion of the Bloch equations t o treat the spinexchange problem. Acad. We have already used modified BIoch equations to treat the effect of slow motion on spinlabel spectra. and H. The Bloch equations that allow for the effects of strong.qn. (2. due to spinexchange collisions. N .474(1973) . 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.. be the outofphase and inphase electron spin magnetizations. One case corresponds to strong but very frequency collisions. Y. : ~ measure the firstorder decay of to ~ electron spin magnetization from each of the states m.56 (1973). J . Devaux.(m). M.170) where K is the frequency of the collisions. Ann..’ = 2 ~ .. so l/T = iK. 9. C. be the three corresponding angular resonance frequencies in a frequencysweep spectrum.168) In these equations T. Sci. Mu. the probability per unit time that one spin label will encounter another in a “strong” collision. From our earlier discussion we see that only 4of all the molecular collisions are effective for spin transfer. 222. one can use the parameter z . McConnell. and let w. such that it experiences a new nuclear hyperfine field.108 2. Reson. 0. The quantity l / can be referred to conveniently as the “spin transfer rate. and the spinexchange interaction affects each of these states equally. 1. Let urn and u. random exchange collisions on urnand v. is the probability per unit time that electron spin magnetization with Larmor frequency om undergo a transition (due to will spinexchange collisions) to a state where the Larmor frequency is w. Scandella. modified to include the effect of spinexchange collisions. Since the populations of the three m states are essentially the same. J . M.3. (where m # m’). Devaux and H. McConnell. Assume for the moment that a nitroxide spin label gives three Lorentzian resonance lines with corresponding transverse relaxation times T. where the nitrogen nuclear spin quantum numbers m are m = 1. that is. Spectra in the intermediate range can be calculated using the Bloch equations. ’’ P. are (2.3.
). M .10). and also into membranes derived from the sarcoplasmic reticulum of rabbit skeletal muscle. Sci. Soc. U. Sci. S.) 2.2977(1976). A . M . 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. I1 0 b other phospholipids.OC(CH. 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.CH.N~ H.2564(1971). Kornhergand H. and thus. 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.. Proc.13. McConnell.4499 (1972). Acud. Nail. Cohn) have been carried out to provide significant structural and kinetic information on enzymes and other macromolecular systems. Section 2.C. 68. .4.N(cH. compared to relaxation due to the interactions of the nuclei among themselves. Clzem. nitroxide spin labels introduced into biological systems can have large effects in enhancing nuclear relaxation.C' rt '(cH.3..4. D. 94. Sackmann. 1.). Many elegant studies of paramagnetic enhanced nuclear relaxation (a field pioneered by M. S. in the presence of ATP. Tduhleand E. in an isotropic sample. Proc. SPINLABEL THEORY : A MATHEMATICAL TREATMENT 109 For example.). BrQletand H.Am. U. 63 P.14) in phospholipid bilayers of egg lecithin and O. A . '' R . the resonance signals are nearly isotropic and sharp (cf.3.60This particular label was chosen since the paramagnetic group is nearest the most flexible end of the chain. The lateral diffusion coefficients so obtained were of the order of D 'v 2 x cm2/sec. Kornberg and McConnel162 and Bralet and McConnel16' used spinlabelphospholipidenhanced nuclear relaxation of nonlabeled lipids to study lateral diffusion of phospholipids in model membranes. and McConnells9 incorporated the phospholipid spin label (1.cocH 0 I CH.2. "' H . Acad. I II 0 CH.CH. Scandella. Devaux. 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.3. For example. McConnell. 13. Nail.oPcH.
The sccond important point is that. For example.” 2. the reader is referred to the books by Berliner’ and Dwek. The Use of Spin Labels as Antigenic Determinants Capable of Reporting Their Physical State 2.4.4. 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. or control. NITROXIDE SPIN LABELS Available space makes it impossible to summarize this large body of work here.110 2. we consider that the examples presented in this section serve to illustrate the various basic types of information that may be obtained by . We have commenced work in other areas of immune function. 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. These examples only concern antibodydependent efferent responses. For leading references. We would like to make two important points regarding the use of stable nitroxides as antigenic determinants.8.4.” 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. 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. When spin labels are used as antigenic determinants they are an essential participunt in the action: their function is to perturb. the system in whatever way is natural. but we have not yet employed spin labels in these endeavors. to our knowledge. However.1. 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. Specific examples of immune responses and our experimental approach towards their elucidation are referred to in Section 2.
64 Further details regarding particular types of lipid structures are given. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 111 using spin labels.COOCH I I II I CH. K. pp.OPOCH. When haptens are mixed with other suitable amphipathic lipids.. and H. such as phosphatidylcholine. o I1 CH3(~H. 321329. 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. Abrahamsson and I.).. Pascher.CN 0 H b IV C%(CH~). M. CHJCH. .. These include the lipid haptens in the manner illustrated by Fig.). eds. where relevant.) O4 P.CH.CO.0.). Among these examples are several that serve to illustrate a characteristic of spin labels which has been underexploited. CH.. G . McConnell. their susceptibility to agents which chemically (or photochemically) destroy their paramagnetic properties.CH. below..(cH.). New York.).NHCO(CH.COOCH CH. I oco(cH.. The structures of these are shown below..cH.).COHN b V All are soluble in organic solvents such as ethanol and chloroform.OPO(CH. and introduced to an aqueous medium by an appropriate method.2. 2. 0 I II CH.4.OPOC%C%N A 111 CH.4.cH.0CO(CH. Plenum. Humphries. M. in “Structure in Biological Membranes” (S. 16. whether they be used as bystander reporters or as central components of the system of interest. Brdlet. but form ordered aggregates in aqueous dispersion. continuous lipid bilayer structures are formed. (Part 11 is devoted to lipid bilayers.).NCH. 1977.2. 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.).CO& I CH.
McConnell. spin labels being exclusively on the internal face of the vesicle membranes. Diagrammatic reprcsentation or a lipid biliiyer containing a spinlabel lipid hupten. NITROXIDE SPlN I. SOC.66 the major structural feature responsible for the vectorial nature of membrane function.)  2. J . McConnell. .3. Lenard. Therefore the membranes of similar vesicles lacking watersoluble spin labels but including compound Ill in the manner illustrated by Fig. This phenomenon has important consequences with respect to maintenance of the asymmetry of biological membranes. I ). ' ~ ) 2.16.2. E.4475 (1972). 16 become asymmetric when treated with ascorbate at 0"C. At 0 ° C such vesicles protect trapped watersoluble spin labels from chemical reduction. Using other lipids. A typical result was that the asymmetry decayed with a halftime of 6. 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 demonstrate l~~ that the rate of insideoutside transitions (flipflop) of phospholipids in vesicle membranes is slow.5 hr at 30°C. Biochemistry 10. Kornbcrg and H. Am. D. Determination of Lateral Diffusion of Spin. by sodium ascorbate (see Section 2. (From BrClet p t ~ 1 . at different temperatures. M. 743 (1977). '' P.) 195.4.C.4. Rothman and J . Chem. Science ( Wushington. even slower rates for flipflop have been reported since these measurements were made. Dcvaux and H. was used to determine rate constants and activation energies for the process.4.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.94. signals received on the external side are believed to be modulated by the membrane to give appropriate messages to the cell. Thc experimental procedurc used to detcrmine thc rate of flipflop involved preparation of small ( 250 A diameter) singlccompartmented egg phosphatidylcholine vesicles by sonication.ABELS FIG.I12 2. We feel safe in assuming that asymmetry is of fundamental importance for membranedependent cellular immune function . M. 66. D. The rate at which equilibrium is approached. 1 1 I 1 (1971). (See Ref. and consequential loss of paramagnetic properties.
4has therefore been used by Schwartz and McConnell to determine these quantitiese9 '* 6y J. A technique similar to that discussed in Section 2. U . then followed the time dependence of the spinspinmediated line broadening to deduce the lateral diffusion coefficient. M . The product is stable and nonparamagnetic. or. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 113 Sackmann.4661 (1978) M.4. Acud.6' Devaux and McConnell created a nonequilibrium distribution of labeled and nonlabeled lipids in multibilayer membranes.9 x lo* cm2/sec. Sci. Sheats and H . For example.2. 837 (1978). McConnell. . the diffusion coefficient for dipalmitoyl phosphatidylcholine is found to be 9. to know the number of such molecules accessible to the exterior of lipid structures. Diffusion constants calculated from such data are similar to those obtained using other methods (cited in Ref. R. S. McConnell. their external surface area cannot be determined by simple calculation. Biorhumistry 17. Schwartz and H . Nail. hv 314 + R Co"(CN)~ + R For this work the EPR spectrometer is interfaced with a computer programmed for double integration of spectra. 68). Pror. M. It is frequently important to know this quantity.4. A . 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. (An important early clue as to the rapid lateral f diffusion o lipids in vesicles was provided by Kornberg and McConnell.5. 2. for work involving immune responses to spinlabel lipid haptens. Determination of the Surface Area of Lipid Membranes As continuous lipid bilayer preparations are rarely unilamellar. 75. at 48"C.62 who studied the dipolar effect of spin labels on the proton nuclear resonance spectra of nonlabeled lipids to infer the rapid lateral motion o lipids.4.) f 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.68 The equation for this reaction is given below. A.
Biochem. McConnell. Nutl. Res. is stable in light and air for several days. and is highly specific for photolysis of nitroxides even in the presence of air and phospholipids. 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.114 2. measured at various times after illumination. Sci.0 and 5. Humphries and H. A .5 % for all preparations of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. J. when spin labels are in rapid isotropic motion their hyperfine splittings are averaged. 522 (1979). 7 2 P.^'^ We are currently producing monoclonal antibodies with specificity for TEMPO. to prepare IgG. we hope. Biorhem. U . The rate of reaction is first rapid and then slow. particularly the low. J . Humphries and H. 74. Commun. 7 3 D. reduction of spinlabel EPR signal intensity is. McConnell. is relatively high (due to superimposition of the contributions from many labels). resulting as it does in the representation of very many different splittings.Ntrimethylamino) benzyl pentacyanocobaltate ions. McConnell. This latter rate is thought to result from slow leakage of radicals through the membrane. As described in Sections 2. This figure is also obtained when such bilayers include up to 50 mole % cholesterol.3 and 2. M.4.7 They have been purified with respect to immunoglobulin class and specificity for spin label^. suffice to convince the reader that immobilization of spin labels.275 (1976). Reference to Figs.6. Commun. M.and highfield lines. M. Photochemical reduction of exposed haptens is induced by an argon ion laser in the presence of excess 4(N. ” G . 16. Antibodies Specific for SpinLabel Determinants Antibodies specific for TEMPO have been prepared by immunizing rabbits either with spinlabeled hemocyanin. Proc. therefore. Biophys. Res. Rey and H.68 The reaction proceeds via an alkyl free radical split off from the pentacyanocobaltate complex. M. K. 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. S. 2. G . 73. M.N.70 or with spinlabeled bacterial flagellin. . Biophys. In order to determine the proportion of external labels. These properties make it more suitable for this application than the ion used by Sheats and McConnell. Acud. Hafeman. Biophys.2. W.6. 86. 3537 (1977). to prepare IgM. Parce. and H. Therefore the amplitude of the peaks (or lines). 6 and 7 will. M.3.248 (1976). McConnell. K. This ion pentrates phospholipid bilayers very slowly.
particularly the high. Although this cross reactivity surprises us.” the EPR spectrum changes dramatically. 17. in contrast to the usual maximum 2Tl. either presented at the surface of a membranes or as constituents of a lowmolecularweight. F 20 G FIG.It is probable that the charge separation in the NO bond is enhanced by an electrostatic interaction within the antibody combining site.2. (From Brclet et a / . this principle has been used to determine the affinity of antibodies specific for TEMPO. such as a hydrogen bond to the oxygen atom. 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. watersoluble species.’* Univalent haptens are employed for such measurements. EPR spectra of large singlecompartmented dimyristoylphosphatidylcholine vesicles containing 2. there is a high probability that the spinlabel immunogen is reduced in v i m before stimulating the antibody response. it probably accounts for the fact that antiTEMPO activity can be obtained. which shows the effect of mixing immunoglobulins.17. These effects are illustrated well by Fig.” except that the hyperfine splittings are remarkably broad (2Tll = 7879 G.and lowfield lines. indicating a marked reduction in motion. and would lead to increased splittings as discussed in Section 2. relative to that previously obtained from samples giving rise to powder spectra. of spinlabel hapten 111 together with IgC prepared from rabbit antiTEMPO hemocyanin serum () or IgG prepared from rabbit antihuman erythrocyte serum (). This would increase the unpaired electron density on the nitrogen atom. 6 4 ) . The vesicles were prepared by dialysis of a detergent solution of lipids. As the signal intensity of the “rapid motion” spectral component is proportional to the concentration of unbound spin labels. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 115 decrease in the amplitude of the lines.5 ”/.4. with TEMPObearing bilayers. When specific antibodies bind to TEMPO groups. The new spectrum is a typical “powder spectrum. of approximately 69 G ) .2. with or without specificity for TEMPO.4.
20G.7. 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.4. and replotted by means of a Digital P D P 8/e computer interfaced with a Varian E l 2 EPR spectrometer. see Section 2.5 mole 7. BiorherniJtry 16. 18. and (iv) the twodimensional concentration of haptens in bilayers.4. Spectra were recorded at 32°C and stored. M.74 74 P. (iii) the cholesterol content of those bilayers. Brulet and H. 1209 (1977). 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). For these three labels. The EPR spectra shown in Fig. (ii) the fluidity of their host bilayers. I FIG. 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. 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. NITROXIDE SPIN LABELS 2. McConnell. 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. Physical Factors Affecting the Binding of SpinLabel Specific Antibodies to HaptenBearing Bilayers Changes in (i) the length of the hydrophilic moiety of spinlabel haptens. ofedch of the three haptens (Structures IllV).116 2. Dipalmitoylphosphatidylclioline bilayers containing 0. have all been shown to affect the EPR spectra of spinlabel haptenbearing model membranes.2. . normalized. integrated.
unpublished observation). by increasing the collision rate of spinlabel haptens. the dominant effect is that due to increasing the rate of motion. When haptens are already sufficiently concentrated to show significant spinexchange broadening in the solid state. New York.” The predictable increase in mobility of the head group is indicated. together with a plot of temperature versus the ratio of the amplitude of the lowfield line to that of the center line. K.). because the hapten is present at a low concentration in thc membrane. eds. Humphries. 1980. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 117 “ w V n LL . 10 2 0 30 TEMPERATURE (“C) ’ FIG. 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. Figure 20 shows the binding of IgG antibodies to liposomes composed of two G. Gregoriadis and A.2. M . (It should be noted that melting the bilayers both sharpens the spectrum. In this instance. Electron paramagnetic resonance spectra of dimyristoylphosphatidylcholine liposomes containing 0. in “Liposomes in Biological Systems” (P. 19.5 mole % spinlabel hapten IV. M. Allison. by increasing the rate of motion. Humphries. The principal phase transition temperature of dimyristoylphosphatidylcholine is 23°C. K . a marked increase in agglutination commences at the phase transition (G. melting is accompanied by an appreciable increase in broadening. 345375. (From H ~ m p h r i e s . ’ ~ ) Figure 19 illustrates the spectral consequences of melting bilayers containing a spinlabel lipid hapten. and broadens it.4. pp. Wiley. ’’ . 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.
Levine.290 (1961). Since ca. lipsomes were removed by ultracentrifugation. The cholesterol content of phosphatidylcholine membranes has a marked effect on the spectra of spinlabel haptens included with these lipids. Binding of rabbit antinitroxide IgC to dimyristoylphosphatidylcholine (0)or dipalmitoylphosphatidylcholine( 0 ) liposomes. NlTROXIDE SPIN LABELS RELATIVE A N T I B O D Y CONC. containing spinlabel hapten I V .20. This is illustrated by Fig. FIG. K.118 2. and spinlabel hapten I V in the molar ratio 50:45:5. 87. In both cases it is apparent that the percentage of antibody bound increases as the ratio of antibody/liposomes decreases. (Previously unpublished data. Wasserman and L. Hapten was 1 mole %of total lipid. G. as a function of antibody dilution. The observed spectral changes indicate that the rate of motion of the head group is increased by increasing the cholesterol content. Immunol.4M total phospholipid. . It is also evident that “fluid” liposomes bind more antibody than “solid” liposomes under these experimental conditions. 21. dimyristoylphosphatidylcholine. After 15 min incubation at 3 7 T . 5 %of the total hapten on these liposomes is on the outermost membrane surface. ’‘ E. J . but that the differences in absolute amounts bound to the two types of liposomes become less significant as antibody/lipid decreases. M.) different phosphatidylcholines at a temperature which is above the principal thermotropic phase transition temperature of one lipid but not of the other. Humphries. the total exposed hapten concentration was of the order of lO’M. and residual antibody was assayed by a WassermanLevine complement fixation assay76 in which the antigen was liposomes consisting of cholesterol. 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.
and replotted by means of a Digital PDP 8/e computer interfaced with a Varian El2 EPR spectrometer.. USE OF SPIN LABELS AS ANTIGENICDETERMINANTS 119 FIG. ’In brief.95 (1980). normalized.2. ~ it is evident that the effect of cholesterol on the physical state of lipid haptens in phosphatidylcholine bilayers is complex. McConnell. Biophys. M. Sci. In addition to its positive or negative effect on lateral mobility of membrane r n o l e ~ u l e s . Biochemistry 19. M. M. U. ’* 77 . Biophys. R. .21. possibly because of the increased rotational freedom resulting from a decrease in their interaction with phosphatidylcholine head groups. L. The temperature was 35°C. as indicated by EPR spectroscopy. L. A. and in each case increased mobility of the spinlabel head group. 76. This phenomenon has been studied recently by Rubenstein ef ~ 1 . Owicki. Narl. Rubenstein. Acad. McConnell. Biochirn. 383 (1980). Antibody binding is favored by inclusion of cholesterol at 2050 mole % in such b i l a y e r ~ . Smith. at 2050 mole %. Liposomes contained 3 mole % spinlabel hapten IV. appears to favor binding. R. McConnell. and H. C. and H. J. M. indicated on the figure. Figure 22 shows the spectral changes observed when the distance between adjacent haptens is decreased. A . R. Similar spectral changes are observed when dimyristoylphosphatidylcholine bilayers are used. and this result is consistent with a homogeneous distribution of spin labels in the plane of the membranes.569 (1980). EPR spectra of liposomes composed of dipalmitoylphosphatidylcholine and the various molar proportions of cholesterol.4. in the range 050 mole %. B. I C. The broadening of the lines is typical of spin exchange. Rubenstein. 30. A fourth type of physical change which affects antibody binding is the twodimensional concentration of haptens in the membranes. Spectra were stored. S. ~ ~ We have discussed three examples of physical changes which control antibody binding. McConnell.  J. Acra599. J. Proc. permits increased mobility of spinlabel hapten head groups. J. ~ ~EPR *~ spectra indicate that cholesterol. 15 (1979). 7 9 B. Owicki and H. integrated. Copeland and H.
NITROXIDE SPIN LABELS 0 5 10 10 0 1 OF IIAPTFN FIG. Samples were prepared as spccified but such that all had the same ourrull concentration of nitroxide ( i c . However. Even so. The data indicate a random. 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. This latter parameter is proportional to the averagc area of membrane available to each hapten. some early work using a monoclonal antibody suggested that for some IgG molecules.120 2. A systematic study of the effect of hapten density on IgG binding has not yet been attempted.") Binding of spinlabelspecific IgM is favored by decreasing the distance between adjacent haptens. hapten density may be important even in the range where the distance between adjacent haptens is less than . EPR spectra of liposomes at 32°C containing rqualumounts of hapten at the various molar percentages (with respect to total lipid) indicated. monomolecular dispersion of this hapten in cholcsteroldipalmitoylphosphatidylcholine bilayers of the chosen composition. 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.22. Spectral consequence of decreasing the spacing of spinlabel hapten IV in dipalmitoylphosphatidylcholine membranes with 50 mole cholesterol. (From Humphries and McConnell. even for the case of membranes for which the rate of lateral diffusion is very high. because of the heterogeneous nature of lipid suspensions. total lipid per milliliter of liposome suspension was varied)." This presumably relates to the ease with which multipoint binding is maintained.
Immunol. Parce. 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. antibody conformational changes are induced by binding to antigens and are required for various functions such as activation of complement. one which does not cross react. Oxidation of the reduced form of a suitable watersoluble nitroxide (i. Of central importance is the question of the significance of multipoint versus singlesite binding of antibodies to membranes. A.e. has been used in some preliminary studies designed to measure antibody off rates from membranes. A full discussion of this subject is clearly not appropriate for this book. Chew. 82 . and H.. Antibodies are polyvalent. J . M. It would be of interest to measure off rates for antibodies bound to lipid haptens on membranes.4. J . Schwartz et aLsl have approached this problem by studying selective hydrogen exchange between nitroxides and reduced nitroxides. but a few points of general interest to biophysical chemists will be mentioned. To what extent. M. 101. 2.3592 (1979). This increase in concentration and anisotropy is favored by multivalent rather than monovalent binding. in some cases. and how. IgG and IgE having two antigenbinding sites and the secreted (serum) form of IgM having ten. 18. 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. do these properties of local concentration and orientation of antibodies control antibodytriggered responses? It has been suggested that. in the immunological sense. Hydrogen exchange between an appropriate nitroxidereduced nitroxide couple can only occur when neither species is bound to antibodies. H. 82. We anticipate that the use of spinlabel haptens and monoclonal antibodies will be particularly useful for elucidation of this important point.2. Schwartz. It is our suspicion that the ease with which antibodies bind to single sites on membranes may. SUC. It is apparent that different nitroxide species differ greatly in their affinity for hydrogen atoms.4. with TEMPO and which is a hydrogen donor relative to TEMPO) by lipid haptens. This topic is controversial. be far less than that with which they bind to the samedeterminants in soluble form. as they are released from the protection of TEMPOspecific antibodies. Am. A h . W . McConnell. 169 (1974). in addition to concentration and orientation. Metzger.8. USE OF SPIN LABELS AS ANTIGENIC DETERMINANTS 121 that between the two binding sites of an IgG molecule. For a discussion see Ref.
Am. G . McConnell. Proc. 179191. under certain circumstances. J . Hafeman. Commun. ~ ’and (iv) twodimensional . and H.7 * ”~ hapten c~ncentration. G. Hafeman. J . W.~’ most cases IgG antibodies have been used. 8 3 N. See Refs. T.M. Biochemistry 19. M. Smith. D. T.). S . Bartholomew. A . and H. 3933 (1978).H. NlTROXlDE SPIN LABELS Regarding the physical state of membranebound antibodies. M. 1980. Sci. We have undertaken several studies of the control of antibodydependent complement activation (or depletion of activity) by spinlabel lipid haptenbearing synthetic membranes. Doepel for skillful assistance in preparation of this manuscript. Tom and H . 8 4 L.McC.and H. 76. Parce. A. have observed no significant aggregation of IgG bound to haptenated bilayers. M. W. 75. and the induction of phagocytosis. F. Parce. Another related area of study has been the binding of antibodycoated synthetic membrane structures by macrophages and neutrophils. S . Lewis. More recently Smith et aLM4 have shown that IgG bound to lipid haptens in bilayers diffuses as rapidly as do the unbound lipids. in “Liposomes and lmmunobiology” (B. Biochrm. . ” J . McConnell. This work has been supported by National Institutes of Health Grants No. PCM 7723586 (H. Biophys. B i d .). Lewis. Res. 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. M . J. superoxide production. Elsevier. Bh D. Henry. Hafeman. 1768 (1979). Biochemistry 19. Parce.’~ using freezefracture methods. McConnell. Six. Chem. pp. M. and H.McC. R. 86. M. Proc. McConnell. D . T. and H. I/. U . Hafernan. 4177 (1979). D. Acad. Esser. M .M. and 1R23 A114813 (G. 522 (1979). ’(ii) membrane ~ f l ~ i d i t y 1 .M. eds. and a respiratory burst which are frequently a consequence of such binding. Sci.). but In Ref. B. J. New York. A . 5 R 0 1 AI13587(H. W.). 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. McConnell. 5376 (1980). M.122 2. 254. 8 5 A .5387 (1980). 8689. G. G . and H. J. J. McConnell. and H. M . affect complement activation. Lewis. Natl. Smith. Acknowledgments We are greatly indebted to Laurie K. W. Parce. Henry. As is true for the case of antibody binding all these types of physical variation do. McConnell.K.5~(iii) cholesterol ~ o n t e n t . Parce.~5~8. 89 J. and M~Connell. Nail. 71 concerns IgM. Acad. Such IgG appeared to be aggregated by subsequent addition of Clq.
The high intensity of such resonanceenhanced bands makes possible the study of much lower concentrations. when sufficiently powerful laser sources ( > 100 mW for conventional scanning. RAMAN SPECTROSCOPY By Margaret R. Inc All rights of reproduction in m y rorm reserved ISBN 0124759629 . VOL. sample size is economical. is a weak Raman scatterer although highly opaque over much of the infrared spectrum. Bunow 3. and the fact that molecules can be studied in any state. this method permits selective study of the neighborhood of biological chromophores. and the sample usually survives its examination unaltered and intact. These advantages over infrared derive largely from the fact that water. surpasses its elder in immense versatility and applicability to studies of biological molecules. no perturbing molecules need be added to the sample. The Raman spectrum is obtained by spectral analysis of the light scattered from a sample illuminated by a laser source.3. in technological development the younger sibling of infrared spectroscopy.1 M or the maximum soluble. whether in crystalline array or aqueous phase or biological cell fraction. which in many cases better represent the biological norm. < 100 mW for resonance) became readily available. The information in this record concerning molecular structure and conformation constitutes an abundance of riches. 20 Copyright 0 1982 by Acddcmic Press. of the frequency of scattered light from the incident laser frequency vL. 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. Introduction Raman vibrational spectroscopy. typically being of the order of several microliters. in conventional (nonresonance) Raman spectroscopy. As in infrared spectroscopy. 0. Furthermore.1. I23 METHODS OF EXPERIMENTAL PHYSICS. this figure may drop to several micromolars in resonance Raman work. Typical concentrations of biomolecules studied in solution are. the natural biological solvent. 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. the spectral record has the form of the intensity I of scattered light versus the displacement v.
representing inelastic scattering. (see Fig. N .. 1). are found at both higher and lower frequencies about the central Rayleigh band. 3. The emitted line thus occurs at a frequency close to v L . Origin of the Raman Spectrum If a beam of light at visible frequency v .excurses to a “virtual” (nonstationary) state. the molecule returns to its ground electronic state but to the first excited vibrational level rather than to the ground vibrational state. Finally.). many weak bands.. In addition. and one emitted. 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. These displacements result from the interaction of light with the changing electrical field of the electronic clouds.6 lines for an Natom nonlinear molecule only by quantummechanical selection rules and by the detectable intensity of the allowed lines.124 3.2. that is. which is usually reported in wave numbers (cm. most of the scattered light is unshifted in frequency. which closely follow motion of the oscillating atoms of the sample molecules. that is. and the Raman spectrum consists of bands shifted to frequencies lower than those of the incident line. The process is essentially instantaneous ( 10. illuminates a sample. The very near neighbors are Brillouin bands resulting from the interaction of light with coherent fluctuations in the sample medium.v.. An antiStokes spectrum is seen at frequencies greater than v. elastically scattered.l 5 sec) and involves two photons. limited to fewer than 3N . 1). the array of vibrational bands composing the Raman spectrum is displaced by from 3 to 3500 cm’ from the central band. one incident. 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. but displaced from vL by the vibrational frequency v. usually in the visible range.l) of displacement. Upon spectral analysis. The spectrum is very complex. and exits to a second intermediate state by creating a vibrational quantum hv. 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. the molecule in the ground state interacts with an incident photon of energy hvL. The interaction of many photons with the sample molecules produces the complete Raman vibrational spectrum. The process just described is known as Raman Stokes scattering. upon emitting a photon of energy h(vL . In the Raman phenomenon.
is the equilibrium position of the vibrational coordinate e in the ground electronic state.. 3. and q. bandwidth. ANALYSIS OF THE RAMAN SPECTRUM W 125 E electronic state ‘he qk FIG.. These quantities are defined and interpreted as follows. . Analysis of the Raman Spectrum Each Raman vibrational band is characterized by its frequency vv. The vibrational frequencies are clearly sensitive to alterations of normal coordinates or of bond strengths.3. wherehv. antiStokes.3. 1).1. and sets of vibrational bands of the proper symmetry and overlap relative to the electronic transition may become resonance enhanced (Fig. Transitions involving a jump of more than one vibrational level are also weak. Relationship between Stokes. 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. This spectrum is too weak to scan more than 500 wave numbers (cm’) from v L . The atoms vibrate with small changes in atomatom distances and angles about their equilibrium positions..3. the molecule may transit to an excited electronic state. intensity. and depolarization ratio. is the energy difference between the first excited and lowest vibrational levels of the ground electronic state. and resonance Raman effects. When vL falls within or near molecular absorption bands. This sensitivity is the foundation for Raman spectral investigations not only of molecular conformation but also of its mutability in differing environments..
which is symmetric in the conventional vibrational Raman effect.: 5:. y.3.!):" = Mzx Mzy (3. 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 . for which the periodic fluctuation in polarizability has the full symmetry of the molecular skeleton. It follows that measurement of the polarization of the scattered light for each Raman line gives direct . 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.). term gives rise to Rayleigh scattering. composes the derived polarizability tensor. as well as proportional to the concentration c of scatterers.3) The indicesj . The a.6 molecular vibrations which may contribute to the Raman spectrum. .2) i where q iis a normal coordinate describing a molecular vibration and higher (anharmonic) terms are neglected. the incident light intensity I .1) azz The molecular polarizability a can be expanded in terms of linear deviations from the equilibrium coordinates of the molecule (3. The matrix of these coefficients. contribute only diagonal elements to the polarizability tensor. as in classical scattering processes: (3. and the fourth power of the frequency of scattered light.3. one can count and classify by symmetry the number of Raman lines expected for a molecule. and z. k run over x. Totally symmetric vibrations. reflections. and the [ ( d a / d q i ) ] . Nontotallysymmetric vibrations have nonzero offdiagonal elements. where the coefficients a j k for the ith vibration are evaluated by quantum mechanical theory. Using grouptheoretical rules.3. and inversions which transform the molecule into itself. qi term expresses the dependence of the induced dipole moment on the 3 N .126 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. 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.
analyzer. 23. ANALYSIS OF THE RAMAN SPECTRUM 127 FIG. The scattered light is analyzed by transmitting light with the field vector first parallel. In Fig. the electric field vector of the polarized laser beam is chosen to be normal to the plane containing incident and observed scattering directions. the exact value reflecting the symmetry group. the ratio of intensities thus recorded for each vibration is the depolarization ratio p. S. and then normal to the plane so defined (0). A.3. 2.75 for nontotallysymmetric vibrations. Schematic diagram of instrumentation for measurement of conventional or resonance Raman scattering: F. and J. These results are refined by calculation. and p = 0. If a sample is a single crystal or oriented polymer. CO. Raman and infrared vibrational spectra are largely complementary. is the depolarization ratio p . may be measured in one of several related scattering geometries. then normal to this plane. this measurement gives 0 < p < 0. a measure of the rotation of the electric field vector of the scattered light. FO.' The polarization scrambler placed between the analyzer and slit (Fig. 2) eliminates unequal grating response to different polarizations. collection optics. focusing optics.75 for totally symmetric vibrations. The depolarization ratio. . For molecules oriented randomly in solution. Spectrusc. measured in the The respective spectra. Claessen. Appl. The ratio p can be measured accurately only for liquids or clear solutions. dispersion or interference filter of laser output. Typically.3. the incident laser beam is polarized out of the plane of the paper ( 0 ) . H.2. multiple reflectibns of incident and scattered light in turbid solutions destroy the polarization. H. information on the symmetry. Selig. and helps one to deduce the assignment of the associated vibration. ratio of peak intensities for each vibration. polarization scrambler. 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. Shamir. Raman spectrum is recorded first with the the analyzer rotated in the plane defined by the incident and scattered beams (). J .8 (1969). Infrared activity requires that a vibration change one of the three components of the dipole moment of a molecule. 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. H.
205. RAMAN SPECTROSCOPY in general. Wiley. and I. and P. B. Rossiter.). 1962. Shepherd. eds. C. in “Mcthods in Experimental Physics: Molecular Physics” (D. When a mode has both Raman and infrared activity. 6 . 33. If a molecule has a center of symmetry. The fourth useful spectroscopic variable is the linewidth. the mixing of electronic and vibrational states allows preresonance or resonance enhancement of the intensities of Raman G.). C. New York. bonding. Part 2. p.’~~’ ’ 3.4. Stoicheff.~. Vol. Biochem. l o S. P.” Van NostrandReinhold. Kolthoff. 127. Durig and W. C. J. Vol. Heyden.. 1977. These lines are broadened when the molecule is placed in solution. Hester. or environment. 1 1 1. in “Physical Methods of Chemistry” (A. R. 1. Vol. 3 . Resonance Raman Scattering When a molecule is illuminated by light at frequency vL near or beneath an electronic absorption band. J . as the symmetry of the molecule is lowered. C. Vol.229 (1977). E. A. in “ Raman Spectroscopy” (H. 1955. 1934. ” N. it can be shown that Ramanand infraredactive modes are mutually exclusive. Plenum.). Herzberg. p. K. E. Akad. Freeman. Elving J. ed.T. “ Molecular Vibrations. New York. Observation of line splitting for dissolved or crystalline samples can be interpreted in terms of inequivalence of molecular orientations. ed. CRC Crit. Raman spectra of crystalline substances at low temperatures exhibit sharp lines. H. 1971. Marx. Harris. 85. Cross. 1972. 2. C. “ Infrared and Raman Spectra of Polyatomic Molecules. Tang and A. 1945. p. Albrecht. London. M. 4.). highly polar chemical groups have strong infrared bands while chemical groups of low polarity and high polarizability are strongly Raman active. Decius. New York. Jr. in “Handbuch der Radiologie” (E. the frequencies in both spectroscopic appearances coincide. 3. Szymanski.. eds. New York. ed. p. more bands are found to be allowed in both types of spectra. B. Placzek. Part 3b.). Weissberger and B. 2nd ed. G. eds. Clark and R. W. New York. J. A variety of introductions to theory and modern instrumentation are a~ailable. 1. in “Advances in Infrared and Raman Spectroscopy” (R. 1970. Wilson. 1974. Vol. Yu. ’ J. Wiley (Interscience). 35 of “Chemical Analysis” (P.128 3. Tobin. Williams.” Vol. New York. “Laser Raman Spectroscopy. Academic Press. reflecting the increased molecular conformational freedom and range of solutesolvent interactions. Leipzig. Verlagsges. “Applications of Laser Raman Spectroscopy. Rev. p. and review articles4’ may be consulted for Several classical details of theory. M..” Wiley. allowing use of infrared spectroscopic compilations. p. ’ ‘ . I . New York.).” McGrawHill.
Rer. Science (Washington.C. Chem. Miyazawa. L.) 188. Sac. 27.7.465 (1976).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.50. 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. J. which require shifts in the excitedstate equilibrium coordinates or changes in the shape of the excitedstate potential well relative to the ground state. Peticolas. Annu. Annu. Phys. (Chem. Bioeng. A.6. Specfrosc.359 (1975). Examples occurring in biologically interesting polyenes and porphyrins are given below (Sections 3. 501 (1977). but it is nonzero only for totally symmetrical vibrational modes. Specfrosc. Behringer. Vibrational modes associated with chargetransfer transitions of ligandmetal complexes in proteins are similarly enhanced (Section 3. 34. Chem. 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.9 and 3. Annu. 28. Rev. Hirakawa and M. Gaber. A. B. 6 . Higher terms of smaller magnitude can usually be neglected. Tsuboi. called A and B terms. j 4 T. An analogous summation is made for Bterm scattering. lnagaki. ATerm Scattering Aterm scattering involves vibrational interaction with a single excited electronic state. Biophys. G. '' A. G. Chem.3. Spiro and B. and T. Mol. Accordingly. This type is usually dominant in resonance Raman scattering. The preresonance and resonance regimes are theoretically distinguishable. l 6 J. Warshel. 1476 (1961).12 The polarizability can now be expressed.7. Reu.1. 273 (1977). Phys. 100 (1974). Albrecht.4.6).4.I3 as a function of two types of terms.286 (1974). Phys. RESONANCE RAMAN SCATTERING 129 vibrational modes associated with the chromophore. Biochem. Rev.13"9 B. Tasumi. 46. M. Aterm scattering depends upon FranckCondon overlaps. Mol. P. The reader is referred to reviews of the theory.7). except that two excited states are included in each term. London) 2. T. Annu. C.'2'6 3. Vibrational stretching modes aligned with bond weakening such as that associated with nn* transitions receive strong Aterm resonance enhancement. Johnson and W. l3 . 553 (1977). Stein. according to Albrecht's formulation.7. F. Spiro and P. The total Aterm scattering for each vibrational mode is found by summing over terms describing all contributing excited states. Y. D. J .
Spiro and T. 56. The mixing of inplane vibrational modes with nn* transitions in porphyrins provided the first example of inverse polarization in vibrational Raman scattering.3. Aton. Chem. Am. the depolarization ratio p may assume a value in the following ranges: (i) 0 < p < 0. Chem. G. C. J . Gill.3)] and for instrumental spectral response. 69. Chem. Phys.’ 3. and of the depolarization ratio.24925 ” T.4. Callender. These plots ’9’ ’’ vL. B. J . 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. A. Kilponen. a is antisymmetric. the vibration is nontotallysymmetric. Proc.130 3. A. Li.303 (1977). ” B. Narl. A. (iv) p = 00. and normalized with respect to a nonresonant reference mode. *’ T. 4438 (1971). the polarizability tensor is no longer necessarily symmetric. (ii) p = 0. Hutley. RAMAN SPECTROSCOPY 3. and B.75 < p < 00. A . Spiro. 2 4 L. (3. C. G. 1 . for each vibrational mode against the variable The intensity profile plots are corrected for the usual v4 dependence of scattering [see Eq. Phys. Honig. less easily. Rimai.6 Vibrational modes that have rotational symmetry are particularly effective in vibronic mixing in producing anomalously or inversely polarized bands. and T. as noted previously. C. J .2.and Bterm scattering can hence essentially be distinguished by the values of the measured depolarization ratios. (iii) 0. Strekas. As a result. Acad. A . Aterm scattering.4. and D. 23 A. The magnitude of Bterm scattering depends on the product of the transition dipole moments of the two electronic states being mixed. Bterm scattering may fall into any of the above categories. R. Nafie. the vibration is totally symmetric. The Polarizability Tensor When vibronic mixing with electronic states is admitted. vibrations with Bterm activity are allowed any symmetry contained in the direct product of the representations of the two electronic transitions. Packer. H . Chem. 197 (1973).3824 (1970). Sci. Doukas.75. Raman Spectrosc. G. Len. 19. by the shape of the excitation profiles in the region of preresonance enhancement. L.248 (1978). Phys. and the Raman mode is polarized. and the polarization is termed inverse. J. S. The two types can also be distinguished. 2622 (1972). Peticolas. BTerm Scattering Bterm scattering involves vibronic mixing of two excited states. 92. . The excitation profile is a plot of the measured intensity. and W. G . L. B.75. C. and the mode is depolarized . Strekas. Johnson. SOC. a is unsymmetric. Albrecht and M. 55. requires total symmetry of the mode.3. R. and the polarization is termed anomalous.
the data can be recorded in digital form or integrated for recording on an xt recorder. F. The gratings of the scanning monochromaters are preferably driven by a stepping motor to allow flexibility for computer interfacing. Spectrosc. 387 (1973).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. Appl. Raman Spectrosc. Appl. and the light scattered at 90” is collected and directed into the slit of a double monochromator. J. Kuriakose.2 ’” 3. instrumentation The basic Raman experimental configuration provides for spectral analysis of scattered light (Fig. Spiro. Weak signals are optimally processed by pulse counting circuitry. W. Nakamura and S. the heliumneon laser supplies a 632. Schwartz.’~’~’ The basic Raman instrumentation can be purchased commercially as a “package” which allows easy interchange of laser source.8nm line. 12. IW. 7.14. 29 T. Opt. Lurix.53(1974).5. currently available holographic gratings have impressively high straylight rejection. Ultraviolet work requires quartz optics and 26 ’’ T. The CW argon ion laser is the workhorse of Raman spectroscopy. J.428 (1976). 169 (1975). 1 .8 nm are widely used. J. E. T. The spectrally analyzed signal is detected at the photomultiplier cathode and amplified. . 2).5. . Frequencydoubled pulsed argon laser lines lie at even shorter wavelengths.5 nm (green). INSTRUMENTATION 131 contain information on the relation of the resonance scattering to the absorption spectrum. and P. a range of longerwavelength lines may be supplied by adding a dye laser pumped by an argon laser or flash lamp. Schrnidlin. G . Edgell. Acta 416.3. The double monochromator arrangement nearly eliminates the intense scattering of laser light unshifted in frequency. Lockwood. as well as on vibrational symmetry and its alteration by environment.0 (blue) and 514. Substitution of a krypton ion laser supplies several lines toward the red.1 nm. E. J . Biophys. two weak argon laser lines at 351. J . Alternatively. 30. A continuouswave (CW) laser beam is focused on a sample. For nearultraviolet excitation. Biochim. Its strongest lasing lines lie at 488. the strongest at 647.1 and 363. 3n . 2. 1073 (1978). G. K . C . 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. Arthurand D. Ruman Spectrosc. Spiro.
Levin. Spccrrosc. H. Nutl. 73.132 3. To optimize resonance Raman signals. Academic Press.~’ 3 1 1. A. p. Johnston. and photodecomposition. 28. Adams. Kiefer and H. and D. H. 32 W . Stryer. G. S . sample concentration c. Suspended samples (such as subcellular fractions or crystals in mother liquor) can be centrifuged to concentrate the sediment.. The signal generally increases nearly tenfold (for equal laser intensities) as the laser line is moved from the red to the blue. Nakanishi.5. 609 (1971). Sample Handling The standard sample container is a meltingpoint capillary holding either a solid or liquid sample. A. R. . Appl. Spectrosc.). 3. W. Moulton. B.3. the sample concentration is adjusted in successive trials so that absorbance does not surpass ~cattering. RAMAN SPECTROSCOPY may require exchange of gratings for those more efficient toward the ultraviolet. 3. W. J.’0. New York. 3 5 T. controlled atmosphere. Small glass or quartz cuvettes are also used. and K.3)] multiplied by the photomultiplier and grating responses.^^ The molecular order and spectral reproducibility of solids is improved by annealing the sample by repeated immersion of the sealed capillary in liquid nitrogen. a result of the vL4 scattering dependence [Eq. Proc. Stationary samples may be thermostatted at temperatures from that of liquid nitrogen to l W C . Sci. 3 3 R. Mathies.3). (3.33s34 Other precautions include beam defocusing and use of lower laser power.2.3. in “Human Responses to Environmental Odors” (A. l(1976). 45. and T. C.or airdriven) spinning plate. Oseroff. These effects are reduced by moving the sample rapidly in the beam path. resonance Raman studies as a rule require precautions against heating. (3. Biochemistry 15. 25. The electronics for gating of pulsed signals and other modifications are generally acquired separately from “packaged” instrumentation. or humidity. Bcrnstcin. J. Spiro. or may be held in vacuum. 1621 (1976). and an adequate. Appl. U. Absorbing (colored) samples suffer heating in the focused laser beam. usually maximal. Turk. Solids are usually pressed into an annular groove on a (motor. Strekas.^. A .1. and L. photoreaction. D. Callcnder.32Absorbing samples in solution can be pumped in a closed circuit through a capillary segment or through a jet orifice past the laser beam. Acud. 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. R. ed. Crouch. Solid samples should be well tamped to maximize scattering and conduction of heat away from the sample area heated by the laser beam.5. using a variety of sample chamber^.324 (1974). Jr. 1974. Doukas.
Optimization of signal/fluorescence.~’ Absolute intensity measurements are uncommon in biological studies. 1977. 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. Biophys. computer averaging of multiple fast scans is preferable to slow scanning. Friedman.phy or gel electrophoresis. Photoquenching by exposing the sample for a period of hours to as intense a laser beam as it can tolerate. Kiefer.5. Proteins may be purified by ionexchange chromatogra. J . E. repeated scanning is preferred. J.68a (1978). Hester. INSTRUMENTATION 133 Slow drifts in incident power. 3. p. Rousseau. a narrowwalled capillary may reduce this circulation.). H. For this reason. Purification by repeated recrystallization or fractionation. and J. 3. twocompartment cell36. 21. Dissolved samples may be passed through activated charcoal or alumina. usually spectral intensity changes are measured with respect either to a spectral band of the sample assumed to be invariant or to an added. London. sample turbidity. L. 3. Because the fluorescence background fluctuates. The fluorescence can be substantially reduced by a combination of the following procedures. The background fluctuation is partly due to convective motion of photoquenched and unquenched molecules in and out of the beam. . Vol. multiple spectral accumulation plus subtraction of fluorescence background will result in goodquality spectra.or alternatively as far to the blue as possible to skirt the longerwave length fluorescence envelope frequently improves the spectrum dramatically. which overwhelms the weak Raman signal. Management of Fluorescence Biological molecules tend to exhibit a fluorescence background. chemically noninteractive intensity standard such as the 983cm’ band of sulfate or 610cm’ band of cacodylate. eds. 36 W. and background fluorescence make small intensity changes hard to quantitate.3. 3 7 D. 30. lipids by silica gel chromatography. due sometimes to intrinsic chromophores and more often to impurities. The sample should be kept cool. J . Difference spectra may be obtained directly by gated.3. 1.such an arrangement has been used to compare cytochrome ~pectra. If the Raman signal can be detected above the fluorescence background in a single scan. Shelnutt. rapid. twochannel accumulation of the two signals scattered from a rotating. Heyden. Moving the laser line to the red to skirt the absorption bands associated with the fluorescence. in “Advances in Infrared and Raman Spectroscopy” (R. then a smallpore filter. M. 2. Clark and R. A.5.
the signaltonoise ratio has been improved less than tenfold. 38 J.9). 4. the free iodine level must be kept low by addition of a reducing agent. Sample purification is in nearly all circumstances the critical procedure. Energy transfer to an acceptor molecule.6. When a conformational marker mode happens to be juxtaposed with a conglomerate of other modes. by chemical modification. the backbone configuration of biological polymers that undertake a regular conformation is apparent from the typical Raman modes.’ The use of vidicon or photodiode array detectors should yield further improvement. Williams. typically. The identity of the mode can at the same time verified by its frequency shift upon such treatment. those requiring modified instrumentation are expensive and time consuming.443 (1976). F.134 3. which must have close access to the fluorescent impurity sites. 3. Of all these methods. Iodide ion is frequently used for watersoluble samples.’’ Similarly. ~ ~ 6. Appl. Coherent antiStokes resonance scattering (see Chapter 3.and SS. . assuming the background is not a function of time or small shifts in laser f r e q u e n ~ y . or by the use of deuterium oxide instead of water as solvent. 30. Thus highly polarizable groups such as C=C. RAMAN SPECTROSCOPY most easily by directing a stream of chilled air or nitrogen on it. Modulation spectroscopy. 5. Since the frequency. useful Raman bands. either it or the interfering modes may be shifted into an emptier spectral “window” by selective deuteration or other isotopic substitution of the molecule. photoquenching is also routinely utilized. Spectrosc. 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.’. and depolarization ratio of a Raman mode may change in response to a change in molecular conformation. Rejection of fluorescence (of 110 nsec lifetime.groups give strong. for biological molecules) by gated detection of the (nearly instantaneous) Raman signal scattered by illumination with nanosecond or shorter laser pulses. Short group frequency tables are found in introductory texts. 7. Even momentary occlusion of the beam reverses the photoquenching effect. Funfschilling and D. Using nanosecond pulses generated by a modelocked argon ion laser. intensity.
2) Base pairing. 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.TABLE Applications of Raman Spectroscopic Analysis to Biomolecules I. bonding scheme sites Structure of membranebound Ca2+ATPase.g. especially of (e.7. bonding scheme Nonheme metalloproteins (Section 3. substrate. chlorophyll Metal ion situation. Helix tilt Structure of rRNA in ribosomesb Polysaccharides (Section 3. hemoglobin.7. sugar chain. and Structure of cartilage sidegroup configuration Possible monitor of helixcoil transitions in plant polysaccharides Proteins (Sections 3.3) Structure of chondroitin sulfate' Sugar linkage.7.7. etc.7.7) Structure of redox or transport Metalligand geometry..1) Membrane fluidity.5) Polypeptide conformation Conformation. base stacking Conformation of nucleic acids in nuclei. ribosomes. 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) .7. bonding 0. 3. its perturbability Miscibility of different lecithins" Nucleic acids (Section 3. viruses. side groups of homopolymers and copolymers Amide I. Electron transport or conformation.carrying mechanisms in energetics cytochromes.4.6) Porphyrin symmetry. and other agents Porphyrins and hemoproteins (Section 3. 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. 111. ahelical content) noncrystalline proteins in native environment Sidechain configuration and Interaction of local region of environment protein with solvent.7.
G . but fluorescence free Extrinsic chromophores in proteins (Section 3. Carey. Dutta. 4751 (1977). of environment membrane fluidity Kinetics (Chapter 3.2809 (1976). Biophys. S. Bansil. 192 (1978). and D. Biophys. J.8) Configuration at site of bound Mechanisms of enzyme activity chromophore Biological polyenes (Section 3. R. Acta 541. ‘T. Maisano. V. J. R. L. Schneider. J . potential for kinetic studies Flavin adenine dinucleotideglucose oxidase binding” R. G . H. M. Young. Carey. enzymes. and H. Osterhout. J . M. photoreceptors. J. . G . V. Prescott. 74. R. and P. M. I. 98. Proc. Loehr. A. Biochemistry 17. N. 535 (1978). Nestor. nonresonance modes nonresonance modes for slower processes ( r > 10 sec) Spectral information from resonance CARS similar to that from resonance Raman. S. R. and G.TABLE(confinued) 1 Useful vibrational modes Structural information Biological insight Example Resonanceenhanced modes involving labeled substrate or active site of enzyme Preresonance. Bernstein. P. conformational transitions in biopolymers Chemistry. Biochemistry 16.25. polarity Polyenes as photopigments. U.. 25. 1. Biochim. Biophys. SOC. Acad.or resonanceenhanced stretching modes of polyene backbone Resonanceenhanced modes for most kinetics (T < 10 sec). K. and T. B. 4146 (1977). 224a (1979).. Freedman. J. R. and T. Lippert. Loehr. Am. Phelps. 108a (1979). Chem. 1081 (1978). E. Biochim.9) Dependent upon molecule and Same insights as for resonance chromophore under study Raman studies. J. Stanley. Yannas. Jr. Schultz. Salares. J. Acta 506. and H. Carriere. I. M.9) Configuration of polyene.7. Sci. R. Spiro. P. R. Thomas. D.8) Kinetics of hemoproteins. Lindsay. J. Biophys. Mendelsohn and J. R. kinetics of papainlabeled substrate complex’ Photopigments of lobster carapaces Dependent on choice of vibrational modes Photocycle of bacteriorhodopsin (see text) Nonlinear phenomena (Chapter 3. Hamilton. B.7. Natl.
1316 (1967). W. 30002800 cm. Yellin and I. 5 0 . Lipids 10. Bunow and I.1.388 (1977). Acad.7. Biophys. W. along with the direction of change of frequency and peak height with increasing conformational disorder. In Chapter 3. and intrachain plus interchain interactions (methylene CH bending modes at 1450 cm. Vibrations associated with the chains are listed in Table 11. Snyder. the information available from Raman spectroscopy is surveyed for several categories of biological molecules. and h1090/h1130 and h1090/h1060 are most commonly used to monitor changes in interchain (6 R. Yellin and I. The sensitivity of the Raman profile to conformation in each case delimits the types of biological questions that may be posed using this technique. Sci. 177 (1977). 165 (1973). Biochim. analogously. Biophys. Levin. Conformational Studies 3. CONFORMATIONAL STUDIES 137 bond energy and polarizability. strong Raman spectrum which is exquisitely conformation sensitive. 1572 (1971). Acta 487. which have a distribution of backbone configurations . local electrical field and symmetry. 68. R. Acta 489. Biochim. 39 40 .3.7. and/or intermolecular order. Raman bands reflecting hydrocarbon chain order can be roughly classified as those reflecting primarily only intrachain conformation (skeletal optical modes associated with CC stretching. Raman spectral correlations are imperfect for heteropolymers. is a function of lipid flUidity. A . 642 (1977). Natl. h2930/h2850. Levin. Phys.and CH stretching modes. J. 4 2 N. 4 1 N. the correlation of changes in Raman spectra with conformation has required a great deal of comparative spectral observation and computation. 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. 44 M. As might be expected.7. A summary of these Raman spectral applications is given in Table I. Chem. Levin. Biochemistry 16.”4244 Peak height ratios h 2 8 8 0 / h 2 8 . S. 3. 11501050 cm. U. such as globular proteins. are readily recognized in the Raman spectrum.I). Larsson. J . the ordered secondary structures.I). Lippert and W.7. L. Peticolas. L. 4 3 K. however. G . Phys. 47. W. ahelix and &structure. 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. The reader is alerted that some of the assignments in Table I1 supersede several erroneous ones found in the literature. Chem. Proc.
Peticolas. Bunow and I . Levin. S. Biophys. R. RAMAN SPECTROSCOPY TABLE Selected ConformationSensitive Modes of Hydrocarbon 11. I J . Hill and I. 191 (1977)]. 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. P. and 1. J . Biophys. Levin.. W.()  + () Terminal methyl group CH stretching m o d e s h ~ d ~ e ~ ' ~ J 2960 (2217) 2935. Levin. Biochim. These two assignments may be reversed [see M. L. R. Biophys. Biochim. Peticolas. symmetric. J . Chem. Biochim. P. and 1. Biochim. (1976). J. C. Sunder. R. Acta 487. w. M. W. 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. Biophys. Biophys.2070 (2126. Asym. Acfa388. asymmetric. Bernstein. Bunow and I . 191 (1978).3. R. R.456 (1976). 70. Acfa 282. M. L. Phys. Levin. Biochim. ' B. Yager. Gaber. Phys. 361 (1975). sym. 22. W. 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.842 (1979).2075) CH asym stretch CH sym stretch. 191 (1977). Biophys. and W. R. Levin. W. Acta433. Spiker. Spiker.C.388 (1977). L. Levin.8. Biochim. modes) a Frequencies and behavior of perdeuterated chains are in parentheses. Jr. W.h 1130 (1249) 1090 (?) 1060 (1145) Alltrans conformers Gauche conformers Alltrans conformers . R.457. W. Lippert and W. Chem. Bunow and I. and H. Lipids 17. I. Acta489. 8 (1972). Biophys. Acta 489. J . Mendelsohn. Jr. mode too weak to monitor.
L. fully hydrated glycolipids. Normalized lateral and intrachain “order parameters ” have been introduced to interpret the peak height ratios of Table I for phospholipid arrays. R.the presence of one very long (24 carbon) acyl chain. Caber. P.^^^^^ also associated with the hydrocarbon chains. Acta455. Biophys. J . which peak at 2850 cm. R.48 Several frequency shifts42. R. Yager.3. Biophys. are equally good monitors of fluidity. A more refractory problem arises in considering the CH stretching region. Biophys. L. J. charge. P. Bernstein. Mendelsohn.) The Fermi resonance background has substantially different contributions ’ ’ ’. Bunow and I. W.191 (1978). Jr.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. J.with overtone and combination modes of the 1450cm.alternatively. 5 1 B. relative to phosphatidylinositol reflects the behavior of the almost totally saturated chains (compare the intensities of C=C bands at 1665 cm. and I . each containing two fatty chains and one sugar residue (galactose in cerebroside and inositol in phosphatidylinositol) attached to the head group. Levin.457 (1976). 15 (1972). Biophys. Acta443. Mendelsohn. (Fermi resonance interaction of a fundamental with an overtone or combination mode of compatible symmetry and similar energy usually results in mode splitting. Mendelsohn. The conformation and packing of lipids in the membrane bilayer is influenced by chain length and unsaturation. and hydrogen bonding capability. Peticolas. Acta 290. Levin. The high degree of chain order in the cerebrosides (note the relatively narrower peaks).7.~’ to a mode of an or external standard. Biochim. The peak height h288.4951 and bandwidth^. 5 2 R. and H. and reference modes which themselves do not vary significantly.613 (1976). Biochim. Biochim. A broad dispersion band is seen in the present case.methylene deformation modes. Biophys. 48 R. Peticolas. 329 (1075). P. W. and W. Acta 413. Spiker and I. S. ~~. 191 (1977). W. CONFORMATIONAL STUDIES 139 plus intrachain order and intrachain order. Levin. Acta489. C. S.202 (1978). Sunder. 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. Biochim.’). Bernstein. M. Biochim. Sunder. Levin. Figure 3 illustrates the sensitivity o f the Raman spectrum to the conformation of the alkyl chains in two different. M. 22. of the lateral order parameter rests on a background produced by Fermi resonance of the methylene CH symmetric stretching modes. Bunow and I. such as the 720cm. Biochim. Biochim. Caber and W. 45 46 .~~~~ peak heights may be normalized to the height of a stable band. Acta 464. Spiker. Biophys. Biophys. and H.560(1976). 5 3 R. Biophys.260 (1977). r e s p e ~ t i v e l y.choline mode in fully hydrated le~ithin. Acta433. ” B. Biochim. C. Biophys. Acta 465. 49 R. and head group bulk.
. nor can it easily be d e c o n ~ o l u t e dThe peak height ratios . R. with respect to the high gauche conformer (9) population of PI (top spectrum) versus the high trans conformer population (t) for cerebrosides (bottom spectrum). which broaden upon increasing chain disorder. L. as well as temperature dependence within these states. brain cerebrosides. Hsu.140 1 8 1 3. G. gel phase. from the gauche/trans peak height ratio (hl. Levin. Bunow and I. " 1600 . to conformation of alkyl chains. Modes in the CC stretching region (11501050 cm') can be treated in a more analytical manner. plus the variable background. Krimm. Since the 2880cm' peak height is the sum of methylene CH asymmetric stretching modes. 1 1400 1200 loo0 .' peak height itself cannot be expected to act as a linear parameter of intrachain order. 395 (1978).~~ in the 30002800 cm. Snyder. Bottom. the 2880cm.region serve actually as phenomenological parameters of lipid order. and S. V tcrn ' i FIG. Vibrational Raman spectra of two different glycolipids illustrating spectral sensitivity 3. of the 54 R. 15°C. The more disordered PI shows broader bands (M.Acfa. 15°C. W. Top. ' RAMAN SPECTROSCOPY I I I Methylene CH stretch: asym /\ syrn J I // 2 3oM) 2800 . 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'. plant phosphatidyl inositol (PI). . unpublished).90/h 130) and an estimate of the gauche/trans conformer energy difference. liquid crystalline phase. A fairly conservative approach allows the direct computation. in the lipid gel and liquidcrystalline states. S. Part A MA. and the band profiles in the skeletal optical region (11501050 cmI). Spectrochim.
R. and Y. in “Membrane Transport Processes” (D.442 (1977). Yellin and I .’~~ The response of lipid lamellar fluidity to the incorporation of additional components including c h o l e s t e r 0 1 .3.^^. Wiedekamm. Bamberg. In this case. 4 ~ * ~ ~ ~ ~ ~ . The hydrationinduced frequency shifts of the amide I and C=C vibrational modes of the headgroup region of different cerebrosides. Biochim. Biophys. Biophys. 1007(1980). M. Wildermuth.490 (1977). allow one to distinguish different molecular packings in hydrated cerebroside bi1a~ets. Biochim. 6 2 W. New York. 8 (1972). S. Weber. 6 3 M. Verma and D. R. Kyogoku. Phys. Shipley. Levin. F. 21. Acta 468. CONFORMATIONAL STUDIES 141 number of gauche conformers formed at a given temperature in lecithins with saturated chains. 6o S . G . Biochemistry 17. Levin. J. P. Lipids 19. Small. ~ ~ ~ Modes associated with the polar head groups of lipids are also observable. p.^' polypeptides. Biophys.245 (1973). Conformation of the glycerophosphocholine head group of lecithin5* and sensitivity of glyceryl. L. Vol. and R. eds. Bunow and I. W. and D. Biochim. Wallach. W. Rand. P. Yu.7. the reference state (alltrans chains) was defined as that at . Larsson and R.41 Using these two sets of peak height ratios for the 30002800 and 11501050 cm. H. M. L.).62 The lipid may melt in a biphasic manner. Lehmann. Lippert and W. 1802 (1978).180°C. Biochim. Raven. Yager. F. W. Toda. that segregated by the added compound and that of independent bulk lipid. F.616 (1976). 55 56 ’’’ . 5 9 E. 5 7 J. Brdiczka. D. Levin.62 N. Curatolo. Acta 464. 32. A. 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’. Acta 426. 1. Gaber. Biophys. Peticolas. 5 8 Y. ~ ~disorder imposed by high and the increased curvature in small lipid vesicles. W. 161 (1978). 6 1 K. which may be interpreted to indicate the formation of two populations of lipid. Biophys. D. P. Acta 326. one can follow the main gel to liquidcrystalline phase transition in saturated the pretransition in saturated l e ~ i t h i n s .74 (1977). 4 ~ antibiotic^. Latorre. Ovchinnikov. C. Verma. P. phosphate. Biophys. Koyama. G. and choline group vibrational frequencies to hydration of lecithin5’ have been studied. J.regions. B. Macco. H. Acta 282. Chem. combined with Raman spectral parameters of chain packing. E. J . Biophys. D. Tosteson. Sakura. and R. Biochim. P. Peticolas. 2. 1978. G. and W. Wallach. L.@ Since the frequency of the 1130 cm’ alltrans skeletal mode is sensitive to chain length for short chains. 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 . Bunow and I.60 *~~~~~~~~ and has been studied by Raman spectroscopy. S.
C. 64 ‘’ S. Biochim. J . T. Harvey. S. F. P. Using this approach. Biochim. 477 (1976). A . Acad. 70 S. C. 73. and H. H. Shore. Carew. Chem. Biochim. The fluidity of natural membranes. S. and R. Sci. 243 (1976). polyenes act as rather useful probes of fluidity (see Section 3. SchmidtUllrich. Wallach.7. RAMAN SPECTROSCOPY In these studies thermal phase transition curves. Kaufman. W. Tu. 67 F.other modes of the perdeuterated chains also lie in spectrally “clean” region^^'. Phys. 6 6 F.633 (1975).53. R. Cancer Res. If the lipid or added components are absorbing (as reflected also in a high fluorescence level). Thompson. sarco. H. H. D. R. Acta 426.64 . or independent determinations of lipid phase transition temperatures may be made using a calorimeter or a microscope equipped with heating stage. Verma. P. F.g. and D.^^ (Table I). P. and D. e. W. 7 1 R. Nail. Wallach. Acra 419. Stanley. U. Harney. Biophys. Biophys. plasmic reticulum. B. Yeh. Biophys. Mendelsohn and J.3490 (1977). constructed from Raman spectra accumulated at a number of temperatures. SchmidtUllrich. Verma. J . one lipid species may be enriched in or substituted by its analog containing perdeuterated chains. P. S.51. P. Acra 506. Milanovich. Proc. F. Wallach. 37. H. and R. P. 6 9 E. Biophys. 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. 6. . W. Lipids 17. Baskin. Milanovich. SchmidtUllrich. invalidating the thermal comparisons. 79 (1976). Y. Verma and D. The methylene CD stretching modes for this species can then be observed free of interference in the 23002050 cmregion50.9). Biophys. H.67and thymocytes68has been assessed by Raman spectroscopy.142 3. and A. are usually compared for different samples of reconstituted model membranes. Verma. This effect may be detected by testing for the dependence of spectral profile on laser beam power. of red blood ~ e l l s . differential behavior compared to that of the bulk lipid. Acra 394. 192 (1978). F. 16. ~ Supplemental analysis of resonanceenhanced ~ ~l Raman spectra of lipidembedded polyene probes has confirmed the interpretation of the behavior of lipid chains. fatty samples may vary from sample to sample. local heating in the laser beam of the compacted. ‘* S . Increased fluidity of membranes of transformed and lectintreated cells has been r e p ~ r t e d . Maisano. Biochim. 3558 (1976).. one should be able to identify a species of deuterated lipid bound to intrinsic membrane proteins by its isolable. 106a (1976). P. Robbins.~ ~ ~~. Wallach. R. and corrections then made. E. R.
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.
TABLEl l . Spectral Regions Containing Conformational Markers for Hydrated I 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 phenomena i DNA and in the melting of RNA; these modes participate 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).
’’
3.7.
CONFORMATIONAL STUDIES
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 ~**~ the state of DNA in c h r ~ m a t i n , ~including specific guaninearginine 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 ~ ~ also highly reare warding. 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 , contacts. A general correlation between the torsional angle $I for rotation 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 2 0 Identification of the disulfide band can be verified by chemical 19 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 pre*~ ~ resonance 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. 48, 2417 (1975). Jpn.
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 . ’ ~ ~ depolarization ratio p , which supplies just The 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. 5482 (1976). 98,
(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 both oxidized and in 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 cor~~, ~~ related 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).
Perutz. H.7. A. s 4 B. Proc. Rimai. T.660 (1975). Nagai. 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. the dynamics of heme restructuring upon substrate or oxygen ligation are being tackled for cytochromes. Am.'" 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. Biophys. Sutcliffe. U. 1 (1975). 1809 (1971). Actu 460. Am. A.) 201. P. to the number expected according to simple grouptheoretical rules. Lutz. 800 (1978). ' ~ . M . H. J. J . Farquharson. Biophys. I s 3 T. representative references are given for this wide fie1d.' 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. Szabo.154 3. Biochemistry 17. 97. Holm. J .150. D. V. T. Herskovitz. Moss. Res. Biochemistry 15. J. G. K. 34. distant heme groups. 62. Biorhrm.It~ ~ *been hypo~ ' has ' thesized that distorted geometry of the ironsulfur complexesfor example. 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. F. C. 831 (1978).' 1. Barron and A. ' . in the cubanelike FeS clusters in oxidized ferredoxin is demonstrated more convincingly by observation of splitting of one of the FeS symmetric splitting modes. Mortenson.'29~'36~141~'42. T. M o l . and G. T."~ 3. Commun.' The coordination of 0. 59. that is. V. Kilmartin. and S. Vallee and R. 93."~ Loss of symmetry.C. H. S. Is' 1. 1 (1978). A. SOC.498 (1968). Biophys. Fairhurst and L. 378 (1976). Bid. and L. Woodruff and S. 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 . Tang. Science (Washington. E. D.15. Chem. Babcock. J. Williams. This splitting is also seen in the spectrum of reduced highpotential iron protein. 15' S. L. and W. R. R. Lovenberg. Soc.Nutl.P. 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.408 (1977).151 Studies of chlorophyll environment in intact chloroplasts are also in progre~s.7. Salmeen. Sci. RAMAN SPECTROSCOPY and then by the other. Biochim. R. Simon. Szabo. Alkins. Loehr. L. L. M. Acad. S.138*147149Similarly. Long. ' ~ 53 . H. inequivalence of FeS (cysteine) linkages. l S 2 M. Spiro. Prog. characterized by their depolarization ratios. Chem. Antanaitis.
Commun. Careyand H. 15' . and with electronic absorption band ~ a v e 1 e n g t h . R. R. 1 5 9 P. Kilponen. M. Chem. yields a wealth of detail on substrateenzyme binding configurations and dynamits. H. 6. 16' A. J . Biophys.3. Heyde. Kurtz. R. T. L. 6. serve to fingerprint the length and isomeric identity of polyenes. B. J. Acud.. Several reviews are available. Bioeng.100.744 (1976). resonance enhanced by admixture of C=C stretching. SOC. Nutl. Biochemistry 15. R. Honig. J .'~" 56*1s7Copperligand modes in blue copper proteins have also been analyzed. Callender and B.9. Doukas. SOC. Res. Lett. the C=C stretching frequency (16001500 cm. R. P. D. " ~ addition. 98. Biol. Chem. SOC. Chem. R. A . P. Masetti.8. 99. Schneider. 15 . I ~ ~ The frequency is also sensitive to solvent p ~ l a r i t y .S. Scheraga.6776 (1971). Rev. J . and H. Honig. B.248 (1978). Loehr. A.4493 (1973).' 58 The reader is referred to recent reviews. Am. Gill. 171 R. and B. II.5677 (1974). Young. M. T. Schneider. and L.7.128 3. 56. E. S. Chem.1'~18~100~128~160 3. Heyde. This approach. Lynn. R. E. Aton. and H. SOC. R. and P. D. L. Biochemistry 15. G. Carriere. E. Sci.15916s and hence is anticipated to be widely exploited. 102. Jr. 1 6 5 R. 0. D. J . and G. H. and D. 93. Warshel and M . 14. Am. Carey and H. Scheule. J. Acc. K. S . Chem. King. U. Am. Kumar. 98. J . Carey. polyene length. Annu. Schneider. R.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. E. Van Wart. M o f . 128. Extrinsic Chrornophores for Resonance Rarnan Studies o Proteins f Vibrations involving the active site of enzymes which contain no intrinsic chromophores can be selectively enhanced upon addition of a chromophoric substrate or inhibitor. P. Carey and H.I ) varies inversely with increasing electron delocalization. Res. Biochem. pioneered by Carey and coworkers. The resonance signal intensity is compatible with kinetic studies. Rimai. Petersen. Chem.3273 (1977).Silman. Loehr. J . Vallee.33 (1977). 5033 (1976). G.7. and I. Thamann. J. Watters.stretching modes. SOC. J .2387 (1976). Chem. K. Zerbi. F. 1661 68 For alltrans polyenes. M. Rumun Spectrosc. that is. and T. L. McFarland. and P. W. R. These are enhanced primarily by Atype vibronic mixing with nz* transitions.726 (1977). Schneider. G. Karplus. Shriver. G. SOC. 122 (1978).2195 (1976). Gill. Chem.18.4187 (1977). Rimai. M. Proc.Y. Li.7. 51. N. Carey. Am. CONFORMATIONAL STUDIES 155 enhanced by illumination in the chargetransfer bands of these respiratory pigrnent~. Biochemistry 16. 95. Dellepiane. J . Biophys. Am. Phys. L. Carey. G.267 (1977).679 (1976). and K. R. Klotz. Am.831 (1973). modes In between 1400 and 1100 cm'. Callender. R."l Heteroatoms bonded in conjugation with the polyene also possess T. M . M. 96. K. Sufra.
Finkelstein and R. W.~~ spectral responses The probably reflect changes in the fit of the polyene among the hydrocarbon chains. Biophys. I. ~ * * ' ~ ~ The intensity of the C=C stretching mode of p carotene decreases dramatically when the lipid matrix of the membrane passes. with increasing temperature. Holz. 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. Isolated cell membranes contain minute amounts of c a r ~ t e n e . 17' 173 .'47'76'177 Studies on this time scale require a signal S. H . and the decrease in dielectric coefficient sensed by the polyene.8. Chem.156 3. Anal. for example. Biochim. B.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. Nature (London) 265. probably by formation of an oligomeric sterolamphotericin B p ~ r e . 48. The polyenic antibiotic amphoterician B acts only upon cell membranes containing sterol. H. B. and L. Alltrans polyenes have been shown to act as Raman probes of membrane fluidity when they are mixed with the hydrocarbon chains of lipid membranes. Membranes 2. Res. P.' 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.659 (1977).'~ enhanced 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. DeKruijff and R. A band several hundred cm' wide of the signal dispersed by a monochromator can usually be observed per pulse. 1 7 6 A. J. RAMAN SPECTROSCOPY resonanceenhanced modes. Commun. ElSayed. Acta 339. H. Terner. Atkinson. and M. Acia401. Bagyinka. 186 (1976). Horvath. Bioehem. Demel. from the gel to the liquidcrystalline state. It can be concluded that amphotericin B is well embedded in sterolcontaining. F. Biophys. Biochim.57 (1974). a nitrogenlaserpumped tunable dye laser. ' ~ ~ The ' frequency and intensity of the preresonance.660 (1977). Verma and D. as the lipid bilayer thins and becomes more 3. Szalontai. Campion.Biophys. A. A. Woodruff and G. Wallach. 168 (1975). 377 (1973).8). Cs. combined with a gated detection system incorporating a vidicon or photodiode array target and optical multichannel analysis. 76. 1 7 ' A. but only surface bound in sterolfree lipid bi1aye1s.
M . A. Sombret.' 78181 Studies of bacteriorhodopsin kinetics on the microsecond time scale have also been tackled by flowing the sample past the beam at a controlled. and M. '" M. 18" . 3046 (1979). These pulses have also been produced by electromechanical chopping.g. 149 (1971). J.'~. J . S.349 (1978). Rerny. A. and J. R.. Oseroff and R.''' but its photosynthetic function as a transmembrane 178 J. Nature (London). ElSayed. J. U. of seconds. Lewis. 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. M. ElSayed. A. Burns.' 78. ElSayed. 1 8 ' J. 20. Terner. ElSayed. enzymesubstrate associationdissociation (time constant z < sec) and substrateinduced conformational changes < z < lo' sec) thus become feasible. S t r u t .L. A.) 195. 6.4) or resonance CARS (Chapter 3.C. A.261 (1977). as in other types of spectroscopy. Biophys. variable flow rate. CrunelleCras and J. '*'M. and M. J . Proc. H. J. Terner. A . 74.369 (1977). Modification of the scan drive has been suggested for faster scanning. 1328 (1977). R. Terner. 45.L. U .9) process. C. Hsieh. Bacteriorhodopsin. ElSayed. Campion. Terner. A. Burns.185 Studies of macromolecular dynamics. A. 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. Raman Spectrosc. and M. B. Terner. Wallart. Campion. consisting of glycoprotein plus retinal bound in Schiff base linkage. Campion. 5212 (1 977).3629 (1979). 4243 (1974). Sci. Biophys.3. using a conventional grating instrument. Proc. 527 (1979). 233. Nut/. Kinetic processes may be triggered. A. C. D. R. 1 8 3 J. using a stoppedflow arrangementIE6)or temperaturejump impulses.8. Osterholt and W . A. Stoeckenius. A. 1. Acad. Biochemistry 13. Merlin. ElSayed. A . and M. Marcus and A. and F. 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. 76. Adu. M. is structurally very similar to the visual pigment rhodopsin"* for which similar studies have been perf~rrned. Natl. Callender. A. Sci. A. Laser Spectrosc. Mol. Hsieh. Terner. by concentrationjump (for example. Hsieh. Science (Washington. combined again with vidicon detection. Rapid scanning on a slower scale. J . C.New B i d .L. and J.IE2lB4 In this approach. 26. Biochemistry 18. A. Acad. can be achieved by computercontrolled scanning over a limited spectral region. D. C. e. S.'" The bacteriorhodopsin studies mentioned above illustrate the dynamic and structural resolution gained by the combination of stationarystate and kinetic resonance Raman studies. 128 (1977). KINETIC STUDIES 157 intensity characteristic of a resonance Raman (Chapter 3.
Acad. Sri. Callender. V. the C=C stretching mode assolE9 P. 4462 (1974). D. Doukas. 194 R. 1' 9 B. Nakanishi. and W. H. Biochernisrry 15. G. Crouch. U. A . Osterholt.band of R570. C. Lozier. and W. J .'^^*'^^'^^ The intermediate M412. M. . Callender. mediates of the cycle are not apparent from the nearly structureless absorption bands at room temperature.158 3. R. a wavelength near the 412nm absorption maximum of M412. R. Stoeckenius. 1 9 * D. and K. K590. H. Becker. A. and T. H. A. 1 9 ' B. appears to resemble 1 3cisretinal. Heyde.' 79*183.9) which has been observed within picoseconds of illumination. 15. Bogomolni. Chem. but retinals can be "fingerprinted" by ~*'~~ their resonance or preresonance Raman s p e ~ t r a ' ~and matched to the appearances of bands in bacteriorhodopsin preparation^. In Fig. Using 5nsec laser pulses at 582. Rimai.192 In the resonance Raman spectrum. the protonation state of the retinal chromophore is differentiated by the frequency of the Schiff base C=N stretching mode.lg3 Substantial stationarystate concentrations in bacteriorhodopsin preparations are seen only for R570 and M412. S . Gill. Stoeckenius. J. visible region absorption m a ~ i m a . C . B. A . Section 3. Nutl. near the 570nm absorption maximum of R570. researchers have observed the C=C stretching mode of the first intermediate.5 nm. J. Lewis. 913 (1974). E.176 lower frequency The of this mode is correlated with the longer absorption maximum of K 9 50 (cf.1621 (1976).1 nm. 1 9 5 A. resonance enhanced by conjugation with the retinal chain. the other intermediates are seen only with kinetic resolution. ' ~ ~ . 4 The structures of the interThe . Biophys. 955 (1974). Bogomolni. for example.5 nm.' 89 The initial rearrangement occurring in the chromophore in picoseconds (isomerization versus electron redistribution) is contr~versial. A . and isomeric retinal intermediates have been thus f identified by the rates o appearance of new. the C=C stretching band at 1570 cm' associated with the intermediate M412. SOC. Porte. Proc. Doukas. Ippen. Ebrey.as a shoulder arising at about 1515cm' on the 1530cm. H. and D . Aton. Lozier. 200. A. A. Chance. 4b).' ~ ' cycle is shown in Fig. 1279 (1968).) E. 93. 15.C. RAMAN SPECTROSCOPY proton pump is better understood than the visual process. 71. Spoonhower. Marcus. Science (Washington. Lewis.6288 (1971). M. R.'82 The kinetics of proton cycling of the retinylidene chromophore have been studied by absorption spectroscopy. 2004 (1977). J . and located at 1643 cm' when protonated or 1620 cm' unprotonated. R.7. B. and L. R. Shank. 4a. and M.'~~ The step at which the Schiff base is deprotonated can be located in the timeresolved resonance Raman excitation profiles. Am. 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. prominent feature in the timethe resolved spectrum is the 1530cm' band. Biophys. Biochemistry 16. when laser excitation lies at 514. Hess. In contrast (Fig.
Spectra adapted from Campion el al. 4 Resonanceenhanced kinetically resolved Raman spectra of bacteriorhodopsin. The 1643cm' band visible in the Fig. an antiStokes Raman line is seen. In order to scan the Raman spectrum. is tuned through the desired spectral interval. A CARS signal beam emerges at frequency v 3 = 2v. phasematching angle. one of the incident lasers.3. It is hoped that the differential resonance Raman studies just described. as suggested by the data of Fig. presage new applications of resonance Raman techniques to other problems in biological kinetics. very high conversion efficiencies ( 104106 times those in spontaneous Raman scattering) are obtainable. In the CARS process.5 and (b) 514. (c) Bacteriorhodopsin photochemical cycle. using excitation at (a) 476. 3. parent compound. 1 7 9 ciated with R570.Mk H release ' FIG. a tunable dye laser.5 nm from an argon ion laser. 4b indicates the protonated state of R570. 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. the subscript on each intermediate is the wavelength (nm) of its visible absorption maximum. 17001500 cm'.v2. showing the isolability of structural information by a combination of excitation profile and time analysis. 4. . 4c has been obtained by similar analyses. The combined results of these studies establishes M412as the first deprotonated intermediate of the photocycle. primarily employing the steadystate flow technique with appropriately fast sampling by the laser beam.v 2 is a vibrational frequency. and the resultant strong antiStokes Raman lines occur not only at higher frequencies than .9. . Whenever the difference v1 . Information about the structure of the other intermediates shown in Fig.9. NONLINEAR PHENOMENA 159 .
4146(1977). G . Proc. Bioeng. R. J. A significant CARS background from glass sample holders has also been reported to be troublesome. illustrating the similarities in spectral profile to the resonance Raman spectrum and the absence of fluorescence background’”.74. Sci. Klauminzer. McDonald.”~ Another Raman process. 28. 3798 (1976). the background emission arising from aqueous solvent is competitive with CARS scattering from the solute. Cramer. Natl. Harvey. Nibler. T. with great potential for kinetic studies.200Since resonance Raman conditions are precisely those suffering from the worst intrinsic fluorescence problems. S. Nestor. and G. Sci. Greenhalgh. M. Toiles. Annu. Biophys. Raman Spectrosc. Chabay.202In a resonance CARS study of highly fluorescent flavin adenine dinucleotide (FAD). S. Black. U .nitrogen NH deformation mode. R. Gilson. depolarization ratios for ferrocytochrome c have also been measured in the CARS configuration. K. Spectrosc. R. 7. andT. A. J. 31. I. Sci. B. under resonance conditions is a good CARS signal obtained. S. A . Spiro.27 (1976). Acad. 90 (1978). Hetherington. 135 (1977).multiple scattering by sample molecules results in loss of incident beam coherence and loss of the CARS signal. U . J . J . Acad. K. W. Hudson. It should be noted that samples must be nonturbid . assignments were suggested for some of the modes enhanced by vibronic mixing with the first nz* transition in the plane of the flavin. RAMAN SPECTROSCOPY most fluorescence but in a beam spatially separated from the incident beams and requiring only a single monochromator for filtering. A.3329 (1976). . W. S. Dutta. G. The tremendous fluorescence rejection and signal strength means that CARS has great potential for biological studies.’” CARS spectral profiles and depolarization ratios differ somewhat from those obtained in analogous conventional and resonance Raman studies. therefore. Proc. 6. Natl. Appl. Laser Focus 14. 20’ J. Hudson. Appl. Klauminzer. U. a “singleshot” spectrum may be obtained. Chabay. Nestor. Phys. and L.253 (1977). If a broadband dye laser is used. but essentially equivalent spectral information can be extracted from the CARS spectra. Nail. 961 Both conventional and resonance Raman antiStokes spectra may be obtained in a CARS experimental configuration. S. only. B.160 3. also nonlinear in response to the applied field. 1 9 9 I. 73. Lett. reflecting hydrogen bonding of FAD to en~yme. *On J. 111. and B.84 (1978). However. 73. The inverse Raman spectrum appears as a ’’’ W . R.’02 A resonance CARS spectrum has been obtained for ferrocytochrome c. Acad. D. is inverse Raman scattering. Spiro. Nestor. ’02 J. T. A . G. allowing a frequency increment in the 1359cm’ band in the presence of glucose oxidase to be interpreted as a shift in the flavin N. Hudson. K. S. B. 203 P. and A. CARS is exactly the technique needed for vibrational studies of particularly fluorescent biological chromophores. Laycock. and G. J. Klduminzer. C. Proc. Rev.
Inverse Raman effects can occur with other nonlinear processes: negative bands observed in CARS scanning of a vitamin B. Biological applications of resonance Raman spectroscopy. P. * 0 5 M. 3. most especially to problems in biochemical kinetics. are anticipated to expand in scope. I . a Use of the microscopein biology is limited because of damage of the biological specimen by laser beam heating. p. . commercial model is available.3.10. J . and also to cellular and subcellular systems including semiordered systems oriented with respect to the beam direction. Vol. in part by using a defocused illuminating beam and a wellcooled microscope stage. The Raman Microprobe A Raman microscope with simultaneous spatial and spectral resolution has found application in minerologyZo5. Heyden. Ira W. Raman Spectrosc. taken precedence over applications of the inverse Raman effect. 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. London. Long. ’04 D.200 3. A. Mathieu.). 33 (1975). I 1. Dhamelincourt. in “Advances in Raman Spectroscopy” (J. 3. 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. 1973. 1. Levin for reading this article and for providing a stimulating research environment. sample have been conjectured to be due to inverse Raman effects. The vibrational spectrum again lies at higher frequencies than the fluorescence Exploitation of CARS for biological studies has. some interesting biological investigations can be envisaged.. however. and then to apply these correlations. Acknowledgment The author would like to thank Dr. If specimen damage can be controlled. ed. Delhaye and P. Further work remains to be done to quantify the spectral correlations for the behavior of biological molecules.1 1.
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Maiman. “Nonlinear Optics. Letokhov. eds. Inc. N. Hellwarth. they have very broad electronic absorption or emission spectra. and also in highresolution spectroscopy. S. H. New kinds of spectroscopy have been devised taking advantage of these properties. and D. Therefore. Similarly.” SpringerVerlag. spectroscopy of high time resolution is of more importance to biological systems than highspectralresolution spectroscopy. 106 (1961). Javan. Since the environment of biological molecules is usually inhomogeneous and aperiodically organized. “Coherent Nonlinear Optics. 1980. 33. 20 Copyright 0 1982 by Academic Press.” Benjamin. Herriott.. McClung and R .4. in many cases. Berlin and New York. Feld and V. This property is utilized in hightimeresolution spectroscopy. The 1960s was a time of very rapid development in short pulse generation by lasers. which is the main subject of this chapter. ~ 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. Phys. 1965. 6. 163 METHODS OF EXPERIMENTAL PHYSICS. Rev. VOL. Bloembergen. Nature (London) 187. ISBN 0124759629 . All rights of reproduction In any form reserved. R. Appl. For example.’ a Qswitched laser with a pulse of several tens of nanoseconds was i n ~ e n t e d . Bennett. in photosynthesis. 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. photon echo. Only two years after the first report on laser oscillation in 1960. New York.the early photoproducts of rhodopsin in visual transduction appear in a picosecond regime. J. A. Lett. The spatial and temporal coherence of the laser makes it a unique light source for spectroscopy.1. * M. PICOSECOND LASER SPECTROSCOPY By Takayoshi Kobayashi 4. W. 828 (1962). R. Introduction The discovery of the laser’ has led to the development of many new fields in spectroscopy. F. and other coherent transient spectroscopies.2 coherent Stokes Raman scattering (CSRS).493 (1960). S. some kinds . 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 addition to the abovementioned properties. J.~~~ of lasers have one more important characteristic for spectroscopic measurementsshort pulse width. W. Temporal coherence is used in coherent antiStokes Raman scattering (CARS).. Phys. Jr.
47 x 10’) v i a x E ~ V (sec). Am. Life utilizes solar energy in two ways. A 308. 95 (1968). J. Kasha. To be efficient in the application of light energy in the visible region. G. Phys. DeMaria.) in the visible region. As picosecond laser spectroscopy was applied to the study of semiconductor and chemical species. The advent of the modelocked solidstate laser7 enables one to study photophysical.8.390 (1926). SOC. and DNA are also being studied by many research groups. J. These improvements led to the application of picosecond laser spectroscopy to biophysical situations. The relationship between the S1 t So radiative lifetime t .67. = (3.e. where solar energy transmitted to the earth is most intense.Ser. * F. ’ . Qswitched lasers were used as powerful pulsed light sources for nanosecond spectroscopy. Phys. from a few tens of microseconds to a few tens of nanoseconds. photosynthetic applications are most prevalent. Collins.. carotenes. R. Lett. Proc.284 (1950). Proc. i( s 1 R. A. Other important biological systems such as rhodopsin. Perrin. or photochemical transient species with rise times or lifetimes in the picosecond regime. Phys. G. Windsor. 994 (1945). Nature (London) 164. W.164 4. A. 3075 (1967).6 Before the appearance of modelocked lasers. D . Left. J . H . 270 (1965). in the transmission of information and in the conversion of light energy to chemical energy. 174 (1966). and xanthenes. Chem. Appl. Ser. biological chromophores should have large capture cross sections in this region. Picosecond spectroscopy offers special advantages for the study of photobiological systems such as rhodopsin and chloroplasts. G . J. Chem. R. Appl. Lewis and M. The chromophores in rhodopsin and bacteriorhodopsin are protonated Schiff bases of retinal and those in chlorophyllprotein complexes are chlorophylls. The chromophores have an electronic structure similar to that of many organic dye molecules. (London).W.” This Qswitched laser increased the time resolution of ordinary flash photolysis5 a lOOOfold. W. chlorophyllprotein complexes in photosynthetic pigments. and the S .658 (1949). R. Porter. Radium 7. Soc. and bacteriorhodopsin in proton pumping pigments. hemoglobin. t So molar extinction coefficient E is given by the equation’ T . 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. Phys. 7. and time resolution were improved by many physicists and chemical physicists. Norrish and G. Soc. Among the biophysical studies using picosecond laser spectroscopy. Porter. the techniques of picosecond laser pulse generation. J. The chromophores and organic dye molecules have an intense transition between the ground state (So) and the lowest excited singlet state (S. photobiological. The lightabsorbing substances are rhodopsin. Novak and M. detection. and H. Heynau. Mocker and R. (London). in visual pigments. N. J. 47. Setser. A 200. i.
the initial photobiological reaction must be completed in a much shorter time than the radiative lifetime.. c Soabsorbance maximum. 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 .of the S. which is 110 nsec. z.is calculated by the equation to be 110 nsec. i. Fluorescence in complicated biological macromolecules is emitted from the lowest excited singlet state S1 because of the very efficient radiationless TABLE List of Abbreviations I. In order for biological systems to utilize efficiently the light energy absorbed by the pigments without losing the energy by photoemission processes. Integration is over the S. In addition to the application of picosecond spectroscopy to the primary photochemical processes in photosynthetic and visual pigments.1.and E of the order of lo5 M .e. t So absorption band. Since many ordinary dye molecules have a bandwidth of the order of 5 x lo3cm. is the wave number in cm..' cm' in the visible region. AMP AR BChl BPh BS CARS CONT CELL CSRS F 11 IR L M ML MONO Nd. there are a number of other important applications to biological systems..4.INTRODUCTION 165 where v. This is the reason why picosecond spectroscopy is invaluable for the study of the primary process of vision and photosynthesis. Some of them are as follows. it must be in the picosecond time scale.
In Section 4. photosynthesis. Comparison of Nanosecond and Picosecond Spectroscopy Flash photolysis was initiated by Norrish and P ~ r t e r who applied the . In Section 4.2.2.2. lasers and Qswitch Nd/YAG lasers have been utilized for excitation light pulses. 4. the time constant of the radiationless process should be in the picosecond range.2.~ technique to photophysical and photochemical processes of various molecules mainly in solution. Because of the interaction between these side chains and the protein constituting pigments. internal conversion. Since absolute rates of transfer are obtained by picosecond spectroscopy. The application of Qswitch lasers improved the time resolution of photolysis experiments by a factor of a thousand.3 examples'of the application of picosecond spectroscopy to biophysical systems. PICOSECOND LASER SPECTROSCOPY S + S. the light energy is transferred to a reaction center without losing the energy by fluorescence emission in the transfer process. Not only Qswitch ruby lasers but also other nanosecond lasers such as N.1. nanosecond range. In Chapter 4. At this particular stage of development. After the absorption of light by antenna chlorophyll. Soon after the success of Qswitch oscillation of ruby lasers.2 the principles of generation and the classification of picosecond light pulses are described. The motion of side chains of biological macromolecules or polymers are known to occur in 10. time resolution was not limited by . In Section 4.1310'4sec from the frequency of infrared or Raman spectra.2. The time resolution of flash photolysis was limited by the pulse width (typically a few tens of microseconds) of the excitation flash. Therefore this process can also be studied by picosecond spectroscopy. In the appendix we discuss several nonlinear optical phenomena useful for generating pulsed light for excitation and interrogation. A list of abbreviations used in this article is given in Table I. Some of the optical elements and detectors used in picosecond spectroscopy are also described briefly in the Appendix. the motion might slow down to the picosecond time scale. The energy transfer should be therefore in the picosecond range. and vision are shown. or for pulse width measurements. lasers could be used for nanosecond spectroscopy. Since the radiative life of S. Nanosecond and Picosecond Spectroscopy 4.1 the comparison of techniques and equipment between nanosecond and picosecond spectroscopy are made. we can determine the structural spacings between the energy donors and acceptors. is generally in the .166 4.3 various methods of picosecond spectroscopy are described.
Excitation Pulsed Light Source for Picosecond and Nanosecond Spectroscopy. Picosecond or nanosecond spectroscopic methods can be classified as either emission or absorption methods. The modulation method can be used only for emission processes exhibiting a single exponential decay. highspeed photomultipliers. in “Ultrashort Light Pulses” (S.‘ the ’ D. By using perturbing pulses with a shorter pulse width than the time constants of the phenomena to be observed.). 4. D. Auston. 204.4. in “Ultrashort Light Pulses” (S. several completely new detection methods were invented. 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. SpringerVerlag.1 . . and that of the timeresolved emission or absorption spectrum.1. 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. ed. There are two approaches to nanosecond or picosecond spectroscopy. Shapiro. the sample is periodically perturbed by excitation light with wellknown phase characteristics. SpringerVerlag. one can determine the time course of the resulting process by observing the intensity of the monitoring light or the emission. ed.By measuring the phase z shift 8. in “Ultrashort Light Pulses” (S. If an emissive species with a finite exponential decay lifetime z is excited by a modulated light source with frequency w.2.2. 1977. 1977. 123. In either case. these devices are not suited for picosecond spectroscopy because they do not have time resolution on the picosecond time scale. Bradley. a pulse technique and modulation technique. L. Excitation light sources only are used for emission spectroscopy. In order to resolve times in the picosecond region. the lifetime of the species can be obtained. Shapiro. 1977. Many sophisticated electronic devices.). In what follows we discuss only the pulse technique. J . Classification of Picosecond and Nanosecond Spectroscopy. In the modulation technique. NANOSECOND AND PICOSECOND SPECTROSCOPY 167 detectors but mainly by the excitation laser pulse width. Berlin and New York. L. Berlin and New York. there are two types of measurement: the observation of the time dependence of emission intensity or absorbance. p.2. while excitation and monitoring light sources are used for absorption spectroscopy. L. singlephoton counters. such as wideband (up to 1 GHz) oscilloscopes.1. 4.’”. In the pulse technique. Berlin and New York.2. yon der Linde. H. ed. a perturbation of nanosecond or picosecond duration is given to a sample. Shapiro. However. The classifications of nanosecond and picosecond absorption and emission spectroscopy are summarized in Table 11. SpringerVerlag. boxcar integrators. 18. light emitted from the species is also modulated with the same frequency and a phase shift 8 given by tan 8 = o . lockin amplifiers. D. p.). p.
. TEA N. Nd/YAG). TRW. flash lamps Modelocked gas (HeNe. and nitrogen lamps with a pulse width of a few nanoseconds are commercially available as excitation light sources. K. . Young.’ Synchrotron radiation can also provide several ten or several hundred picosecond pulses. G . excimer lasers. 1979.’ parametric oscillation. Ndiglass. G . fast photodiode. synchronously pumped dye lasers Picosecond continuum: nanosecond laser: nanosecond flash lamp Subpicosecond continuum Su bnanosecond (1 0. 191 (1968).’ Nanosecond flash lamps such as hydrogen. Heard. 65 (1964). Soc. Phys. streak camera. ((1966). N. lasers and dye lasers pumped by the lasers Modelocked solidstate lasers (ruby. 9. Kunz.I6 hydro ’ C. Berlin and New York. dye lasers pumped by the above lasers. Lu. Neeland. variable delay. sampling scope Qswitch ruby and Xe flash lamp: Nd. l 5 E. repetitively pulsed lasers. and D. PICOSECOND LASER SPECTROSCOPY Classification of Emission and Absorption Spectroscopy in Nanosecond to Subpicosecond Time Region Monitoring light sources for absorption Time domains Nanosecond ( 1000. I ” H.” SpringerVerlag. Model 31A and 32A. l 3 “Handbook of TRW Fluorimetry” Manual of T R W Nanosecond Fluorimetry Equipment.168 TAHLE II. B14li. 4. Xe flash lamp Ar ion) lasers. and photomultiplier Subpicosecond (1 0. Kerr gate.1 psec) second and higher harmonics of the picosecond pulse. lasers. such as nitrogen.users 1. Evtuhov and J . Snitzer and C. . H.!. such as Qswitch ruby14 and Nd/glass’ lasers. Inc. streak camera Picosecond ( 1000. deuterium. modelocked solidstate lasers (ruby. l 4 V.. synchronously pumped dye lasers Passively modelocked dye lasers. “Synchrotron Radiation Techniques and Applications.I nsec) Excitation light sources Detector systems Photomultiplier. Kerr gate. Nd!YAG). N.1 nsec) Sampling oscilloscope. Nd/glass. variable delay. ed.rrrs2. and photomultiplier Variable delay and photoinultiplier .I pscc) Echelon and O M A . air breakdown and H. very widehand oscilloscope.YAG lasers. variable delay and photomultiplier.’’ Singleshot solid lasers.Am. ’. wideband oscilloscope: boxcar integrator.
l9 ’’ H. J . 1260 (1960). Soc..05 0. ed. Berlin and New York.01 200300 4060 (0. K . Bazhulin. In a fourlevel system. ’O . 32 (1978).310) x 103 (10SO) x 106 5 1 13 0. and dye lasers” pumped by flash lamps or nanosecond lasers have been powerful light sources for nanosecond spectroscopy and laser photolysis. 1 (1976). P. Ewing.” SpringerVerlag.11 0. J.4. (nmy 337. Wavelength of laser oscillation. Trans/. G . and energy are typical of commercially available lasers or homemade lasers. Rhodes. genI7 and Nd/YAG lasers”. Nd/YAG is yttrium aluminum garnet doped with Nd3+. “Excimer Lasers.11 0.1 0. In a threelevel laser system.. Knyazev. ed. laser oscillation takes place between a lower level and an upper one where the latter is populated by relaxation from excited levels.. Schafer.05 % (by weight) C r 2 0 3 where only the chromium ions participate in the laser oscillation phenomenon. Lasrrs4. Petrash.1 156. Phys. Phys. Pulsed Lasers for Nanosecond Spectroscopy Typicalb repetition rate (Hz) Typicalb peak power (MW) Typical singlepulse energy’ ( J ) Pulse width (nsec) Laser Gas lasers N. 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.2 I060 1001 < 10 0.. N. A. JETP (Engl.3 347. with 0.6 0.O.3 310 15 2030 2030 15 15 . Berlin and New York. 1068 (1965). peak power. laser oscillation takes place between an upper and a l7 P. The chemical composition of ruby for use in laser oscillation is AI.030. NANOSECOND A N D PICOSECOND SPECTROSCOPY 169 TABLE 111. its laser oscillation is interpreted in terms of a fourlevel laser.20.530 193 248 308 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. and G. 1973. Ch. 1979. G . Ruby laser oscillation is interpreted in terms of a threelevel laser.02 0. Danielmeyer.71 61. laser H.2. Dye lasers pumped by excimer lasers as well as excimer lasers2’ themselves are also useful light sources for nanosecond experiments.)20.04 ’ Repetition rate.3 694. “Dye Lasers. 22. I.” SpringerVerlag. F. Today 31.
with the final duration of the pulse theoretically limited by the gain spectrum width of the laser medium used (Nd/glass. . shortening the pulse width each time it passes through the dye solution.1. 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. and pulse width of these nanosecond lasers are listed in Table 111. The oscillation wavelength. and a laser gain medium.2. 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. As the light beam passes through the active medium.2. Kr ion.170 4. The operating principle of the modelocked laser can be explained in terms of either the time domain or the frequency domain (Fig. The timedomain explanation is illustrated in Fig.CAL RESONATOR RESPONSE CURVE FREQUENCY MODELOCKED LASER OUTPUT  LkJduL TIME (b) FIG. The gain of the active medium of a threelevel system is more sensitive to temperature than that of a fourlevel system. The laser pulse increases in intensity when it passes through the active medium. 1). the beam then passes through the dye.2. ruby. Mode locking in frequency (a) and time domains (b). the beam passes back and forth many times. 1 . 4. Picosecond Light Pulses 4. Nd/YAG. l b and is given as follows.2. a passively modelocked laser is obtained by inserting a saturable dye in a cavity composed of mirrors. Pulse trains from a real GAIN CURVE (MATERIAL RESPONSE) OPT. the former is populated by exciting Nd3+ from the ground level to levels located above the upper level. Experimentally. or dye solution). Generation of Picosecond Pulse. PICOSECOND LASER SPECTROSCOPY lower level. pulse energy. Ar ion. peak power. In the cavity. an active medium.
the samples must be circulated using a pump and a flow cell. in glass matrices of organic solvents at low temperatures. The longitudinal modes are separated in frequency by c/21. 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.2. P. 103.4. and S. namely. Several typical picosecond light sources which can be used for research in biology are listed in Table IV. Each laser material (gain medium) has its own gain curve. Shank. 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. Shank. Each transverse mode in a laser cavity has a set of longitudinal modes in the frequency region where the laser gain overcomes resonator loss. Repetitively pulsed lasers are more appropriate for obtaining more precise data since it is easy to average data at high repetition rates. L. in “Picosecond Phenomena” (C. 4. Berlin and New York. With a lasing medium of an adequate bandwidth.2. They can be classified as follows: (i) singleshot lasers and (ii) repetitively pulsed lasers. This E. . the ruby laser. When the repetitively pulsed lasers are used for photoreactive samples. 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. The Nd/YAG laser and passively or synchronously modelocked dye lasers pumped by an Ar or Kr ion laser are repetitively pulsed lasers.2. Ippen. lppen and C. This is due to spreading of the pulse width caused by laser material and cavity dispersion. the output will be a welldefined function of time forming the modelocked laser pulse train. a picosecond pulse train with a constant time separation of c/21 can be produced. the ff ashlamppumped dye laser. If a set of longitudinal modes is maintained in a fixed phase and fixed amplitude relationship. the output of the laser varies in the distribution of the phases and amplitude of the longitudinal frequency components. in polymer matrices.). 1978. There are various kinds of singleshot modelocked lasers. la. SpringerVerlag. E. V. Shapiro. The mechanism for modelocking a laser as visualized in the frequency domain is illustrated in Fig. P. which undergo irreversible photochemical reactions. p. V. NANOSECOND AND PICOSECOND SPECTROSCOPY 171 resonator do not achieve the theoretical pulse width limit. The geometrical configuration of the laser resonator and gain medium is responsible for resonator loss. eds. Without the mode locker. Photoreactive samples in the crystalline phases. the Nd/giass laser. Classification of Picosecond Light Pulses.2. 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.
Ippen. In the following section.3. Shapiro. Ippen. 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. Lett. 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. 1978.2.5 Wavelength of laser oscillation. PICOSECOND LASER SPECTROSCOPY TABLE IV. peak power.001 5 x IO~ 610630 10Hz 1000 0. Berlin and New York. L.1 MHz 100 0..3 347. Repetition rate. 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. V.172 4. p. in "Picosecond Phenomena" ( C .5 1060 530 1064 532 610630 I10Hz 110 HZ 0. The former is necessary for the assignment of the transient species and the latter for the determination of the rates of the corresponding processes. The continuum is generated in nonlinear optical processes such as parametric fourwave mixing and/or selfphase modulation.000 5001000 1000 20100 210 50 5 30 3 20 1520 58 46 3050 2040 0. E. Phys. P. P. we give some details of several different experimental schemes summarized in Table V. V. eds. Appl. E.).2 Typicalb repetition rate Typicalb peak power (MW) Pulse width (psec) single shot single shot single shot single shot 10005000 100500 500010.. 373 (1974). and energy are typical of commercially available lasers or homemade lasers. P. (nm)" 694. Shank. C. Shank.5 0. 4. Shank and E. 103. detection . The reason why so many different schemes have evolved is that there are various picosecond lasers. SpringerVerlag. Ippen and C. and S. V. 24.
TRS S 5b systems.2.3. The principle of this method is as follows: Monitoring light intensity is probed before and after excitation. Absorption Spectroscopy for Time Dependence Measurement Using a Repetitively Pulsed Laser. VD. 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. In the block diagram shown in Fig.4. The fundamental pulse after passing BS is reflected by M2. and M1. The monitoring light signal is averaged over many laser shots. By changing the variable delay time of 315nm used for the excitation of a sample. the delay time dependence of the absorbance change is determined.1. TABLE V. A cavity dumper attached to the dye laser is operated at 10 Hz in synchronization with the Nd/YAG laser for amplification. averaging is easily performed by means of a repetitive pulse. Readers interested in a particular application can skip to the relevant section. 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. It is focused by L3 on a sample cell. This is an example of a repetitively pulsed light source. absorption or emission (A. 4. and M3 and focused by L1 into a continuum generation cell.2. The second harmonic generated by SHG is for an excitation light source and is reflected by BS. 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. 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). The second harmonic (315 nm) of the laser output is used as an excitation light source. TRS Method number. VD with a stepping motor. 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. and it is difficult to eliminate the effects of longterm fluctuation . and methods to resolve on a picosecond time scale. 2a.
2. The second harmonic generated by SHG2 is reflected '* M. and (b) ML Nd/glass laser. M. R. Lett. The third or fourth harmonic can also be used as the excitation light source. Chem. The single pulse is amplified by AMP after being extracted from a pulse train by SPS.174 (a) 4 4 4.3.22 In the block diagram. Block diagrams of (a) repetitivepulse and (b) singieshot picosecond spectroscopy equipment to measure the time dependence of absorbance of intcrmediates. 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. . and R. 2b. 1 (1971). P. Echelon Method for the Measurement of Time Dependence of Absorbance Using a SingleShot Laser. and focused on the sample cell by L1. illustrated in Fig. This is the big disadvantage of using a singleshot laser. 176). 9. Topp.2. 4. uses an echelon to convert time resolution to spatial resolution. a modelocked Nd/glass laser is used as a singleshot light source. M1. M2. VD. The experimental system can be modified to be a dualbeam spectroscope in order to minimize the effects of intensity fluctuations (see p. P. The second harmonic generated by SHGl is for the excitation of a sample and is reflected by BS1. Phys.2.PICOSECOND CONTINUUM AMP UI SPS YL LASER FIG. Picosecond lasers used are (a) Arlaserpumped M L dye (rhodamine 6G) laser with a cavity dumper and a Nd/YAGiaserpumped dye amplifier. This method. of the laser intensity by averaging with this method. Rentzepis. Jones.
transmissive and reflective. and M11. Both the monitoring and reference beams are detected by a vidicon or TV camera attached to the monochromator. They are subsequently focused at a small angle to each other in the sample cell by lenses L1 and L6.4. (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.3. The monitoring beam passes through BS2 and then is reflected by M6 and M7 and focused on the sample cell by L6. The echelon method was invented by Topp et a1. These echelons are used to convert a monitoring pulse to a pulse train with appropriate interpulse separation in time and space. There are two kinds of echelons. M8. and then focused by L7 together with the monitoring beam on the entrance slit of a monochromator (MONO). they can give timeresolved information about the sample. For example. 3. M5.cos O]/c for the transmissive echelon. The reference beam is reflected by BS2. A single pulse is converted into a pulse train passing through or being reflected by echelons. When the step thickness of an echelon is d. The generated continuum is focused by L3 on a scatterer (SC) and collimated by L5. NANOSECOND AND PICOSECOND SPECTROSCOPY 175 by M3 and M4 and focused by L2 into a continuum generation cell (CONT CELL). The echelon used in picosecond experiments is simply a stack of glass plates or blocks.2.sin2 0)1/2 . It was also utilized to get time resolution of absorbance change on a picosecond time scale using singleshot lasers. After passing through an echelon the continuum is split into monitoring and reference beams by BS2. Both excitation and monitoring pulses travel nearly equal optical distances. The light that travels through the different steps of the transmissive echelon experiences varying retardations. M10. the normal angle of incidence of the monitoring light passing through the echelon is 6. in the case of an echelon with steps of 3 mm ( a ) TRANSMISSIVE ECHELON (b) REFLECTIVE ECHELON FIG. M9. as shown in Fig. If the light pulses passing through the steps of an echelon can be resolved in space.22 obtain time resolution to of emission intensity in picosecond region. and the refractive index of the glass is n. then the interpulse separation is given by Ar = 2d cos O/c for the reflective echelon and At = d[(n2 . . The situation is similar when a reflective echelon is used.
''' .2. Nagakura. or photographic plate. This fluctuation is caused by the amplitude instability o laser pulses which induces fluctuation of the angular distribution of the f picosecond continuum intensity in the optical medium where the incident laser is focused. for example. 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. a silicon vidicon. transmissive echelons are appropriate for experiments requiring high time resolution. 4.43. 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. Phys. the absorbance changes with time on a picosecond time scale after an excitation pulse. up to about 1 nsec.g. Them. A Nd/glass laser is used as the light source.Resolved Absorption Spectra. a dualbeam (monitoring and reference beams) method was de~ised. 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.. and then focused by L1 to excite a sample cell. The intensity of each pulse in the monitoring pulse train is decreased or increased by transient absorption or bleaching of the sample. A pulse train separated both spatially and temporally. With the transmissive echelon. while reflective echelons are suitable for experiments with relatively slow kinetics. VD. 29. The fluctuation of the spatial distribution of the monitoring light pulse may give artificial data if a singlebeam (monitoring beam) method is applied. 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. Phys. 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. then l ~ g [ Z " ~ ( ~ ) ~ ~ ~ ( i ) / Z ~ gives) Z ~ ~ ( ~ ) ] ~ ( z the absorbance change at delay time At(i) corresponding to the ith echelon. Chrm. Therefore. 4. Netzel and P. A block diagram for the measurement of picosecond timeresolved absorption spectrum is shown in Fig. Lett. To get a highquality transmitted and reflected image. To remove the artifact caused by the changes in the intensity distribution across the echelon steps. e.3. Spectroscopy for Time. Kobayashi and S . The second harmonic generated by SHGl is reflected by BS. formed by reflecting or passing through echelons from a monitoring pulse. The second harmonic generated T. L e f f . If. PICOSECOND LASER SPECTROSCOPY thickness. respectively. L. M . each step of the stack must be in optical contact and each plate surface must be parallel to all the others.3. TV camera. 337 (1974). Rentzepis.176 4. T.429 (1976).'~" Figure 2b shows the equipment for dualbeam picosecond absorption spectroscopy. the intensity distribution over the echelon steps after passing through the sample cell is different from that before passing through the cell. can be observed by a detector which has spatial resolution. and M 1.
S. The other light beam. Twodimensionalmode OMA is useful for dualbeam spectroscopy for measuring timeresolved absorption spectra.4. and T. is focused on the lower part of the entrance slit of the monochromator and refocused on the B region of the OMA. A block diagram showing the experimental system for multishot emission kinetics is shown in Fig. absorbance is given by l o g [ Z ~ ( n ) Z ~ ( I ) / Z l ( I ) Z ~ ( Ia) ] as function of A. Ohtani. Photobid. Block diagram of an experimental system for measuring the timeresolved absorption spectrum at a fixed delay time with a singleshot laser. Opt.2. 2b. Shichida. The A region covers the top half of each channel of the photocathode of the vidicon. H. T. Suzuki. 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. 2162 (1971). Kobayashi. Yesaka.4. 32. first applied to the observation of picosecond phenomena by Duguay and M a t t i ~ k The~ Kerr shutter used in picosecond emission . 4 can be modified to a dualbeam spectroscope similar to that of Fig. Mattick.23bIn the twodimensional mode. Multishot Emission Spectroscopy for the Measurement of the Time Dependence of Emission Intensity. the system shown in Fig. by SHG2 is focused into a continuum generation cell by L2.2.~ Z 3 b K. T. Y. 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). Duguay and A. 809 (1980). 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. A. T o minimize fluctuations. If the counts of a channel corresponding to wavelength I are 1. the plane of the photocathode can be divided into two regions A and B. A p p l . Either a photographic plate or a vidicon with an optical multichannel analyzer (OMA) can be used as a detector. 4. F V I DlCON A PICOSECOND CONTINUUM c CONT CELL L2 BS FIG. A modelocked Nd/glass laser is used as a light source.(I) and ZB(I) for the A and B regions. 10.4.3. and the B region covers the bottom half. Nagakura. . Yoshizawa. passing through a pinhole which is not exposed to excitation light. Photochem. This method utilizes a Kerr shutter. 1060 nrn NANOSECOND AND PICOSECOND SPECTROSCOPY 177 4 530 nm . H. 5a.
5. and the relaxation time of the Kerr medium. such as a photomultiplier (PM). The second harmonic generated by SHGl is reflected by BS. Ippen and Shank” have succeeded in operating a CSz gate at the Kerr resolution limit. which does not have picosecond time resolution. 1060 nm PICOSECOND LASER SPECTROSCOPY * 530 nm * VD r UME I L SPS LASER FIG. A modelocked/Nd glass laser is used in both (a) and (b). VD. The time resolution of the system is determined by the width of the pulse exciting the sample. Ippen and C. P. the width of the shutter pumping pulse width.The measured Kerr response time of CS2 was determined to be 2. The fluorescence is detected by an ordinary photodetector.0 k 0. The second harmonic generated by SHG2 is reflected by M3 and M4 and then focused by L2 on a sample cell. Phys.92 (1975). Appl. 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.3 psec. Shank. 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. 25 E. In the process they made a direct measurement of the Kerr lifetime.4. M1. 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. Lett. Using subpicosecond pulses from a modelocked CW dye laser. 26. spectroscopy is opened at several delay times after excitation of the sample. V. .
Kobayashi. then S(At) = F(At). Busch. F ( t ) must be obtained by deconvolution or convolution/simulation. G. Phys.At). 0. there are two disadvantages to the method. However. The emission from the sample is focused by an ellipsoidal mirror on the entrance slit of a monochromator. One is that a streak camera is much more expensive than the Kerr shutter necessary for multishot experiments for emission kinetics. M. L. Olson. in order to accumulate data without the effect of the jitter to improve the signaltonoise ratio.5. and P.2. E.2. ’’T.3. M2. Kobayashi. 5b. 3570 (1978). E. Deconvolution can be done by applying a Laplace transformation to S(At) and g(t . and M4. J . VD. or a time 26 G. it is necessary to have a standard time. Another gate method which can be used instead of the Kerr shutter is the optical pulse gain amplification m e t h ~ d . Phys. 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. Appl.2. J . 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. The timedependent spectrum is observed by the combination of the streak camera. M3. The sample and the entrance slit of a monochromator are located at the focal points of an ellipsoidal mirror. and then focused on a sample by L2. R. F ( t ) is the convolution of the excitation pulse shape and a function of the intrinsic fluorescence decay. Lerr. twodimensional emission information from the sample is monitored by the vidicon. Jones. If the width of the gate function is narrow enough compared with the time constant of the fluorescence decay.3. Phys. When the time constants are not sufficiently long for the effect of convolution to be neglected. A modelocked Nd/glass laser is used as a picosecond light source. The other is that the camera has a jitter that makes it difficult to average the data. This method is much easier than the multishot method explained in Section 4.** The main portion of the second harmonic generated by SHG is reflected by M1. Streak Camera Method to Measure the Time Dependence of Emission Intensity Using SingleShot Laser.At) of the Kerr shutter. ’’T. Rentzepis. and P. Rentzepis. an image intensifier.4. S.4. Therefore. where C is the constant of proportionality. Appl. 27. P. Chem. . 31 18 (1979). S(At) is given by the equation S(At) = C S l d F ( t ) g ( t . ’ ~ * ’ ~ 4. Degenkolb. 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 . M. Greve. 450 (1 975). K. and a vidicon. 50.” To get a standard time.At) dt. 69.
and M2. . 100 psec). 4. 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. 6. The emission spectrum can be detected either by a photographic plate or by a vidicon attached to an OMA coupled to the monochromator. and focused by L4 on the entrance slit of a monochromator (MONO). By modifying the methods.3. SingleShot Experiment to Measurement TimeResolved Emission Spectra. we can obtain both types of data at the same time. A block diagram of the equipment necessary to obtain timeresolved fluorescence spectra with a singleshot laser is shown in Fig. passed through the shutter. 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. M1. PICOSECOND LASER SPECTROSCOPY reference.6. 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.3. The second harmonic generated by SHGl is reflected by BS. In the diagram. 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. The timcresolved emission spectrum of a sample at different delay times can be obtained by moving the variable delay. To acquire twodimensional picosecond absorption spectroscopic data.180 4.2. 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. 6 . Emission from the sample is collected by L3. TwoDimensional Detection of Absorption Spectra as a Function of Delay Time After Excitation.2.28The 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. The second harmonic generated by SHG2 is reflected by M3 and M4 and then focused by L2 on a sample cell. a Nd/glass laser is used as a picosecond light source.7. 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. 4. VD.
APPLICATIONS TO PHOTOSYNTHESIS AND VISION 181 dependence measurements of absorbance described in Section 4.3.O) takes place following the equation .2. The new OMA from Princeton Applied Research EG & G.2. The exit slit width is one of the factors that determine time resolution of the monochromatorstreak camera system. to form carbohydrates (CH.2. as well as the spectrum. A twodimensional streak camera is now available from Hamamatsu. Instead of using a vidicon in a onedimensional mode. In the reaction center.3. and the intensity distribution perpendicular to the slit gives the spectrum. it can be used in a twodimensional mode.Photosynthesis 4.3. 4. This is also done using a photographic plate.8.5. The twodimensional image on the screen gives both spectra and time dependence. Applications to Photosynthesis and Vision 4. Photosynthesis takes place in chloroplasts.4. TwoDimensional Detection of Emission Spectra as Functions of Delay Time after Excitation.3. OMA 11. 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. where the energy of light absorbed by antenna pigments is transferred to a reaction center. 4.1. can be easily operated in a twodimensional mode. at several delay times after excitation. whole emission spectra can be measured at a delay time using one laser shot. In this mode. An understanding ofthe process ofthe application . It is possible to obtain data on the time dependence of emission intensity.1 Introduction. 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. the intensity distribution across the direction parallel to the entrance slit gives the data for time dependence.3.3.2. The perpendicular slit is not for monochromatizing but for getting the total spectrum and for limiting the height of the spectrum. 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. 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.3.I. reduction of CO.
3. P. Windsor and M. Porter et d Z utilized the 9 secondharmonic pulse (530 nm. W. Porter. C . Barber. 3 1 P. Picosecond Study of the Energy Transfer Process between Antenna Pigments by Emission Spectroscopy. Avouris. T.). Kaufmann.. allophycocyanin (APC) at 660 nm.232 (1978). 369. and J. 3 0 M. 4. for Details of the study are given in the following subsections. Amsterdam. Acfa 501. (a) Observed and (b) calculated time dependence of the fluorescence intensity of Bphycoerythrin (BPE) at 576 nm. SpringerVerlag. G. Tredwell. and chlorophyll a (CHL) at 685 nm. 0. was studied with the use of the emission spectroscopic technique for red algae by Porter et dZ9 later part of the process. primary electron transfer. ed. Biophys. 6 psec) of a modelocked Nd/glass laser to excite the accessory pigments in the intact alga of porphyridium cruentum. From Porter ct a/. 1978. H. 7. Elsevier.e.182 4. 126. in “Advances in Laser Chemistry” (A. Rentzepis. G. ed. Biochim. was studied The by Windsor and Rockley3’ and Rentzepis et d 3 ’ bacteriochlorophyll. in “Lasers in Physical Chemistry and Biophysics” (J. Rphycoerythrin (PRE) 29 G. J.2. Degenkolb. Berlin and New York. p. the transfer of energy from antenna pigments to a reaction center. i. M.). PICOSECOND LASER SPECTROSCOPY 0 30 TIME (psec) 100 FIG. Rphycoerythrin (RPE) at 640 nm. Zewail. JoussotDubien. W. F. J . The rise and decay kinetics of emission from Bphycoerythrin (BPE) at 576 nm (which is excited by the picosecond pulse).1.” The preceding part of the primary process of photosynthesis. . K . Rockley. p. Kobayashi. Searle. and E. 1975.
Sci.4. it decays in 246 a 16 psec. J. and J. M. while a negative absorbance change at 610 nm is due to the appearance of P' and it appears in 220 _+ 14 p ~ e cKinetics indicate. 7b. 72. Photosynthetic bacteria are often used in studies of photosynthesis because they have relatively simple structure and the reactioncenter chromophores are easily removed with detergents. and chlorophyll a (Chla. Assuming that the energy transfer between chromophores takes place by longrange interaction in the order BPE to RPC to APC to Chla. Proc. and P. Windsor.90.32observed that the 864nm band due to bacteriochlorophyll bleaches within 10 psec of excitation with a 530nm pulse. Netzel. The induced spectral change measured 240 psec after excitation has all the characteristics expected of the difference spectrum between P'X. W. 7a. Leigh. R26. R. Cogdell. Dutton. W.)188. CHL in Fig. With the use of a picosecond absorption spectroscopic system with echelon.C. Acad. shown in Fig. The results. therefore. This study is the first direct observation of energy transfer between chromophores in antenna pigments. Picosecond Absorption Spectroscopic Study of ReactionCenter Oxidation in Bacteriochlorophyll Photosynthesis. 8. Porter et al. J. and W.3. Rentzepis.33and Rockley et al. T. APC. Parson. Science (Washington. Leigh. L. and the spectrum of oxidized species P + becomes evident.1301 (1975). M. The experimental results of the time dependences of the fluorescence intensities are shown in Fig. of 12 psec for RPE. that P + . D. respectively. J.3. Kaufmann et al. P. 2251 (1975).) 182. Science (Washington. 24 psec for APC. The fluorescence from the latter three pigments all showed finite rise times. and 50 psec for Chla. Netzel. S. and 175 psec.29calculated the time dependences of the fluorescence intensities. is very often used. P. Kaufrnann. The experimental results obtained by these two groups are quite similar to each other. The intermediate state PFdisappears after several hundred picoseconds.118.34. The mean fluorescence lifetimes of BPE. A . G . Rockley. Natl.and PX. to the maximum emission intensity. 4. are in good agreement with the experimental observations. A mutant which has no carotenoid. allophycocyanin (APC) at 660 nm. 7) at 685 nm were monitored by a streak camera. Rentzepis. Netzel et al. K. 32 33 .~~ T. The time dependences of absorbances at 680 and 610nm are shown in Fig. APPLICATIONS TO PHOTOSYNTHESIS AND VISION 183 at 640 nm. U.C. After this very preliminary experiment. A positive absorbance change at 680 nm is due to the immediate appearance of the intermediate PF after picosecond excitation. and Chla were 70. M.3. RPC.34did independent picosecond experiments over a wide spectral range. All of the fluorescences except BPE fluorescence appeared to follow an exponential decay law.238 (1973). S. 34 M . The spectral change observed immediately (within 10 psec) after excitation with a 530 nm picosecond pulse is due to an intermediate state PF. L. L. D.1.
whereas BChl monomer has no absorbance in the region. 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. 610 nm 0 200 400 600 ( p%C) 800 TIME AFTER EXCITATION I ~~ ~ ~ ~~ ~~ 0 400 TIME AFTER EXCITATION 200 600 (pSeC) FIG. Rentzepis. Dutton et al. B. 8a. In order to clarify the electronic structure of P'X. Dutton.184 4. FEBSLetr. 60. From Rockley et d f 4 forms directly from PF.35 investigated the 1250nm absorption band of the reaction center of R26 mutant. M. PICOSECOND LASER SPECTROSCOPY AA. indicating that P+ is formed directly from PF. and the rate of disappearance of P' (filled circles) measured from the absorbance increase at 680 nm after picosecond pulse excitation. The absorbance at 1250nm increased immediately after the excitation of neutral reaction center with 10 psec laser 35 P. The oxidized dimer of BChl (BChlLBChl) formed by the treatment with ferricyanide has absorption maxima at 1250 nm. .34The timeresolved absorption spectrum 20 psec after excitation is very similar to that observed for chemically reduced PXafter 20nsec pulse excitation. Kaufmann. J. L. Chance. (b) Semilogarithmic plot of Fig. K. and P. From the result.275 (1975).8. (a) Rate of formation of P+ (open circles) measured from the absorbance decrease at 610 nm. PF disappears in 246 & 16 psec and P + forms in about 220 & 14 psec.
2.[BChl'BChl J BPhlX < I0 psec J c. However. 905 (19341935). From these experimental results. It has been suggested that the PF state could be symbolized as [BChPBChl I] where I represents another reduced component. .2. 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.. 316 (1933).1.p*X = c. As mentioned above. whose structure is approximately a half of a carotene is present in the eye..4. light energy absorbed by antenna chlorophyl is transferred to reaction center and utilized 36 3' G. In photosynthesis. Nature (London) 132. The expected difference spectrum between [BChlLBChl BPh] and [BChPBChl BPh:] resembles the observed timeresolved spectrum of R26 at 20 psec after excitation. Wald. 18. Vision 4. APPLICATIONS TO PHOTOSYNTHESIS AND VISION 185 pulse at 530 nm.3. Therefore I is concluded to be BPh.[BChliBChl 1 100250 psec BPhlX As can be seen from the above discussion.37 first showed.appears several hundred picoseconds after excitation. Physiol.. the physiological purposes of the photosynthetic and visual processes for light energy utilization are quite different from each other.3.3. G. Some of the photoreceptor molecules used in the very fast first steps of visual and photosynthetic processes are similar to each other. the carotenoids. A class of molecules. picosecond spectroscopy was very usefully applied to the clarification of the mechanism of the primary process of photosynthesis. P'X. Wald. and.X c2[BChlBChl BPhlX c. Introduction. The concluding photochemical steps of bacteriochlorophyll obtained by the above experimental results are3' c2P. J . are present in most normal plants.P+X = c. as Wald36.[BChl'BChl BPhlX c. retinal. Cen.PFX = c. 4.
Applebury. whether hypsorhodopsin or bathorhodopsin is the first intermediate after the absorption of the photon. Vol. M. L. 69. Sci. and P. rhodopsin. 1972. 74. 146. Nature (London) 267. Busch.. PICOSECOND LASER SPECTROSCOPY for. A. driving the entire photosynthetic process and then finally converted to chemical energy.2802 (1972). Alfano. U . Rentzepis. hypsorhodopsin could not be observed after 530 nm excitation of bovine rhodopsin in LDAO (i. Proc. Green.40 The second harmonic (530 nm. observed by irradiating cattle rhodopsin at 77 K. J.39 After the observation of hypsorhodopsin. Sundstrom. 4. Rentzepis. S. A. A . Lifetimes of these intermediates are very short. 179 (1977).38 and hypsorhodopsin. In visual process. Peters. T. L.2. Applebury. Yoshizawa and Y . Several intermediates are formed successively after the photophysical event. 4 3 K. VII. Applebury. Sci. Nature (London) 182. Picosecond spectroscopy equipment was used to study the very fast roomtemperature kinetics of appearance and disappearance of intermediate species absorbing at 561 nm.). on the other hand. Peters. G . 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. Two of the intermediates observed at low temperatures are bathorhodopsin. Aton. Ammonyx LO). Dartnall. ed. Nail. M. Proc. M. 1604 (1958). Acad.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. Kito. 31 19 (1977). 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. and R. 40 G.e. P. A . Callender. it was concluded that bathorhodopsin is formed within 6 psec after picosecond pulse e~citation. 6 psec) of a modelocked Nd/glass laser was used as a picosecond light source to excite bovine rhodopsin solubilized in Ammonyx LO detergent. K . Berlin and New York. U . Rentzepis. E. and P. the question arose. it was observed that hypsorhodopsin is formed from squid rhodopsin solubilized with digitonin with use of the second harmonic of the T. Nail. The absorption of the photons by the photoreceptor. and M . H. Picosecond Absorption Spectroscopic Study of the Primary Process in Vision. R . M.43 However. p. M. From the observed instantaneous rise of absorbance at 561 nm. H. Yoshizawa. 38 39 . seen at 4K. L. 4 1 V. Monger. Acad. Nature (London) 269. A. SpringerVerlag. initiates the primary process in visual excitation. R.3. 645 (1977). Lamola. T. in “Handbook of Sensory Physiology” (H. 4 2 B. S.186 4. B. Part 1.2.~’ Furthermore.
Nagakura. FEBS Lett. and 630 nm are shown in Fig. 44 Y. T. and both the rise time of bathorhodopsin and decay time of hypsorhodopsin were 50 f 20 psec. T. hypsorhodopsin.4. 4 s Y. The lifetime of the excited singlet state and the rise time of hypsorhodopsin were both found to be 15 5 psec. and S. 335 (1978). 80.313 (1979). H. in the detergent (LDAO and digitonin). H. 4’ T. FEBS Lett. 106.found that ~ ~ ~ the primary process of cattle rhodopsin is almost the same as that of squid rhodopsin. 4 6 T. Photochem.3. T. 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 . 480. respectively.570. 440. ’’’ p5ec + hypsorhodopsin * 2o psec + hathorhodopsin. The data obtained for 430. Yoshizawa. The kinetics is observed to be triphasic at these wavelengths. 570. Photochem. 27. Kohayashi. Ohtani. 600. Shichida. a first phase appears at 020 psec and a final one at 80400 psec. or in the protein (cattle and squid).e. Yoshizawa. 214 (1977). Nagakura. Phoiobiol. 32.e. APPLICATIONS TO PHOTOSYNTHESIS AND VISION 187 modelocked ruby laser as an excitation light s o ~ r c e . 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.. 9. 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 . ~ ~ ~ ~ ’ From the analysis of the depth of the dip observed in the time dependence curve of absorbance change at 570 nm. Ohtani. ’ 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). Kobayashi. and S . 207 (1980). T. The species corresponding to the kinetic stages are assigned to the excited singlet state of rhodopsin.and octylglucosidesolubilized solutions after excitation with a picosecond pulse at 530 nm. Kohayashi.It~was . i. 4 ~ ~hypso intermediate was found to The ~ ~ convert into bathorhodopsin with a time constant of 50 10 psec at room t e m p e r a t ~ r e .. Kobayashi. Shichida. 550. and bathorhodopsin. The time dependence of absorbance at seven different wavelengths (430.440. i. Phoiobiol. . ~The~discrepancy between the formation time constant of ~. and 630 nm) was observed for bovine rhodopsin in LDAO.The~time constant ~ *~~ is <20 psec at room t e m p e r a t ~ r e . most cattle bathorhodopsins are formed by the following process at room temperature: rhodopsin (So) h S.
Ltd.188 4. Pergamon Press. irrespective of the excitation wavelength.9a. The formation and decay times of cattle hypsorhodopsin are 15 & 5 and 50 L 20 psec. hypsorhodopsin is the main first intermediate of the visual process in bovine and squid rhodopsin. 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. Is ’ psec. Squid and bovine bathorhodopsin are both formed primarily via their respective hypsorhodopsins. of the nature of the detergent. Reprinted with permission from Kobay~hi. Absorbance change at 440 nm for octylglucoside sample is shown by (c). and of the protein. while those of squid rhodopsin are <20 and 50 k 10 psec. 0 I I I I 400 100 200 TIME I p s e r l 300 FIG.~’ Copyright 1980. From this fact it is concluded that. respectively. There may be a small amount of bathorhodopsin formed by the following process : rhodopsin (So) h v S . 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.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 . .
Reprinted with permission from K o b a y a ~ h i . Ltd.08  a 0060 0 0 0 04  0 002 0  I J FIci. Pergamon (b) LDAO sample. ~ ’ Copyright 1980. . 9c.016 012 2 001 00. Time dependence of the absorbance change at 570 nm for (a) octylglucoside and (b) LDAO sample. Reprinted with permission from K ~ b a y a s h i . O r FIG. ~ ’ Press. Time dependence of the absorbance change at 630 nm for (a) octylglucoside and Copyright 1980. 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. 9b.
The dye molecules then relax to the lowest excited state and emit fluorescence. Giordmaine. and then the transition from the state to the final state takes place. M. For example. 4 8 J. Dye molecules used for pulse width measurement by twophoton fluorescence techniques should be highly fluorescent. When the laser beam has noisy peaks with picosecond pulse widths and the total pulse width is much longer than the picosecond pulse. The laser beam is split into two beams of equal intensity which are then reflected by mirrors M1 and M2. Appl. and should have no singlephoton absorption cross section and a high twophoton absorption cross section at the wavelength of the laser. Lerr.l . Phys. l .190 4. 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. Nonlinear Optical A. In the latter process. Multiphoton Absorption. 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. an intermediate electronic excited state is actually excited by one photon. L. or fluorescein is used for the pulse width measurement of ruby lasers. PICOSECOND LASER SPECTROSCOPY APPENDIX. and it should be smaller than the twice the photon energy. 216 (1967). Rhodamine 6G. Nonlinear Optical Phenomena. rhodamine B. At a high laser intensity the absorption cross section is not constant. rhodamine B or rhodamine 6G is used. Twophoton absorption has been used for pulse width measurements of picosecond lasers. The laser pulse optically pumps the dyes into an excited state with energy twice the photon energy. and K. W. On the other hand. Shapiro. Rentzepis. but depends on the light intensity. I . 10. . 11. the width of a picosecond pulse can be measured. Simultaneous twophoton absorption is different from a twostep absorption. 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. 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. P. A.48 Using the simple optical system shown in Fig. for the pulse width measurement of the fundamental of Nd/glass lasers or Nd/YAG lasers. S . Wecht. This phenomenon is called a twophoton or a multiphoton absorption process. Two or more photons can be simultaneously absorbed by a medium a t sufficiently high light intensities. In order for twophoton absorption to take place efficiently in organic dye molecules. and Detectors Related to Techniques of Picosecond Spectroscopy A . Optical Elements.
49 In this case. Nonlinear processes such as second.2. 5 0 P. the ratio should be 3: 1. Mitschele. Shank. One beam passes through a fixed delay and the other through a variable delay. Block diagram ofapparatus for measurement of pulse width by twophoton fluorescence. The oscillating wavelengths for various picosecond lasers are approximately 1060 nm for a Nd/glass laser. They are focused on the same spot on a nonlinear crystal to generate the second harmonic. P. The delay time is changed shot by shot when a singleshot laser is used. The experiment can be performed in the following way. 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. 83. 694 nm for a ruby laser. The twophoton fluorescence intensity is monitored by a camera or vidicon. . Third. Ippen and C . J. AND DETECTORS 191 CAMERA or VIDICON FIG. Berlin and New York. Higherharmonic generations can be used to measure the pulse width of modelocked lasers. ed. C. 122 (1970). L. Second. M. Appl. SpringerVerlag. and A .APPENDIX. Saxman.1. in “Ultrashort Light Pulses” (S. 49 E. Shapiro. The laser beam is split into two beams of equal intensity. It is continuously changed by sliding a stage with a stepping motor for a repetitively pulsed laser. Rentzepis. the peaktonoise ratio is 2:1. Lett. Higher ratios can be obtained by applying the threephoton fluorescence method. third. while if the pulse has no substructure and it is a single picosecond pulse. and 610630nm for an Arlaserpumped rhodamine 6G laser. V.10. p. and FourthHarmonic Generation. Two mirrors are adjusted such that these two beams are coincident at the center of TPF cell. it is necessary to convert the wavelength to the visible or nearUV regions to excite biological macromolecules. and fourthharmonic generation are effective means of generating shortwavelength radiation. In many cases. 17. The incident laser beam is split into two beams with equal intensity. 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. Phys. C. 1977. NONLINEAR OPTICS. 1064 nm for a Nd/YAG laser. OPTICAL ELEMENTS.).50 A.
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. C R C Press.06 0. Picosecond continuum light can be utilized as a monitoring light source for picosecond absorption spectroscopy. Commun.3.. It sometimes extends several thousand wave numbers to both the Stokes and antiStokes sides of the oscillating laser wavelength. Chem. A. and near IR. When a modelocked ruby laser is used.6943 1.1. Cleveland. p. Nakashiina and N. Picosecond Continuum Generation.6943 0.6943 1. 1977. carbon tetrachloride. ed. Mataga. all of the visible region. .192 4. cyclohexane. 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. ethanol.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. PICOSECOND LASER SPECTROSCOPY TABLE V1. Lett. Copyright The Chemical Rubber Co. heavy water." When intense laser light is focused into a cell containing various organic or inorganic liquids or solids. and the spectral region of the continuum is broader than that obtained with normal phosphoric acid or isophosphoric acid. Ohio. Kobayashi.487 (1975) T.06 0. In Table VI.). In order to generate continuum light with picosecond width. the halfwidth of the continuum pulse is close to that of the pumping pulse. 35. often used for higherorder harmonic generation are listed. " '* N . To obtain the monitoring continuum light in the visible region. liquids and glasses or solids are usually used for selfphase modulation or fourwave mixing media. 28. Phys. polyphosphoric acid gives a more intense continuum than other liquids. Inc. ' ~ ~ ' ~ The continuum covers the near UV. However. CRC Press. nonlinear crystals.06 0. An intense continuum with very wide spectral range is obtained by focusing the fundamental of ruby laser into phosphoric a ~ i d . @ i f . the second harmonic of a Nd/glass laser or Nd/YAG laser is usually used. extreme spectral broadening can be observed. 497. Usually water. or phosphoric acid are used for continuum generation. J. Presslcy. 147 (1979).
C R C Press. can be used to obtain an excitation light source at different wavelengths from the fundamental or higher harmonics of lasers. TARLE VII. then the energy is dissipated and the light may not be as monochromatic as the fundamental.') 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. p.APPENDIX. Copyright The Chemical Rubber Co. '' .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.). J. One of them is selffocusing which may enhance the conversion efficiency but tends to break the beam up into several filaments.4. In order to get highly intense Raman light. ed. If the abovementioned effects occur. Ohio. 527. Stimulated Raman Scattering Light Pulse. it is important to keep in mind that stimulated Raman scattering with picosecond pulses is accompanied by other nonlinear optical phenomena. In Table VII. 1977. C R C Press. making accurate control of the geometry of the interaction difficult.1. Cleveland. OPTICAL ELEMENTS. producing considerable spectral broadening. NONLINEAR OPTICS. From an experimental point of view.. Inc. the shifts of the stimulated Raman scattering from the pumping laser are given for various kinds of materials. Stimulated Raman Scattering from Various Liquids" Frequency shift (cm. Pressley. Selfphasemodulation can also occur. The stimulated Raman scattering process.AND DETECTORS 193 A. which is related to the thirdorder nonlinear susceptibility.
5.1 psec. + wi. There are two kinds of parametric emission processes that can be attained by ordinarypower ruby.1) (A4 where oL. while the latter is related to the thirdorder nonlinear suceptibility." esu) or chlorobenzene (1. 0 1 + 0. 0. which is of the same order as that of nitrobenzene (1. The former process is related to the secondorder nonlinear susceptibility. + w. 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.3) A. Optical Elements and Detectors A. and idler. In order to get intense Raman scattering.6 x l o . + wi. ('4. This process is represented as ('4.2. rea. One is a threephoton parametric emission process and the other is a fourphoton parametric emission. Optical Kerr Effect.1.6. which is much shorter than that of nitrobenzene (30 psec).. it is necessary to use a short cell containing a highly Ramanactive medium. Nd/YAG. electronic hyperpolarizability of isotropic molecules. The three main causes are anisotropic polarizability of anisotropic molecules. A." The two processes are written as WL t 20. Saturable Dye Solution and Cell.6 x lo" esu).1.41. the relaxation time of an anisotropic refractive index is different. signal. it is necessary to use a lens with a somewhat longer focal length than that used to obtain picosecond continuum. and electrostriction. while the nonlinear refractive index of CS2 is (12) x lo" esu. and wi are the frequencies of the laser. The reason why CS2 is most often used for the Kerr shutter is that the relaxation time of the anisotropic refractive index is 2. . 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 optical Kerr effect has several causes.194 4. Fourphoton parametric interaction can also be obtained by mixing two waves at different frequencies. spectively. PICOSECOND LASER SPECTROSCOPY the other effects mentioned above must be reduced. Kodak A9860 or A9740 dye in ethylene chloride solution is generally used as a saturable dye for Nd/glass and Nd/YAG lasers. rnnitrotoluene (50 psec) or chlorobenzene (68 psec). Depending on the causes of the Kerr effect. When a Kerr shutter is used in hightimeresolution experiments. +0 2 + mi. A.1. or mixing process. Parametric Interaction. or Nd/glass lasers. To reduce the probability of selffocusing.2.
Recently synchroscan streak cameras have become commercially available as new group 3 detectors. OPTICAL ELEMENTS.4. Three groups of detectors are used for picosecond spectroscopy. NONLINEAR OPTICS. These cameras can be operated synchronously with a repetitive pulse. time resolution is obtained by the optical system which makes the correlation between time and space. A. The lenses should be AR coated to eliminate the possibility of satellite pulse generation. repetitivepulse experiments can be easily carried out using group 2 detectors. Cavity Mirrors. which have picosecond time resolution. 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. AND DETECTORS 195 The light transmission at 1060 nm of the solution is adjusted to 6270%. To avoid degradation the dye solution should be circulated.4’quinocarbocyanine iodide (cryptocyanine) in methanol solution is used for mode locking in ruby lasers. Streak cameras. Detectors. If it is operated in a synchronousscan mode the resolution time becomes about 10 psec because of a jitter in each scan. such as vidicons and TV cameras. Therefore. Single.2. When the first two groups of detector are used.3. A.Pulse Selector. Both can be flat mirrors if two lenses are put inside the cavity to compose an inverted Galileian telescope. The camera has a time resolution of a few picoseconds when it is used in a singlescan mode.2. A. Multishot experiments must be performed when a detector of the first group is used to obtain a single kinetics curve. The first group includes various photomultipliers and photodiodes that have neither spatial resolution nor picosecond time resolution. Detectors of the second group are free from jitter.2. One of the cavity mirrors is flat and the other is usually concave. This is to reduce both the loss of Q value by light diffraction and the thermal lensing effect of the rod. .l‘diethyl4. High voltage is applied either by an optically triggered spark gap o r highvoltage pulse driver. 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. with spatial resolution form the second group. They are also useful for protection against damage of the highly reflective mirrors of the cavity by multiplying the diameter of the beam. to reduce the thermal lensing effect. l. slightly divergent from one side to the other. The output mirror of a cavity usually has a reflectivity of 4060%.2.l’DiethyI2. constitute the third group. Detectors.APPENDIX. 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. which is cross polarized to the entrance one. A singlepulse selector is usually composed of two crosspolarized GlanThomson prisms (entrance and exit polarizers) and a Pockels cell between them.2’quinodicarbocyanine iodide (DDI) or I.
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I97 MI. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS By Joseph Schlessinger and Elliot L. l n c All right\ of reproductinn in . Nature (London). W .. L.5.ti>) lorm re*ervcd ISBN o .1. Microscopic motions take place over distances corresponding to molecular dimensions and consist of molecular rotations and deformations. These include the reception and transmission of biochemical “signals” (from other cells and the environment) which control metabolism. and M . transport of molecules along or through the membrane. Anderson.Y 20 . to form specialized regions on the cell surface (e. This part describes fluorescence methods for measuring cell surface motions. and other cellular functions. Brown. Introduction Fluorescence measurements can reveal structural and dynamic properties of animal cell plasma membranes which are crucial to their physiological function.I H ~ s ~ ~ ? . The plasma membrane not only separates the cell interior from the surroundings but also participates in many active processes. Motions of membrane components can be classified as microscopic or macroscopic according to the distances over which they extend.695 (1977). S. between membrane enzymes and substrates. C‘op>riglit @ 19x2 by Acadcmic Prc\s. J . In fact the situation is more complex. 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. or to share membrane components between progeny cells during cell division.g.270. differentiation. “coated regions”’). V D L . For example. the flexibility of the hydrocarbon chains of phospholipids has been mapped by R . for example. G . The mobility of some membrane components is constrained by interactions with structures either within or outside the membrane. rHODS OF t X P E R I M t N 7 A L PHYSICS. and the control of locomotion and morphology. These processes often require that cell surface components move relative to each other to permit interactions. Goldstein. Elson 5. At least some of these interactions are likely to have functional importance. 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.
Auchet. new methods were required for measurements of motions over a range of microns on cell surfaces. New York.). U . 7. Frye and M. M. and T. Axelrod. Elson. W. Tews. J . ’ 1055 (1976). J. 319 (1970). Sci. U . J . Biophys. L. A r i a 367. K. Biochem. Biophys. Elson. Vol. Science (Washington. D. Proc. R. 438 (1974).C. . W. 16. Koppel. J. Elson. and J. Schlessinger.198 5. Zagyansky and M. Science (Washinglon. l6 D. Kornbergand H. Koppel. Kasai. Koppel. J . Bahr. Acta433. 1315 (1976). E. however.) 196.) 184. D. 11 1 I (1971). Po0 and R. W. Peters. Webb.282 (1974). Webb. l 5 D. D. Science (Washington. W. Biochim. Biophys. Webb. Edidin.C. J .307 (1977). D. Webb.215 (1976). Liebmann and G. M. Macroscopic motions extend over distances large compared to molecular dimensions.^ Using spin label and nuclear magnetic resonance (NMR) methods. F. Schlessinger. J. Lardner. Biochemistry 10. D. K. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS Kornberg and McConnel12*3and other^.457 (1974). Aria 433. E. A. and W. D. l 4 J . in “Biological Membranes” (D. Petit and M. McConnell. A . Edidin. 1183 (1974). Schlessinger. In contrast to measurements of microscopic motions. 1973. 16. Axelrod. As is discussed below.~ A photobleaching method was first used to measure the lateral mobility of rhodopsin in amphibian retinal disk mernbranes.) 195. M. Schlessinger. S. E. A.C. Y . S. and G. Natl. Proc. Koppel. Zagyansky. Changeux. M. Science( Washington. Edidin. Edidin. R. W. Kornberg and H. respectively. Y. L. C. H. P. E. J .466 (1976). Poste. Biophys. E. 18. Cell Sci.C. Wallach. G .209 (1976). H. Chapman. D. Acad. Rotational mobility is often taken as an indicator of the “microviscosity ” of the membrane lipid bilayer. Academic Press. Nail. P. 73. Cone. and W. A68. D. this analysis in terms of a single viscosity parameter may be inadequate to describe the constrained rotations of asymmetric fluorophores in complex anisotropic membranes. L. and E. Biochim. which were adapted from methods previously used in bulk solutions.) 185.”’’ Translational motion over tens of angstroms can be measured using R. D. L. Nuture (London) 247. Acad. L.2564(1971). 2. Macroscopic lateral motion of membrane proteins was first observed as the intermixing of histocompability antigens in mousehuman heter~karyons. Wu. Wahl. Indications of segmental flexibility in membrane proteins have appeared in measurements of fluore~cence. W. McConnell. J.~. Biochim. eds. D. Entine. and E. D. Axelrod. Eur. Jacobson. and W. J. Acad.332 (1971). A. M. Axelrod. Jacobson. D. p. Chapman and D. Elson. Peters. Sci.~ Magnetic resonance and fluorescence methods have also frequently been used to measure rotational motion. they showed that the flexibility of the chains increases from the head groups to the center of the bilayer. l 3 K . D. D. P. V. Biophys. 91.’~~ This approach has now been generalized to fluorescent labeled molecules and provides a versatile procedure for quantitative measurements of macroscopic lateral mobility. E.2409 (1976).
A useful compilation of fluorescent probes can be found in Azzi. Berlin. an array of stationary molecules should display a defined relationship between the polarizations of absorbed and emitted light. The factors that control microscopic and macroscopic mobility in membranes are not yet well understood. 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.5. If the molecules rotate randomly during the interval between excitation and emission. 179 (1974). M. 819 (1978). Similarly.2. Biophys. A fluorescence fluctuation method. this method has found few applications. Rev. Annu. D. Interactions with membrane proteins and larger molecular aggregates may further impede all types of motions. Rotational motions are indicated by measurements of steadystate fluorescence polarization and of decay of fluorescence polarization and transient dichroism. Fernandez and R. Azzi. Rev. Consequently. The scope of this paper is limited to a description of fluorescence methods that are useful for studying dynamic behavior on cell membranes. The structure of a molecule fixes the relative orientation of the transition moments. Q. Stryer. Annu. Nafure (London)264. The viscosity of the lipid bilayer provides a lower bound to the forces resisting the motion of molecules embedded in it. is also briefly described. 3.’Oa 5. but a detailed description of probes is not considered in this paper. light emitted as fluorescence is parallel to the emission transition dipole. cholesterol increases the apparent viscosity and suppresses the gelliquid crystal phase transition of the bilayer. Edidin.411 (1976). MOLECULAR ROTATIONS IN MEMBRANES 199 fluorescence energy Up to now. 8. L. For example.20 These studies have shown that the viscosity of the bilayer is very sensitive to lipid composition. Rev. Bioeng. which may be useful in some limited conditions. this relationship is Is l9 2o S. A dependence of rotational diffusion rate on membrane viscosity has been demonstrated experimentally. A. The most versatile and convenient method for measuring macroscopic lateral motion is based on fluorescence photobleaching.2. Viscous resistance should vary linearly with the viscosity of the bilayer for both macroscopic and microscopic motions. These interactions are likely to be more specific than viscosity effects. 47. Some specific fluorescent probes are considered as examples. Biochem. They could influence differently microscopic and macroscopic motions and could vary among different types of membrane components. . however. M.237 (1975). Biophys.
which are defined as so that P = 3r/(2 + r).2) The steadystate level of fluorescence depolarization may be measured in a timeindependent experiment. typically on the scale of nanoseconds. This arrangement is shown in Fig. (5. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS attenuated to a degree which depends on the extent of rotation. The interpretation of fluorescencepolarization measurements in molecular terms requiresthat transition moments. which is fixed in a laboratory frame of reference. .)or perpendicular ( I . EXCITATION DETECTION FIG.2.200 5. Geometrical arrangement for measurement of fluorescence polarization.. The transition dipole is described in polar coordinates. The (monochromatic) excitation light beam propagates in the y direction with vertical ( z axis) polarization. 1.) t o the direction of polarization of the excitation light. the rotation rate can be deduced from the extent of depolarization of fluorescence excited by planepolarized light. Fluorescence is detected perpendicular to the excitation (x axis) through an analyzer which is oriented either parallel ( I . be related to the plane of polarization of the incident excitation light. 1. ) to the polarization of the excitation light. 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. Therefore. Using more complex apparatus one can observe the timedependent decay of polarization or anisotropy. if the mean fluorescence lifetime is comparable to the time required for molecular rotation. The fluorescence polarization is usually characterized by the polarization parameter P or by the fluorescence anisotropy r. fixed relative to the threedimensional coordinates of the molecule.
say p l . specified by p. 5. then _ _ _ _ _ cos 4 = cos 4o e . For nonspherical particles C(Y) may vary with r. In general. (5. J . and q is the viscosity of the solvent..o is the angle between the axis and some reference orientation at time t o and d. as where k is Boltzmann’s constant.2.1. G.5.g.f 2 = f 3 = 8nqR3 where q is the viscosity of the solvent.f 2 andf3. 33 (1934). which depend on the size and shape of the molecule./q.” p .23 a different form: in ro/r = 1 + 3 ~ . Then. Adu. 7 . Protein Chem. is related to the frictional coefficients for rotation about the other axes. For a sphere of radius R . Weber. J . More generally. A slightly more complex relationship must be used for nonspherical particles which require more than a single relaxation time to describe their rotational behavior. / p .2. (5. SteadyState Fluorescence Polarization The steadystate anisotropy of the fluorescence of spherical particles is determined by the relative magnitudes of two characteristic times. Perrin. is the limiting fluorescence anisotropy in the absence of molecular rotation (e. Radium Ser. is the value at time t. the relaxation time for rotation about one axis. Radium Ser. F. for example.3) Here r. the rotational relaxation times are related to the rotational frictional coefficients. 7 .4) where c(r) is a function of molecular shape and the location of the transition dipoles in the molecular framework.2. Phys. T is the absolute temperature. More precisely.. Hence if d. Weber” showed that ro/r = 1 + c(t)Tz. in a solvent of high viscosity). 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. 1 (1936). the latter quantity measures the time required for the average projection of a molecular axis on its initial orientation to decay by e’.415 (1953). the rotational relaxation time. Phys. MOLECULAR ROTATIONS IN MEMBRANES 20 1 5. 23 F. 8. the fluorescence lifetime zo and the time required for molecular rotation. A molecule of arbitrary shape can be described in terms of three rotational frictional coefficients. 22 .2. 7 . where the overbars indicate average values. Perrin.
202 5. Mol. Proc. E. 231 (1972). ~ ~ . 2652 (1974). 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. S. Litman.4) after determining the absolute temperature T and the fluorescence lifetime z. Ann.^' 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. Nail. 1655 (1975). Parola. M. 4529 (1976). 250. B. however. W. M. Sci. Cogan. Biol. liposomes. Weber. and Y. and cell membrane^. 35 P. 603 (1974). Sachs. 3 7 M. Chem. 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. 166 (1975). Robbins. Biochemistry 15. (Leipzig) [5] 12. Inbar. Shinitzky. 169 (1929). Barenholtz. Fuchs. (5. M.245 (1973). Immunol. 117 (1974). Biol. Shinitzky. Lentz. and T. 3 4 S. Perrin. Y. Biochim.2. Inbar. G . Shinitzky. Barenholtz. 29 F.2. U . B. Biochemistry 15. A.2. J . FEBS Lett. 34. J.^'^' In a few cases the fluorescence polarization method has also been applied to measure the rotational relaxation time of fluoresceinconjugated Concanavalin A (Con A. Phys. Y . 30 B. and S. The fluorescent probes most commonly used are perylene or DPH (1. Eur. and L. R . Biochemisiry 12.4521 (1976). and L. 2 7 K. Shinitzky. 28 M. 521 (1973). Gitler. Toyoshima and T.247 (1973). and T. Phys. and T. Mol. Dianoux. Rudy and C. R. 249. Lipids 12. Sachs. Lentz. 5. Stubbs. 26 U.3. 81. 3351 (1975). Wobschall. Biol. and E. Jacobson and D . Barenholtz. R. J . Inbar and M. phase transitions and degree of artificial3’*3 1 and order in micelles. 72.6) This simple approach has been used to study the “microviscosity” of 3236 membranes. 38 M.5hexatriene). A. 85.5) This equation has been extensively applied to the measurements of the microviscosity of membranes. J. P. Biol. Shinitzky and M. C.6diphenyl1. Shinitzky and Y . 36 G. Typically. 24 2s ’’ . Barenholtz.2766 (1976). Inbar. Biochemistry 15. Acad. 32 M. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS For spherical particles. Osawa.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 .~ ~ The membrane microviscosity can be calculated by applying Eq. W. Chem. Thompson.521 (1973). Weber. (5. Thompson. Blout. Biochemistry 12. Biophys. Acta 288. and the steadystate polarization of asuspension of the cells or vesicles is measured.38 M . Nishida. E. Shinitzky. B. Gitler. 3 3 M. J. A. a small. a mannose binding protein) both in aqueous solution and bound to cell membrane^. hydrophobic fluorescent molecule is dissolved in the lipid bilayer of a vesicle or cell membrane. C. J. Chem.
MOLECULAR ROTATIONS IN MEMBRANES 203 5. Nanosecond Time.2. and L. Biophys.5.. H . Then the behavior of the fluorescence anisotropy is correspondingly complicated. Moreover. and make possible a more realistic analysis of the effects of bilayer "viscosity" on the motions of embedded particles. the nanosecond timedependent fluorescence depolarization method can in principle resolve the different components of zi and pi. however. DeToma. 16. the situation is more complex. J.2. Multiexponential functions must be used to represent both the total emission: (5.2. The observed fluorophore may display several fluorescent processes with different lifetimes zi. Frequently.571 (1976). R. . i (5. The measuring system most commonly used is a simple modification of a singlephotoncounting f l ~ o r o m e t e r .39 The decay of total emission and emission anisotropy are measured by collecting I . Consequently c ( r ) of Eq. (5.2.2.(t) 39 J .2. Brand. Easter. then both the decay of the total emission and the decay of the emission anisotropy of the molecule can be described as monoexponential functions. P. For a system of this type the average fluorescence lifetime is defined as and the average rotational correlation time is defined as follows: (5.7) and the rotational correlation time: r(t) = C Pie"Pi.4) is constant and the equation is linear in r.2.10) While the steadystate depolarization measurement can give information about the average rotational relaxation time ( p ) using the average fluorescence lifetime (z).Dependent Fluorescence Polarization When a fluorescent molecule can be described as a spherical rotor in a threedimensional liquid. 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. detect possible asymmetric restrictions to rotation in the system. ~ ~ Sample fluorescence is excited by pulses of polarized light from a thyratrongated air flash lamp.8) This leads to a nonlinear Perrin plot.
FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS and ZL(t) (see Fig. 4 5 R. 55. Kinosita. Bid. J. and A. E. 4" 4' .44 5.timedependent fluorescence polarization of DPH was The ~ ~ . J. Kinosita. After appropriate deconvolution from the time profile of the excitation pulse. 1). they proposed a model which allowed them to estimate the effective viscosity of the bilayer. 4 6 R.204 5. New B i d .~' Phase shift methods are also in use. L. and L. 2163 (1977). Kawato et ~ 1 separated the anisotropy decay curves into two phases. Weber. 2319 (1977). Chem. Ikegami. Eiophys. A. the dichroism decayed.289 (1977). FEES Lert. and L.A general theoretical discussion of the analysis of restricted rotation by fluorescence anisotropy decay has also appeared. R. S. 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.3.46 He exposed rod outer segments to 5nsec pulses of polarized light. Cherry. J. Brand. 252. 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.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 ~ e n t l y . a . A. This exposure selectively bleached rhodopsin molecules whose absorption transition dipoles were parallel to the axis of polarization. A . 4 2 R. G. J. Eiol. K. Cone.4081 (1977). I (1975). Dale. E. 20. 236.2. Phys. 7500 (1977).45 Measurement of rotational diffusion of a membrane protein was first accomplished by Cone. ~ ~ . Chen. 4 3 S. ~ A. ~ ~ measured in paraffin oil. Dale. ~ ~ fast phase occurring in about one nanosecond and a much slower phase. 35 (1972). Roth. the timedependent anisotropy v(t) can be calc~lated. Ikegami. Nuture (London). From the L. Brand. J. 66. ~ ~ ' 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. Biochemistry 16. As the rhodopsin molecules randomly reoriented because of rotational diffusion. and A. Based on the temperature dependence of the anisotropy relaxation data. Chem. Excitation and observation must be repeated many times and averaged to reach adequate precision. Chem. thereby inducing a dichroism in the rhodopsin. Kawato. 44 K. Kawato. Chen. 252. S. in egg lecithin and in dipalmitoyl phosphatidyl choline v e s i ~ l e s . who made use of the natural photochemistry of rhodopsin in retinal disk membranes.
5 msec for the band3 protein. and G. In a few cases the limitations embodied in the assumptions have been ignored and data therefore misinterpreted. These experiments yielded a rotational relaxation time of 0. Ntrfurr (London). Moreover. A. 245. and D. Measurement of the transient dichroism of triplettriplet absorption of labeled proteins yielded the rate of rotational motion. Parish. This method was used to study the rotational diffusion of band3 proteins in human erythrocyte membranes. this time was not affected by removal of the erythrocyte spectrin network. Since tripletstate lifetimes are in this range. It is possible to divide these assumptions into two categories. Cherry et u1. Chapman. R.5.2. Busslinger. Special Features and Limitations The interpretation of steadystate fluorescence polarization data of membranes in terms of microviscosity is based on a few crucial assumptions. 389 (1976). The anisotropy factor measured in this experiment was defined as (5. M. First. The initial dichroism was produced by polarized laser light pulses of 12 psec duration. G. 249 (1973).4. 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. the conceptual limitations.2. which is readily excited to a triplet level.11) A l l( t )and A . RaziNaqvi. Nature (London) 263. . 5. Their approach was to label membrane proteins with probe molecules that have an excitedstate lifetime in the millisecond range. It is clear that even a bilayer composed of a single lipid species is not an isotropic fluid. Cherry.45.2. A very different result was obtained when a similar experiment was performed on bacteriorhodopsin in purple membrane fragment^. Biirkli. NeM B i d . J. R. a major constituent of the erythrocyte membrane. Schneider.4a have developed a general method based on transient dichroism for studying the rotational relaxation of membrane proteins. the conceptual and the experimental. From this experiment the rotational relaxation time of bacteriorhodopsin was calculated to be greater than 1 msec. The fluorophore rotates in an anisotropic environment in which the rotations can be restricted to varying extents depending on the 4 7 K. J. J. GonzalezRodriguez. It is assumed that the fluorophore rotates in the bilayer (which is a twodimensional fluid) as it does in isotropic fluids. Chcrry. (t) are the timedependent absorbancechanges for light polarized parallel or perpendicular to the plane of polarization of the excitation light. they used as a label eosin isothiocyanate.^' In this system the induced dichroism did not decay during the period of observation. 4 8 R.
Shinitzky. (5. thereby restricting the motion of the probe molecule. A second problem originates in the typical complexity of the mechanism of fluorescence decay. de Laat. This can lead to serious problems of interpretation. From our experience we conclude that most fluorescent hydrophobic molecules tend to enter very rapidly into the cell interior. The location of the probe in the cell is often unknown and is difficult to ascertain. A . In an isotropic medium without quenching processes the decay kinetics of a fluorophore can often be described by a simple exponential decay. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS location of the fluorophore in the bilayer. (5. NBDphosphatidylethanolamine and rhodaminephosphatidylethanolamine. Acad. P. 74.2. It is often assumed that the fluorescent hydrophobic probe is inserted in the lipid matrix of the plasma membrane. 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. The situation is even more complicated in a cell membrane which is composed of different types of lipids. (2) Mixed population of cells. W. The fluorescent probe may bind to different species. Partial quenching or chemical reaction during the fluorescence lifetime would yield a complicated multiexponential decay. Sci.206 5. and M. glycolipids. 4458 (1977).4)] instead of the individual components of the fluorescence decay processes. 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. S. At least one million cells are necessary for a single fluorescence polarization experiment. U . Proc. van der Saag. 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. The measurements of fluorescence polarization arc usually performed on suspensions of cells which have been labeled with a fluorescent probe. and glycoproteins. which are sensitive to the viscosity and anisotropy of the fluid. Primary cultures are usually composed of heterogenous populations of cells at different stages of their cell cycles or differentiation.6).49 49 S. This includes carbocyanine and merocyanine dyes and also fluorescent derivatives of lipids. Narl. Experimental assumptions that can lead to misinterpretations can be summarized as follows: (1) Localization of fluorescent probes. Use of synchronized cell cultures can reduce this problem. T.2. It is very common to use a single average lifetime (t) in the Perrin equation [Eq. 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. . proteins.
MOLECULAR ROTATIONS IN MEMBRANES 207 (3) Segmental flexibility in measurements of receptor rotational relaxation times. singlelamellar vesicles.transition are determined for both large. But this time domain is adequate for lipid probes which have rotational relaxation times in the nanosecond range. In order to compare the rotational relaxation times of Con A binding sites on normal and transformed cells. 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 .'^ 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.” Measurements of fluorescence polarization have also provided reasonable values of the phase transition temperature and degree of order in micelles and erythrocyte membranes. Therefore the significance of measurements of apparent . ~ The authors interpret the differences in the polarization of the ’. which is in the nanosecond range.20. Fluorescence polarization studies on artificial bilayers have precisely detected the gelliquid crystal phase transition in phosphatidylcholine dispersions. multilamellar liposomes and small. Neither steadystate nor nanosecond timedependent fluorescence depolarization measurements are adequate for studying the rotation of proteins in the cell membrane. The parameters of the phase.4548 Most values are on the order of microseconds or longer. is too short compared to the relaxation time of the proteins (microseconds or more). The fluorescence lifetime.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. 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.^"^' They found that the rotational relaxation time of fluoresceinCon A in solution is 58 nsec. These values are at least 100fold shorter than the values obtained for protein rotations on cell membrane by other methods.309 The results are qualitatively similar to those obtained by other methods. When fluoresceinCon A is bound to the cells the polarization data yield relaxation times of 701 60 nsec. 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. The major advantage of the steadystate fluorescence polarization method is its simplicity. 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^.5.2.~~ fluoresceinCon A on the cell surface as an indication that the Con A receptors can rotate at different rate^.
rapid measurement. uniform in speed and direction. The varying time course of the fluctuating fluorescence is analyzed statistically to yield D and V. and simple. Basic Concepts Lateral motion may result either from random Brownian diffusion or from a systematic driven process. sensitivity. such as flow along the cell surface.208 5. Two methods based on these ideas have been developed.2128 (1974). the number of fluorophores and consequently the fluorescence undergo transient spontaneous fluctuations about their mean values. fluorescence polarization methods are suitable for studying the rotational relaxation of lipids but not of proteins in membranes. 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. In one. . The second method. Shinitzky. with diffusion coefficient D.1. Sci. 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. The method of decay of transient dichroism seems to be a promising general approach for studying the latter. Nevertheless.3. Therefore the mean number of fluorophores in the observation region is constant. 71.48 5. Null. the concentration of fluorescent molecules on the membrane remains in overall equilibrium throughout the experiment. D and V can be calculated from the measured time behavior of the fluorescence. The concentration of fluorescent molecules is minimally perturbed by measurement. A . fluorescence photobleaching recovery (FPR). and simple flow. Once the size is known. In principle other molecular properties could be used. generates an initial nonequilibrium gradient in the concentration of the fluorescent molecules by exposing them to an intense pulse of light. Acad. Therefore their number is estimated from measurement of the fluorescence emitted from the observation region. 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 ~ ~ ~ c h o l e ~ t e r o lon 38 and ~~ membrane fluidity cannot now be definitely known. Generally. Inbar and M . Macroscopic Membrane Motions 5. but fluorescence has the advantages of specificity. In these experiments the specified molecules are distinguishable and detectable by their fluorescence. Proc.3. called fluorescence correlation spectroscopy (FCS). with velocity V. U . S. Then the recovery of the fluorescence in the region due to redistribution of the bleached and '' M. We consider both diffusion. This irreversibly photobleaches a portion of the fluorophores in the observation region.
'1s'2 and Jacobson et a l l 3 We call our approach fluorescence photobleaching re~overy. it is convenient to use the same microscope optical system and laser excitation source for both phases. (a) Fluorophore concentration profile induced by a brief pulse of an intense Gaussian laser beam for different bleaching energies.5. 2. Although other experimental arrangements have been tried. We shall then give a briefer description of FCS and compare the two approaches. Principle of the FPR method.'~''(See Fig.2." Edidin and Zagyansky. Subsequent bleach on the same spot would show increased fractional recovery.3. the irreversible photolysis of a portion of the fluorescently labeled molecules in a small region of the cell surface and. 2.. second. MACROSCOPIC MEMBRANE MOTIONS 209 unbleached fluorophores is analyzed to yield the mobility parameters. 5.The diffusion curve shows an immobile fraction 1 .Fluorescence Photobleaching Recovery An FPR experiment consists of two major phases: first.) Total Energy 0 2 4 6 t/T 8 1012 14 FIG.3. .A/B. The photobleaching pulse must be short compared to this recovery time. During the recovery phase the laser excitation is attenuated by several orders of magnitude relative to the bleaching pulse to minimize further bleaching. We shall consider FPR in some detail since it has proved to be more convenient for measurement on cell surfaces. (b) Schematic recovery curves for diffusion and flow. the monitoring of the fluorescence recovery due to redistribution of bleached and unbleached fluorophores. The observation region is defined as the area illuminated by the excitation light and is typically a small fraction of the cell surface (< 1%). Fluorescence photobleaching experiments have been performed by Peters et al. The recovery time is determined by the size of the illuminated area and the rate of motion (Dor V).
The beam from a krypton or argon ion laser is focused through a spatial filter. Microscope geometry for FPR experiments. a beam splitter that supplies a small portion of the radiation to a photodiode intensity monitor (MON). The appropriate laser line to excite the fluorophore is selected by a prism in the laser cavity. In order to observe and photograph the cell. The mirrors (M) and the field diaphragm (FD) can be moved to present an image to viewing eyepieces or a camera (C). The field diaphragm limits the volume “seen” by the photomultiplier.65). a spatial filter (SF) to ensure a reasonably regular Gaussian intensity profile. through a field diaphragm (FD) and lens L3. 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. or light can be supplied by the illuminator (IL). .3. (usually water immersion 40 x NM 0.210 5.l6 The illuminated area on the fluorescencelabeled membrane is usually a spot of 1p radius. vertical illuminator.  FIG. The beam is passed through a filter (PG) to remove plasma glow. the lens (Ll) may be removed to spread the laser illumination. and a scatter aperture (A) and lens (Ll) to control the size of the beam as it enters the vertical illuminator. The fluorescence radiation is collected by the same objective lens (L2). and onto a sensitive photomultiplier tube (PMT) that is connected to photon counting electronics for measurement of the timedependent fluorescence intensity. Dotted lines indicate removable components (see text for notation). 3. and the objective lens L2 of the microscope onto the specimen membrane. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS The apparatus that we have developed for the photobleaching experiments is shown in Fig.
The duration of the bleaching pulse ranges from 5 msec to 3 sec. (5. The “amount” of bleaching induced in time To is expressed by a parameter K : K = aTOZ(O). also allow alignment of the field diaphragm with the center of the visual field and visual identification of the area selected for quantitative observation.t). OD 4 or 3) is momentarily removed from the laser beam.4) For a Gaussian intensity profile.3. Z(r) is given by Z(r) = (2p0/72~ ~ ) e . etc. t ) of unbleached fluorophores at position r and time t in the absence of transport can be calculated from  dC(r. a solenoidoperated neutral density filter (FF. The bleachingfilter solenoid pulse is synchronized with the chopper wheel.3. For a bleaching pulse which lasts a time interval To short compared to the recovery time. MACROSCOPIC MEMBRANE MOTIONS 211 Eyepieces El and E. 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). To supply the photobleaching light pulse for FPR experiments.l o cm2/sechas been limited by autofluorescence of the cells to 2200 fluorophores per square micron.’ The sensitivity for observations on membrane proteins with diffusion coefficients 10.2) where Co is the initial uniform fluorophore concentration. The fluorescence light intensity is measured by sensitive coldoperated photomultiplier to reduce dark current. For the fasterdiffusing phospholipids.aZ(r)C(r. bleaching times as short as 4 msec are preferred with brighter bleaching intensities. t)/dt = . Special efforts and/or multiple labeling may improve this sensitivity. For slow diffusion. 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).5. Then the concentration C(r.3. where W is the halfwidth at e’ height and Po is the total laser power.~ ” ’ ~ ’ . The electronic system includes photon counting electronics. the fluorescence concentration profile at the beginning of the recovery phase (t = 0) is given by C(r.0) = CoeaTo’(r). . The entire apparatus is mounted on a table with pneumatic supports that eliminate essentially all building vibrations.1) where I ( r ) is the bleaching intensity. (5. multichannel digital recording.3. and simultaneously the highvoltagebias potential is removed from the photomultiplier dynode chain to avoid damaging it. recorders. (5.3) (5.3.
5) r0 = W2/4D (5. as well as expressions for F ( t ) for diffusion. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS Axelrod et al. the . The values of yD and yF versus K . 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. Simultaneous diffusion and flow would yield recovery curves of intermediate properties. For pure flow. F ( t ) has a sigmoidal shape with zero slope at t = 0 and then a relatively rapid approach to the final value (see Fig..3. ' 5. 2).2)YD. or for simultaneous diffusion and flow. Fluorescence Correlation Spectroscopy The experimental apparatus and theoretical analysis of FCS and FPR measurements are similar in many respects. (5.time required ~ for half of the observed fluorescence recovery to occur: difusion = (w2/4zl. and simultaneous diffusion and flow are presented in the paper by Axelrod et al. YD = T1/2/ZD.' If the fluorophore is attached to a variety of molecules with a range of diffusion coefficients.3. More complicated expressions are required for arbitrary K values.' 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'. 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.8) where yD and yF are constants which depend on K . flow.3.the recovery curve reflects a weighted average D which depends on the distribution of individual diffusion coefficients. It is often simplest to determine a transport rate from T ~ . Theory was also developed for uniform flow at velocity V.6) and F ( t ) is the fluorescence intensity at time t. The major differences between the two methods arise from the smallness of the typical spontaneous Auctua . (5.3.7) POW v0 = (w1Tl/2)?F7 YF zl/2/TF? (5. Values of D and A / B are determined by curvefitting procedures described elsewhere.3.212 5." For pure diffusion.3. 2) of fluorophores. F ( t ) has a finite slope at t = 0 but approaches its final value very slowly.
(3) Lateral motion over micron distances is measured directly. Unlike F(t). Therefore the fluctuating fluorescence signal in an FCS experiment must be analyzed statistically to yield precise values of transport coefficients.~’’~ Development of g(t) requires that the fluorescence signal be observed over periods of time long compared to an individual fluctuation. Biophys. 5 3 D. E. L. the amplitude of g(t) yields directly the average number of independently diffusing fluorescent units in the illuminated area. This is appropriately done by computing the autocorrelation function g(t) of the measured fluorescence. Magde.311 (1975). 5 4 E. L. and W. This contrasts with FPR. W. Bioeng. ganglioside analogs. however.4. E. Rev.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 may also be used .2.3. (4) Mobility of specific fluorescently labeled components is measured. 4. the narrow depth of focus of the microscope makes possible a determination that the fluorescent marker is on or near the cell surface. Magde. (2) Since the observation region is small (. Webb.~~ to measure chemical kinetics in suitable systems although this has not yet been demonstrated in practice. L. 5. Magde. Biopolymers 13. Elson.1 pm radius). Up to now these have included surface antigens. and W. Elson and W. the mobilities of components in different regions on the same cell can be measured and compared. 1 (1974). in which a single recovery allows an estimate of D or V. D.3. and lipid probes. W. It is not necessary to infer rates of macroscopic transport from measurements of microscopic motions as in magnetic resonance or fluorescence depolarization. ” 52 . The time dependence of g ( t ) is identical to that of F(t) [Eq. Furthermore. 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. Rev. W. 29. lectin binding sites.5. Webb. Elson and D. Phys.705 (1972).6)] in an FPR experiment (for K G 1) for diffusion or flow. Lett. (5. E. 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. L.29 (1974). Biopolyniers 13. Elson. Webb. A detailed discussion of the measurement and interpretation of the autocorrelation function has been pre~ented. hormone receptors. Annu.
thereby enabling a correlation of physiological events and morphology with dynamic properties. on living cells. the currently available and simplest interpretive theory assumes irreversible firstorder photobleaching. In FPR. which  55 E.~ ~ ~ . ~ FCS requires no assumptions about the photochemistry of the observed fluorophores. . it is necessary to assume (and verify) a rate law for photobleaching.. in preparation. The requirement for a statistical analysis of spontaneous fluctuations. to develop interpretive equations for more complex mechanisms of the bleaching kinetics. which requires the destruction of a substantial portion of the fluorophores in a small region of the cell surface. In terms of diffusion coefficients this extends from to cm2/sec. in which a diffusion coefficient can be determined from a single recovery curve. 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. As previously indicated. FCS avoids observable perturbations of the measured system. FCS is less useful than FPR in the lower part of this range. creates difficulties in applying FCS to living systems. This is in contrast with FPR.214 5.Second. This capability could be useful in studying aggregation e q ~ i l i b r i a .g. (As indicated below. 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. however. It is straightforward. At least in principle the FCS method has three significant advantages over FPR.55 Third. however. Indeed. L. 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). diffusion or flow). FLUORESCENCE METHODSFOR STUDYING MEMBRANE DYNAMICS ( 5 ) A wide range of mobilities is accessible.) (6) The methods are quantitatively precise and in principle capable of distinguishing among various mechanisms of transport (e. In practice FCS is useful for measuring > D > lo* cm2/sec. while FCS on the same system would consume more than two hours. however. First. An FPR measurement of the diffusion coefficient of a typical membrane protein ( T ~ 20 sec) would last approximately three minutes. For slower diffusion coefficients in the range processes it is usually necessary to use FPR. however. Unlike a photobleaching experiment. it directly determines the average number of fluorescent diffusing units (differentially weighted by excitation and emission coefficients in heterogeneous mixtures52). (7) Transport rates are measured in individual cells (under physiological conditions).
3046 (1978). and 482 nm. Biochemistry 17. as erythrocytes or plant cells do. Webb. 18. the duration of the bleaching pulse 1 sec. Biochemistry 16. . 129 (1977).This heat is rapidly dissipated owing to the high thermal conductance of water so that a thermal steady state is reached within sec. Wu. 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. krypton laser lines 568.58 Direct detection of photochemical effects in the bleached region is very difficult because the number of photolabile molecules is so small (5002000). W. Consequently.     (1) To minimize cell damage. These results have been confirmed in similar experiments by Fahey and Webb. the concentration of fluorophores lo00 molecules/pm2. FPR is usually the preferred method over the entire range of mobilities.g. Jacobson. The laser power density during the bleaching pulse is approximately W/pm2. we choose fluorophores excitable at wavelengths at which most cell components do not absorb. Therefore it is essential to evaluate the possibility that the bleaching pulse in an FPR experiment damages the cell or the plasma membrane.5. and the extinction coefficient of the fluorophore at the wavelength of the laser line lo5 M cm.2. F. K. E .. 4 . Biophys. and D. P. 520. 3936 (1977). A greater than 100fold change in D was detected in dimyristol phosphatidylcholine multilayers at 23"C. 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. Papahadjopoulos.'.03"C and during the fluorescence recovery phase 1050C. during a typical bleaching pulse 0. the temperature at which the gelliquid crystal transition has been shown to occur by many other methods.56 Although these increments are probably negligible. MACROSCOPIC MEMBRANE MOTIONS 215 typically show enough membrane activity to distort FCS measurement even of relatively fast processes. the maximum temperature rise is small. These parameters yield a heating rate of 105"C/sec. e. J .3. Fahey and W. 5h 57 D. 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.8. 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. Axelrod. Therefore information on this matter is mainly indirect but in its cumulative weight provides a persuasive argument against significant adverse photochemical effects.
toxin. and changing the fluorescence probe from rhodamine to fluorescein did not alter the measured diffusion coefficients or the fraction of mobile fluorophores. Jacobson. Exp. They have also tested the effects of freeradical quenchers and light intensity on the incorporation of trypan blue. Hou. and W. but rather is bound to a large protein ligand. R.61 5. E. Up to now they have detected no photochemical damage in the bleached area. Wolf. (3) Increasing the laser intensity tenfold [into the range 103102 W/(pm)’]. Henkart. Schlessinger.60FCS experiments do not require an exposure at high laser intensity as do FPR experiments. and P. F. Webb. Fahey. 116.2 to 520. Elson. D. The fluorophores and resultant free radicals are in the aqueous phase.~’ Similar values were also obtained by the two methods for the diffusion coefficients of fluorescent lipopolysaccharides in artificial bilayers. Koppel. 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.1. 179 (1978). D . Elson. Biochemistry 16. Barak. E.8 nm. changing excitation wavelength from 568. They are therefore exposed to rapid quenching processes. such as an antibody. D. 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. 6 o D.4. 305 (1977). Measurement of the mobility of the lipid probe diI (3.216 5 . W. Therefore there were no perceptible effects which depended on excitation wavelength or intensity or on the identity of the fluorophore. Y. Jacobson et al. Cell Res. C . . 6 1 K. E. ) 195. W. (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~. L. 3476 (1977). They have used scanning electron microscopy to examine the irradiated area after bleaching with different light intensities.4. L:S. E. a standard test of cell viability.3’dioctadecylindocarbocyanine iodide) in different cell types confirms the 5 9 P. W. and J. hormone. Webb. Applications 5. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS (2) In most cases the fluorophore is not directly attached to a cell structure. J. Science (Washington. as would be expected if substantial photochemical processes were occurring. Wojcieszyn. L. or lectin. not embedded in the membrane. Blumenthal.
Nutl. Schmitt and F. U . Yahara. Poste. and E. Schlessinger. S. G. S. ' ' . Po0 and R. Tews. Wiegandt. P. Sci. Sci. Biochem.C. 319 (1970).C. D. W. Jacobson. Proc. MIT Press.14 108.50. Massachusetts. 179 (1974).1090 0. L. van der Saag. Acad. K. Schlessinger. Bioen. Cone. Yamada. Wu. Sci. Reidler. J. Science (Washington. 2409 (1976). S. Science (Washington. G. D. Acta 367.Zagyansky. Proc. Zagyansky and M. Axelrod. Nutl. and W.3. E. de Laat.) 184. 691. Rutishauser. U. K. Koppel. <lo"" 5 x 2 x 10~0109 2 x 2 x 10'O 4 3 x I 0. Acud.) 196. C.. 1183 (1974). Schlessinger. W. Nature (London) 247. M.TABLE Lateral Diffusion of Cell Membrane Components I. Jacobson. D. Acud.448 (1978). J.60 0. D. L.8 0. A. Cell Sci.) 195. Lardner. H. M.4 x lo" 10"<3 x 0. 1217(1976). and G. M. E. See K. Petit and M. A . S. D. A . A . Rev. J. Sci. Peters. 0. Nature (London)272. in "The Neurosciences: Fourth Study Program'' (F. Annu. Natl. 0. Yamada. Biophys. D.q.209 (1976).C. Biophys. Webb. ' S.7 00. W. W. L. Schlessinger.). D. E. Elson and J. A. Elson.457 (1974). ' R. Liebmann and G. Y.240. Schlessinger. L. 77. Acta 433. S.5 (continued) Shows two distinct components. Elson. Axelrod. Science (Wushington. M. 74.8 0 . and T. J. Frye and M. Biochim. Schlessinger. and J. 73. and G. and H. Edidin. E. A. Pastan. 73. V . Edelman. Y. Koppel. 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.) 185. Entine. 7. 282 (1974). K. S. Acta 433. D.30. Peters. Edidin. Eldridge. M. Elson. Proc. Bahr. I Proc. V. in preparation. Edidin. A . E. J. eds. and I. A.C. < 10'0a 108. ' P. Webb.30. Diffusion coefficient (cmz/sec) lo@ 109 Membrane component Lipid probe (diI) Analog of ganglioside GM. M. Edidin. 307 (1977). Biophys. J. J . 1978. Biophys. I.25 109. Biochim. W.438 (1974). L. D. J. Cambridge. Elson. and E. 1110 (1977). Elson. T. U . Edidin. Acad. Edidin. Johnson and M.466 (1976). L. . Natl. Science (Washington. E. L. 215 (1976). Worden. W. 1526 (1980). Webb.
Schneider. J . Sachs. Smith. Podleski. I 4 x 1010 3 x l o . A. and J. W. Aria 288. 9. A. L. Elson. Schlessinger. I P. K. TABLE Rotational Diffusion of Cell Membrane Components 11. Cone. M.s I.2909 (1977). 74. Inbar. Koppel. 550(1976). ( M. A. U. I. K . P.4 Nanosecond time> 700 nsec dependent fluorescence polarization  h * G. P. and T. L. Cherry. Radda and D. P. C. RaziNaqvi. R. E. Metzger. L. Busslinger. W. E. G. S. Eur. J. Biochem.8 0 0. Webb. 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 . Pastan. K. Parish. f M. Nature (London) 263. Natl. and G. Pastan. Auchet. Nail. 75. Schlessinger. E. ' R. W. N ~ t w (London). Chapman. New Biol. Shechter. W. Elson. scc) 3 x lo" <lo'* 3 x 1010 < 3 x 10lz Mobile fraction 0. and D. D. 5353 (1978). ' J. 34. FEBS Lett.' " " ' D. Acad. Arad.50. e New Biol. 9.8 0 0. Nail. d e 70160 nsec  f. Gitler. J . 23 I (1972). Yamdda. G. Kasai.S. ' R. 245. Proc. Cuatrecasas.TABLE(continued) I Membranc component Acetylcholine receptor (diffuse) Acetylcholine receptor (patch) IgEFc receptor Fibronectin Insulin receptor E G F receptor Diffusion coefficient (cm'. Sci. Bio[. Biirkli. J. Webb. and A.287 (1970). R.Wdhl. and L. " J. M. Y. and L. FEBS Leti. GonzalezRodriguez. 35 (1972). A . 389 (1976). Ravdin. Changeux. 332 (1971). Axelford. 4594 (1976).Rudy and C. Proc. Sci. Mol. Cherry. .3 110 b 20 psec 2 < 20 msec 0. U. A . Biophys. J . Sci. Willingham. Schlessinger. L. and I . M. 247 (1973). Proc. Nature (London). 236. Inbar.8 References f. Nature(London)264. R. Acad. Webb. 73. U . S. 81.8 0. and E. M . J. Sachs. Schlessinger.5 msec ~. 18. J. M. Barak. G. Hammes. J . Elson. Shinitzky. 249 (1973). M . Shinitzky. W . Biochem. W.S. S. 245 (1973).
A .63The formation of a lipid boundary layer could influence the mobility of both the lipid and the protein. Vanderkooi. 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. C. Sci. S . U . H. 4a). Griffith. 70. 0. 480 (1973).62is similar to that of diI. A. and H. Nevertheless. and G. J . R. The similarity of diI diffusion in different kinds of cells suggests that the viscosity does not depend strongly on cell type. J .4. Reidler. Its diffusion coefficient is only a factor of 2 smaller. Wiegandt. 62 h3 . P. E. C. Natl. in preparation. Eldridge. A . L. FPR curves for (a) the lipid probe diI and (b) proteins on cell membrane labeled with TNBS and rhodamine antiTNP antibodes.5. In order to study this phenomenon using fluorescence methods. The behavior of the fluorescent analog of the ganglioside GM. Capaldi. Interaction of real membrane lipids and of diI with hydrophobic membrane proteins could also limit their rate of motion. it may be wrong to assume that the major factor limiting the mobility is membrane viscosity. Acad. Note complete and fast recovery for diI and incomplete recovery with slower diffusion of the labeled proteins. it is necessary to develop fluorescently labeled lipids which retain their capacity to bind to membrane proteins.APPLICATIONS 219 I I 0 80 160 240 TIME (sec) 320 FIG. existence of a fluid membrane matrix in which membrane molecules are free to move over wide areas of the cell membrane (see Fig. Jost.4. Proc. Evidence for the immobilization of membrane lipids by hydrophobic proteins has been obtained in studies using spinlabeled fatty acids. It is not certain that only fluid dynamic factors limit the lateral mobility even of so simple a molecule as diI. Elson. Schlessinger.
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. G. Null. Using the same approximation. Saffman and M. . 4b). C. Nevertheless. 10 (1970). It is noteworthy that in retinal disk membranes rhodopsin is the major protein constituent and cannot have interactions with cytoskeletal elements. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS The mobility of membrane proteins is more complex. Acud. Actu 211. a viscosity of 12 poise is calculated from the diffusion coefficientsof diI and the fluorescent ganglioside analog in the cell membrane. The mobile membrane proteins fairly consistently diffuse about 50100fold slower than the membrane lipids. ~ ’The . Biochim. The calculated viscosity would be 20 poise. A . Delbruck. and D. N.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.~ assume that membrane proteins have reasonable size (radius of 50 A) and that the diffusion coefficient is 2 x 10. nonspecific collisions of membrane proteins with each other. It is approximately ten times faster than the diffusion of insulin or epidermal growth factor receptor complexes on 3T3 cells. It is impossible at this time to give a quantitative interpretation of the difference in diffusion coefficients of diI and the mobile labeled proteins. Proc. S. The calculation suggests that the lipid viscosity does not control the lateral mobility of the hbeled surface proteins. The membrane proteinsoccur in both mobile and immobile classes (see Fig. 72. G.l o cm2/sec. 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. 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. The diffusion coefficient of rhodopsin is 5 x cm2/sec8. U. 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 . which is approximately tenfold higher than the viscosity estimated for the lipid matrix by many different methods. 3 I I 1 (1975). then the mobility of membrane proteins in other cell types seems to be limited by interactions in addition to viscous drag. One possible interpretation is hydrodynamic: the proteins are larger and therefore move more slowly because they experience greater viscous drag. and   64 65 P.220 5. data necessary to d o this are not yet available. Fleischer. McConnell. Sci. An analysis of this possibility could be carried out using the theory of Saffman and D e l b r ~ c kLet ~us . S . Nielsen.which is approximately 20 times faster than nonselected proteins and antigens on cell membranes and IgE receptors on rat mast cells. Biophys. we feel that the presence of these unsaturated lipids will not affect the qualitative validity of this discussion.
Bid.4. Annu. Mol. 70 J. ~ ~ . '' A. 152 (1971). Sci. Pastan. Biochim. S. Hynes and J . Sci. 87 (1977). 6. W. and E. Acad. G. Henderson. Rev. it was found that depletion of spectrin did not affect the rotational relaxation of band3 proteins in the erythrocyte ghosts. U. Yamada and J.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. Nature (London).48It is not clear what interactions impede the motion of this protein. The fast relaxation times measured for fluoresceinCon A on cell membranes by steadystate fluorescence polarization37338 for ANS and DNS and  '' R. R. Cell 3. 113 (1974). 1.S. A . 233. Biophys. L. 39. W. 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. M.47This difference is consistent with xray diffraction data which indicate that disk membrane is a planar fluid array of rhodopsin m~lecules.417 (1969). Yamada. R. 1. Hammes. Bye. M. 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 .~' bacteriorhodopsin is packed in a crystallike arrangement while in the purple membrane. S. Webb. Pastan.222 (1976). Natl.2909 (1977). Hynes. K. bacteriorhodopsin does not rotate rapidly in the purple membrane. Stoeckenius. New B i d . 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. Proc. 0. In addition. E. J . 74. Unlike visual rhodopsin. U. . A. Worthington. Acta 456. Barak. M. K. Proc. Yamada and I . 7 1 J. Acad.~ ' The rotational diffusion of cell membrane components is in many respects similar to their translational motion. 7 3 R. 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.5. Sci.3492 (1974). Its rotational relaxation time equals 20 psec. Weston. L. 71.48 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. K. Lipid probes rotate at a rate which seems to be controlled by the viscosity of the lipid matrix (in the range of 110 poise). G . A similar value was obtained when the viscosity of the amphibian disk membrane was calculated from the lateral diffusion coefficient. Bioeng. Blaurock and W. Schlessinger. 0. which corresponds to a viscosity of 2 poise. Biophys. M.73 (1976).Elson. Trends Biochem. A . Blasie and C. Again rhodopsin in amphibian disks is an exceptional protein. Natl. 68 69 66 K.
P. 7 6 P. Roth. Y . Nevertheless. Acad.2. some of which might be mobile.S. 5353 (1978). 43. ~ Lateral motion ~'~ of the hormone receptor in the plasma membrane would enable the hormone to interact with various effector molecules.261 (1976). 74 75 . CI.70. 5. 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 . 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.222 5. Sci. Natl. 1877 (1976). U . Proc. Natl. and P. 7 7 C. Ce/lBio/. Chang. A . Cyclic Nucleotide Res. B i d . Proc. restricted rotation of the probe molecule or the flexibility of a segment of the protein. Shechter. 75. Schramrn. J . Willingham. 9 ' Y. Sci. A.4410 (1976). J . DeMeyts. Shechter. 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. U.79The fluorescent analogs retained substantial binding a ~ t i v i t y ~ ' . Proc. Cuatrecasas. 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 . Cuatrecasas. Jacobs. Kahn. 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. 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. 7 8 J. 15. and 1. Chem. 3. Orly and M. Accid. Schlessinger. Ref). S. 7 9 3T3 fibroblasts were labeled with the fluorescent derivatives of insulin or EGF and the FPR method was employed to study their mobility. S. I (1973). S . 169 (1974). W.~ ~ 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 .4. M. Natl. P. fluorescent derivatives of those two hormones were prepared. Pastan. and others located at fixed sites.Biochem. 73. Annu. R. Adu. Cuatrecasas. R. A . Sci. K. A . ~ ~ . 251. Acad. 2135 (1978). Schlessinger. 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. J . and J.J. Perkins.and biological ~' potency. P. Bianco. Furthermore.
80~81 The large glycoprotein known as fibronectin appears in an immobile structure on cell surface^. 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. internalization. in the mechanisms of hormonal activation of certain cellular functions. This pattern is consistent with the apparent involvement of fibronectin in cellcell and cellsubstrate adhesion. It seems likely that the behavior seen in this study is involved in the regulation of receptor levels on the cell surface and also. Proc. Azide. Y.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. Addition of exogenous fibronectin to transformed cells partially restores the morphology.S . The presence of immobile fibronectin fibrils did not impede the diffusion of a lipid probe. and I . Con A binds to fibronectin and is largely immobile in areas rich in fibronectin. Natl. adhesiveness. Therefore oxidative phosphorylation and some cytoskeletal elements do not seem to be responsible for the properties of the bound fibronectin. I Schlessinger. A .B2These observations prompted a study of how fibronectin binds to cell surfaces using fluorescence microscopy and photobleach methods. These static properties of fibronectin are consistent with its apparent structural function as an adhesive or anchoring component. The amount of this protein decreases substantially after transformation. APPLICATIONS 223 receptor immobilization. M. Acad. Natl. Shechter. Proc. M. andI.4. Sci. Acad. U .^^*^^ 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. and parallel alignment typical of normal fibroblasts.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). 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 . Sci. 75. S. perhaps. vinblastine. U.1217(1976). or a combination of both.5. C. In contrast. 2659 (1978). Yamada. A . Yamada. and cytochalasin B did not alter the immobility and the cell surface distribution of the fibronectin molecules. 73. Therefore the fibrils d o not form a barrier across the lipid phase of the plasma membrane. Pastan. ** K . In another study Schlessinger et aZ. Willingham.. . Therefore the immobilization of the hormone receptor complexes is presumably the consequence of receptor aggregation. S. or various surface antigens. a fluorescent ganglioside analog. S .
86 T. Vaz. G . J. C. Cantor and T. Cantoni and D. Biophys.2.1. M. 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. and W. 366. B.” L. Tao. R. J .5. E. Biophys. Fluorescence measurements of rotation and macroscopic translation of macromolecules in solution have proved useful in a number of applications. Science ( Washingron. 1971. Harper. Vol.224 5. 28. 545 (1980). D. 8 5 J . L. 176 (1981).C. The original demonstration of the FCS method concerned coupled translation and chemical reaction of ethidium bromide with DNA. 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.53 More recently. C. Y.). when we combine measurements of microscopic and macroscopic motions to provide a more complete picture of the dynamic state of various cellular structures. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS the mobility of cellular components. Recent Developments 5. L. 29. 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. 2807 (1979). In particular. Biopolymers 18. New York. in “Procedures in Nucleic Acid Research ” ( G . S T D. 5. The latter is blocked by a fast shutter except during the bleaching interval.281 (1979). eds. Bartholdi. p. N. R. The bleaching and monitoring components are recombined into a single colinear beam after passing the shutter. 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. both steadystate and timeresolved measurements of fluorescence depolarization have been used for some time in studies of protein^^^*^^ and nucleic acids. M. Jovin. 31. Acad. Barisas.86This provides a sensitive and versatile complement to the transient dichroism method described in Section 5.84The newer techniques of FCS and FPR have rarely been applied to macromolecular solutions. Stryer. Sci.5. The incident laser beam ~’ is split into a weak monitoring component and an intense bleaching component.) 526 (1968). Ann. Koppel. 3) by an arrangement of beam splitters and a s h ~ t t e r . Borejdo” workers have used FCS to study interactions among muscle components. Borejdo. 83 . Davies. 162.3. 2.
89 90 . Wolf.225 (1981). S . 9 7 M. and M . and M. 9 2 M.000 named a n k ~ r i nUsing a cell fusion method. Biochirn. Cone. 9 8 D.89 Another is a pattern photobleaching method. J . Elegant biochemical work has demonstrated that the band 3 protein. E. Lepock. Biophys.2. S. R.L. which has the advantage of allowing measurement of slow or anisotropic diffusion on cell surfaces. Proc. 94 C. Sci. U . recent work has provided evidence that these effects have no detectable influence on measurements of lateral diffusion on cell surface^. 255. A. 77.^^ 5. J . Schindler. Koppel. 85. A. H .5.3314 (1979). E.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. J . 99 R. McConnell. Veatch. A. 9 1 J .^^. 5 . Fowler and V. the erythrocyte anion transporter. Sci.^^. 3114 (1979). Edidin. A model for constraint of the lateral mobility ofmembrane proteins by steric interactions with a labile cytoskeletal matrix has been D. is linked to the spectrinbased erythrocyte cytoskeleton through a protein with molecular weight 215. Proc. E. 96 V. ~ ’ ~ ~ * Measurements of the rotational mobility of band 3 protein. Biophys. and H.^^ Fortunately. Stenbuck. Nature (London) 285. U. M. Biol. S. Koppel. Natl. 30. Schindler. 9 3 D. Biophys. Acad. Natl. J. E. Biophys. Bennett and P.2540 (1980).5 . and D. Acad. A . R. it has been shown that displacement . Sheetz and D. J .99 This confinement could be due to steric interactions with thespectrinmatrix. E. Dragsten. 289 (1979). Sheetz. 344 (1978). W. Res.2043 (1980).~~ of ankyrin from spectrin enhances the lateral mobility of erythrocyte integral membrane proteins. Thompson. Commun. Cherry. The erythrocyte provides the most advanced example up to now. 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 .g0 A potential problem in the interpretation of FPR data is the possibility of photolysisinduced crosslinking of cell surface components. J. P. 215 (1978). 76. U . Kruuv. M. 5641 (1979). Nafl. M. Sheetz. A . Supramol. Wey. Natl. Biochem. B. Origin of Constraints on Membrane Protein Mobility Only recently have the structural bases of these constraints begun to emerge. Acta 559. A . P.96 More quantitative studies using FPR have confirmed and extended these ~ t u d i e s . Clark. M . Golan and W. 2537 (1980). This type of crosslinking has been experimentally demonstrated under excitation conditions quite different from those of FPR measurement^. Struct. U . and D. Chem. A . Proc. Wallach. P. One is suitable for simple analysis of diffusion on a sphere. E. R. and P. 8. y 5 V. Acad. Proc. S . J . Koppel. Acad. Sci. Sci. however. RECENT DEVELOPMENTS 225 Several new bleaching geometries have been proposed. 33. 510 (1980). F. Bennett. 77. Smith. Edidin.
J. 78. Sci. Y. 3469 (1 980). U .Receptor Mobility There appears to be a common mechanism for the internalization of cell surface polypeptide hormonereceptor complexes. J. A . 75. J. A. 75. Acad. 3576 (I98 1). l o b A. which may be related to the triggering of cellular responses. Shechter. Pastan.. . Acad. Pastan. A.Zidovetski. S . Schlessinger. finally. Sci.lo2 5.L. J. Yarden. and J . Proc. Orci. Natl. Maxfield. Webb. Willingham. 805 (1978). Proc. l n 3 F. 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.macroglobulin. 1. has been detected by measurements of phosphorescence anisotropy. This involves the binding of the hormone to initially diffusely distributed mobile membrane receptors. Sci. U . lo' Y. Acad. The origin of mobility restrictions in the plasma membrane of nucleated cells is less clear. Narl. Shechter. 6981 (1981).'" This reduction can be reversed by treating the cell simultaneously with both colchicine and cytochalasin B. Narl. Proc. 77. S.lo6 l thyrotropin (TSH). Narl. S. A.lo4*o 5 nerve growth factor (NGF).'" 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. Mol. 78. C. W. Proc. Sci. E. the pinching off of vesicles containing the hormonereceptor complexes. S . M . Acad. P. Sci. and T.lo8 l o o D. E. M. A . M. and M. Sci. Cohen. "* R. F. FLUORESCENCE METHODS FOR STUDYING MEMBRANE DYNAMICS proposed. Shechter. Y . Carpentier. submitted (1982). Neufeld. Proc. L. Cell Endocrinol. N u t / .5. S. U . Levi.226 5. Koppel. U . Acad. Cell 14. Schlessinger. Gorden.3. Avivi. and I."' 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. D. 78. C. Javin. U. the aggregation and immobilization of these receptors. AmbesiImpiombato. Elson. l o ' W. 5025 (1978). ( . which then cluster mainly over coated pits. Proc. A . Tramontano. and M. S . Natl. Y. 5. Schindler. Acad. and. Y. Sheetz. Willingham. Henis and E. R. Hence the integrity of both microtubules and microfilaments is required for the effect. I . and L.S. Schlessinger. ' ' ~ epidermal growth factor (EGF).' O 3 An early phase of the aggregation of EGFreceptor complexes. personal communication. 1 P.A. and J. Schlessinger. This process has been seen for i n s ~ l i n . 1072 (1981)."' and a. Schlessinger. J.2135 (1978).
F.C. in press (1 982). W. S.5'cyclic monophosphate (CAMP) show quite different mobility properties. too slow to be involved in the stimulation of cAMP synthesis. S. L. M. W. D. U . Reidler. Koppel.5."' Pretreatment of the thyroid cells with 8bromoCAMP induces the clustering of TSH receptors in the absence of TSH. Science ( Washington. Hekman. This work was supported by NIH grants GM 21661 and CA 14454 and by NSF grant D M R 7504509 (to W. ' l o A. In contrast to Preceptors. Wolf. N a f l . C. I . and D. A . D.''~The response to the agonist is. Tramontano. Quite different behavior is seen for the receptor for TSH on thyroid cells. Webb. D."' Acknowledgments We are especially grateful to our colleagues. Avivi. Webb).). Barak. Fahey. . Acad. P. Sci. cAMP reduces the potency of TSH to induce the production of cAMP in thyroid cells. M. largely immobile state but to be released and mobilized by the agonist ( )isopr~terenol.5. D. and J. J. The Padrenergic receptor on liver cells has been shown to be present in a patchy. L. Helmreich. AmbesiImpiombato. Proc. who have worked with us on the various studies from our laboratory discussed in this paper. 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. Elson. Ambesi. Moreover. . and E. TSH receptors are mobile and randomly distributed over the cell membrane. Axelrod. E. RECENT DEVELOPMENTS 227 Two receptors which stimulate production of adenosine 3'. D. J. '09 Y. W. submitted (1982). however. Schlessinger. Henis. Eldridge.
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Blundrll and L. 20 Copyright @ 1982 by Academic Press. “The Use of XRay Diffraction in the Study of Protein and New York. STRUCTURE DETERMINATION OF BIOLOGICAL MACROMOLECULES USING XRAY DIFFRACTION ANALYSIS By Eaton E. Such models provide the basis for our understanding of the mechanism of enzyme catalysis. K . L. Johnson. and many other macromolecular functions. they serve to direct and organize other types of studies of the molecules involved. The Determination of Crystal Structures. which is concerned with the shapes and interactions of molecules rather than with their atomic structures. ’ ’ ’ 229 MFTHOIX O F EXPERIMENTAL PHYSICS. 1968. B. P. New York. crystals of virus particles weighing several million daltons have been analyzed in nearly atomic detail. VOL. G. since it is better known and covered in many textbook^. New York.1. Inc. this volume. describe data collection technology.’ We touch only briefly upon the closely related technique of small (. By atomic structures we mean models in which the positions of the individual atoms are known to within several tenths of an angstrom. Nucleic Acid Srructure. Part 8. Stout and L. ligand binding. including such important ones as hemoglobin. Vol. H . Holnies and D. 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. Jcnsen. C.” 3rd ed.6. Cochran. immunoglobulins. H. 111. Recently. Blow. Prutein Crystallography. Mario Amzel 6. Lipson and W.” Wiley (Intersciei~ce).100 atoms)molecule crystallography. N. “The Crystalline State. 1966. and DNA. Moore. 1966. M . Lattman and L.^. while correspondingly complex fiber structures have also been studied. and then present some case studies that illuminate the versatility and limitations of the methods. London. No clear limits to size and complexity are yet in view. is the subject of Part 8 of this v01ume. In addition. H. For more detailed material on this subject see Blundell and Johnson’ and Holmes and Blow. 1976.~ T.^ Smallangle xray diffraction analysis.” Academic Prcss. In this part we present briefly the principles of singlecrystal and fiber XRDA. All Iighh if reploduction in any form reserved ISBN 0124759629 .” Macniillan. “XRay Structure Determination: A Practical Guide. Bell.
An XRD experiment differs in several ways from this prototype. The loss of phase information. A lens placed behind the specimen focuses the diffracted radiation into an image. in principle. equally spaced planes appears to be reflected from these planes. sometimes called “the phase problem. such as s or x. specimen is illuminated by a collimated beam of radiation. The method also differs from conventional microscopy in the characteristics of the specimen. In a typical microscopy experiment a thin. Position vectors in direct or diffraction space will be denoted by lowercase. given the phase and amplitude of the scattered rddiation. such as A. Diffraction by a General Object We begin by defining some of the notation used in this paper. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In some ways XRDA might better be termed lensless xray microscopy. The goal of the method (XRDA) is the conversion of this pattern into an image. 6. boldface letters. The process is an intricate one. an XRD specimen should perhaps consist of a single molecule held delicately in front of the beam. Matrices will be written with uppercase. Much of this paper is devoted to the discussion of techniques for dealing with this problem. This pattern becomes the only observable in an XRD experiment. The contrast in such an experiment would be vanishingly small. F = A + iB. Highquality xray lenses do not exist. as we discuss below.2. However. with the computer playing the role of the lens. available xray detectors measure the amplitude but not the phase of the radiation. reconstruction of the image is. The situation can be improved by using periodic specimens such as crystals or fibers. which is diffracted by fluctuations in scattering density within the specimen. usually aperiodic. the structure factor. To preserve the analogy. We frequently write this vector. In the absence of a lens the distribution of radiation scattered far from the specimen is termed the Fraunhofer diffraction pattern. by concentrating the diffracted radiation into certain defined directions. We show below that.230 6. boldface letters. These increase contrast both by increasing the number of molecules in the beam and. We can break F into components in two ways: F = I FI exp(ia) = F exp(ia).” is the central difficulty in the practice of XRDA. as F. 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 most conventional approach to the theory of XRD in crystals is through Bragg’s law. It states that a beam of x rays incident upon a stack of parallel. a simple process. The use of F for 1 F (is conventional in the protein crystallographic literature. as a .
This artist has represented the continuous density fluctuation by four shades of grey. When the angle of incidence differs from 0 no Bragg Incident beam \\. when nA = 2d sin 8. For constructive interference this distance must be an integral number of wavelengths. the Bragg angle. For this object only a path difference of 2 produces constructive interference. is the angle between the incoming beam and the planes. (From Blundell and Johnson.. Bragg’s law.1) Here n is any positive integer. and 8. = 2d sin 0. (a) Geometrical representation of the diffraction condition for a stack of = cqually spaced planes. d is the spacing between planes. This is illustrated in Fig. thus nE. la.2.A 2 ’ Reflected beam e d \ El/ FIG. + .6. I .’) (b) Diffraction by a sinusoidal density fluctuation of wavelength I/s. thus i= ( 2 / s )sin 0.2. Rays “reflected” from adjacent planes have a path difference 2d sin 8. (6. DIFFRACTION BY A GENERAL OBJECT 23 1 mirror reflects light.
2. the amplitude of the diffracted wave.5) . Equation (6.2) is not difficult to obtain. If the origin of the spatial wave is shifted to xo.2. phase) of that wave. This approach can be extended to analyze a general object if one considers diffraction from a spatial.. ) (6.3) or p ( x ) = po exp(2nis0 * xo) exp( .232 6.2. (6. It is well known that any object can be represented through a Fourier integral as a combination of such spatial waves (Fourier components). We can then write m o ) = Po.2nis * x) d V . The situation is diagrammed in Fig. then. and both are periodic along this axis. (6. we have p ( x ) = po exp[2nis0 (x . Experimentally.2nis0 * x). is proportional to the amplitude of the spatial fluctuation. only differences of phase can be measured. Note that only one angle of incidence 0 is permitted for this function. m (6. and therefore F(so) = po exp(icr). and therefore F(so) = po exp(2niso * xo) exp(ia).2. and we omit a derivation for lack of space.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. It is perhaps not surprising. sinusoidal density fluctuation p(x) = po exp(2nis0 * x).. 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. From the diffraction by an isolated spatial wave one can thus determine the period (through d).x] . XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES reflection is observed. so that m p(x) = / / [ G ( s ) exp( . and the origin (i. and so CI in the above equations is generally defined to be zero. It is also clear that F(so).4) Thus translation of the spatial fluctuation by xo produces a phase shift of 2nx0so in the diffracted radiation. lb.e. that diffraction from the density fluctuation occurs when a slight variant of Bragg’s law is satisfied: 1 = (2/lsol) sin 0. the amplitude.2.
6.3. 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. 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. 1 it is clear that the vector triangle OAsOB is equivalent to the one formed by the incoming and reflected rays. 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.2nis * x) d V . Diffraction is observed along the direction of OB.2). The textbook by Goodman6 provides an excellent introduction to Fourier theory and diffraction.” McGrawHill.2. denoted s.2. and is / . OA denotes the direction of the incident beam. The signal recorded at any point represents the diffraction from one Fourier component.6) to give a result which is used extensively below. 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.2. drawn from the center of the sphere to a point A which is taken as the origin of all vectors s.4. “Introduction to Fourier Optics. 1968. Consider any s which terminates on the surface of the sphere. the wave vectors of the various Fourier components of the object. Figure 2 shows the vectors OA. rotated in the same way.6) The Fourier inversion theorem can be applied to Eq. W. (6. and s drawn in a sphere of radius 14 (Ewald’s sphere). G and F are identical. If the object is rotated they are. To measure the full. The vectorss are defined with respect to the object’s coordinate system. By comparison with Fig. DIFFRACTION BY A GENERAL OBJECT 233 The amplitude and phase of G(s) indicate the weight and position of the Fourier component. perforce. . and draw from the center of the sphere to its terminus the vector OB. Goodman. OB. Strategies and instrumentation to achieve this goal are discussed in Section 6.2. 03 (6. We can thus identify the diffraction pattern of an object with its Fourier transform and write m p ( x ) = JJJF(s) exp( . ’J . Clearly. For any given orientation of the specimen only certain of the Fourier components satisfy the condition in Equation (6. apart from a constant of proportionality. New York.
W.234 6. periodic specimens which produce simplified diffraction patterns (see James’). Vol. ( a ) A sphere of radius IjA is drawn so as to pass through the origin of the reciprocal lattice A.2. . The Ewald construction. James. 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.’ Copyright @ 1966. London. John Wiley & Sons.” Bell. 1957. 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. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES FIG. We next discuss crystals and fibers. (Holmes and Blow. Reprinted by permission of John Wiley & Sons. Inc. Inc. 11. two additional mathematical R. “The Crystalline State. The Optical Principlcs of the Diffraction of XRays.) We have thus far dealt with diffraction from completely general specimens. however. and have kept the discussion general as well (for a more complete discussion of the Fourier approach to diffraction see Goodman6). 19).
6.) t devices must be introduced: the application of the convolution theorem to diffraction’ and the projection theorem. If F and f are a Fourier transform pair and so are G and g. (6.9) This theorem has wide application.8a) F * G = F(fg) and that FG = F ( f * g ) . . * M. Rossmann. the convolution theorem states that (6. Image of a point after Fourier reconstruction with wavelength cutoff at I/s = 1 A.2. G. In Fig.u ) du. 3* and represents the image of a point object after wavelength cutoff. 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. As an illustration we consider the effects of the nonzero wavelength of x rays on our ability to form an accurate image.2. “The Molecular Replacement Method. ed.” Gordon & Breach. 1972. .Y is the distance in angstroms from the point.8b) where 9 represents the Fourier transform and the asterisk represents convolution. This transform is shown in Fig.. (6. (Reprinted from Rossmann and Blow38 with permission. which is defined by f ( x > g(x) = Jf(u)e(x * . DIFFRACTION BY A GENERAL OBJECT 235 FIG 3. New York. The observed image is therefore the convolution of the true image with the transform of the solid sphere. thus all diffraction from such components must remain unobserved. 2 it is clear that no vector s of magnitude greater than 2/A can touch Ewald’s sphere.2.2.
z ) dx dy dz. (6. .10) demonstrates that that projection o(x.e. where the precession camera. especially for crystals. We now discuss the projection theorem. Application of the Fourier inversion theorem to Eq. threedimensional image down the normal t o the central section. y. instead of any value between 0" and 360". Thus 1/smax the wavelength of the highestfrequency component in the synthesized electron density function. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In protein crystallography wavelength cutoff is not usually the factor limiting the amount of diffraction data available. They are thus signed.4. ( to show that such projections have structure factors of phase either 0" or 180". z)exp[2ni(sx + ty + uz)] dx dy dz. the projection down z). we rewrite Eq. If a(x. where smax the radius in reciprocal space beyond which no data are obis is served. Setting u equal to zero and rearranging.3) automatically provides photographs of particular planes in the diffraction pattern. (Section 6. ~ ( xy) = o x. real numbers. It is easy . (6.. 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. we have F(s. y) represents the integral over z (that is. y. 0). Phases for such reflections are particularly easy to determine.236 6. t . and has a closely corresponding form for crystals. (6. y)exp[2ni(sx + ty)] dx dy. t. This theorem is of great practical importance. t . Resolution is given by l/smax. u ) = / / / p ( x . since it is frequently very simple to record diffraction from such central sections.2. which states that the image synthesized using only a central section from the full diffraction pattern is the projection of the full. so that centrosymmetric projections are of great interest experimentally. 0) = ss exp[2ni(sx + ty)] S p(x. This gives rise to the crystallographic definition of resolution in an image. y). y) is the Fourier transform of the central section F(s.2. This result is independent of the projection axis chosen.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. To derive this. then a(x. sometimes it is expedient for data collection to be limited by the investigator. which is the form most commonly used.7) using the components of x and s: F(s. Many crystals have projections which are centrosymmetrici.2.
3.. n. c* = a x b/V. (6. reflections. Diffraction by Crystals A lattice is a regular grid of points in three dimensions. and c. and h. the corresponding reflection. in fact. It is clear from Eqs. the Fourier transform of p. Also. b.8b) one can show that the Fourier transform or diffraction pattern of a crystal is the product of F. where m. The reciprocal lattice is thus intimately related to the geometry of diffraction. and p are any integers and a. pM. 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.1) that a a* = b b* = c c* = 1. These relations are the source of the name reciprocal lattice. is a section from the threedimensional lattice defined by h. it is the set of points at the termini of the vectors H = ma + nb + p c .6. This net. and c are any noncoplanar vectors. (6. The crystal can be regarded as the convolution of a single unit cell’s worth of molecules. b* = c x ajV. 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.2. and some discussion of its characteristics is essential for understanding xray photographs and data collection in general.3. The reciprocal lattice is an important tool in dealing with the geometry of Bragg diffraction.3. the Fourier transform of H. which is defined by the vectors a. b. and the Fourier component giving rise to the reflection. It is easy to show that h is itself a lattice (called the reciprocal lattice) with unit cell vectors a* = b x cjV.1) where V is the volume of the unit cell and x denotes the cross (vector) product. 7 (which is discussed more fully below) reveals that the Bragg reflections lie neatly arranged at the points of a twodimensional net. with a set of 6 functions at the lattice points. Using Eq. Inspection of Fig. the unit cell vectors. but we shall only consider specific examples as they arise. CRYSTALLOGRAPHY 237 6. (6.3. which is zero except at points of the reciprocal lattice. it is a unification that a given h will serve to label a reciprocal lattice point. the diffraction pattern of a crystal is given by FM(h).1.   . and translations. 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.3. Crystallography 6.. Formally. As we stated above.
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.3.3.3.3b).2. must vanish.5) . and I’ are measured in reciprocal angstroms. where the triplet hkl is often called the Miller indices. k. (6. (6. This rather abstract derivation has shown us that only certain welldefined Fourier components exist in a crystal. k’. = From the crossproduct relations in Eq. and fractional coordinates. Eq. Of course. Similarly.1) all terms containing a * b*.238 6. (6.3.3a) (6..3.3. and so diffraction can only be observed when one of these satisfies Eq. If one rewrites (6. The latter notation is universal in crystallography. (6.2nih x). etc. Using Miller indices.4) which is the usual inner product form.2a) (6. and 1 times along c. and Fourieranalyzed it accordingly.3b) or x = xa + yb + zc.3. the inner product h * x is independent of the choice of formalism. + ky + / z .5) to reflect the periodic nature of the crystal.2b) h = + kb* + lc*.3. (6. in the second h. Eq. Recalling that aa*= bb* = cc* we have h * x = hx 1. the position vector x can be written as x = x’d. In the first case h’.3.  (6. (6. + y$ + z‘i. where a caret denotes a vector of unit length. we have h x 9 = hxa * a* + kyb * b* + Izc .c* + terms in a . etc.2b).b*.3. Thus the hkl component repeats h times along the a axis.2). XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES The vector h can be written as h or = h’d* ha* + k’6* + l’i. one finds p(x) = 1 h F(h) exp( .* (6. k times along the b axis. and 1 are in units of the reciprocal lattice translations and are always integers.
The diffraction pattern of one unit cell is the sum of the diffraction patterns of all the atoms in the cell.5): F(h) = /JJ p ( x ) exp(2nih ..3.” Wiley. New York. have been tabulated (Ref. One can also apply the Fourier inversion theorem to (6. (6. Vol. Formally. Although symmetry is one of the most important fields of study in crystallography. and lo). 9.3. It is worth noting that almost all biological molecules are handed (i. Birmingham. IV). h and x are expressed in integer and fractional coordinates by convention.” Kynoch Press. Buerger.e. we do not attempt a general discussion. Diffraction patterns for each atom type. and are usually denotedf(s). and we interpret their electron density functions in terms of atoms. England. called the structure factor equation. we pass over this entire topic by noting that symmetry operations which superimpose the whole crystal on itself do exist. CRYSTALLOGRAPHY 239 This is the equation used to calculate electron density function images. one can write that (6. Nowf(s) represents diffraction by an atom situated at the origin. and x j is the coordinate of its center.6) relates the diffraction pattern to the continuous electron density in the crystal. but deal with particular examples as they arise. I. Equation (6.x) d V . 1952.3.6.) This precludes the existence of mirror planes or other inverting operations in their crystals. These groups are termed space groups.3. unit cell (6.its diffraction pattern is phase shifted by the factor exp(2nih * xo). Again. 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. 9. Crystals are made of atoms.3.8a) International Tables for XRay Crystallography. . are nonsuperimposable upon their mirror image. It is therefore convenient to have a formula. The sum of the diffraction by all atoms is therefore F(h) = I‘ cf.(h) exp(2nih j * xj). often called atomic scattering factors.f(s) is also.3. “XRay Crystallography. J . and the symmetry operators are the composition of the translational symmetry of the lattice with operations that relate the molecules within one unit cell. 1942.7) where p j is the electron density function of the jth atom. Since atoms in this model are spherically symmetric. lo M. Vol. which expresses the diffraction pattern in terms of atomic positions and types. (refs. If the atom is shifted to point xo. Again.6) giving the structure factors as a function of the electron density.
and it is commonly named after him. a synthesis using the observed F2(h)which have a phase of zeroas coefficients can always be calculated. the location of heavy atoms that have been bound to the protein molecules. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES where xj is the position of atom j . can be related to the rootmeansquare excursion of the jth atom. Phys. The more general form for this factor. 46. We show below that the Patterson function is the autocorrelation of the electron density function and represents a complete. Rev. These locations are essential data for the procedure that provides phases for the protein diffraction pattern. The quantity B is usually called the temperature or DebyeWaller factor. and forms the basis of the process for refining such models in order to minimize differences between observed and calculated structure factor magnitudes. (Some texts say. The final form of the structure factor equation (6. The Patterson Function Electron density maps can be calculated from the structure factors F(h) using the Fourier synthesis described by Eq.3. The contribution to the structure factors is different from resting and from vibrating atoms. L. In this nomenclature one deconvolutes the ) Patterson function to recover the electron density.) Until about 1960 the Patterson function was the dominant tool for solving the phase problem in crystallography. Equation (6.3. To take into account the effects of atomic thermal motion (James.' Chapter 5) each f j ( s ) must be multiplied by the factor exp( Bjs2/4). . which allows for anisotropic atomic motion. However. is rarely used in protein work. Normally the phase of F(h) is not known. the structures solved typically had a limited number of atoms.8a) is based on diffraction by atoms at rest.8a) is (6. usually determined experimentally. Patterson" in 1934. loosely. (6.3. This synthesis and its significance were first discussed by A.240 6. 6.Patterson.2. but in fact it is the convolution of p(x) with p( . where the value of B j .x. In protein crystallography we use the Patterson function for a problem of similar complexity. As we shall see.3. it can provide clues leading to a structure determination. Properly interpreted. L. alternative description of the observations. It is well known that the atoms in a crystal are undergoing thermal motion. '' A.8b) This equation enables us to calculate the diffraction pattern from a preliminary atomic model. 372 (1934). that the Patterson function is the selfconvolution of the electron density function.3.5) when the phase of F(h) is known.
In crystallographic nomenclature the Patterson function P(u) is defined as 1 P(u) = 7C F2(h)~ 0 ~ ( 2 *nu).5) for the electron density we can use the convolution theorem [Eqs.2.9). and we cover it as briefly as possible.3. p(x) can be expressed as = 1 Pj(x j  xj). (6. Substituting in (6.9) The function P(u) is centrosymmetric [P(u) = P( u)]. we obtain P(u) = 1 C [F(h)F*(h)] cos(2nh h * u).3. As shown before [Eq. the topic can be tedious. which depends heavily upon symmetry. CRYSTALLOGRAPHY 24 1 Use of the Patterson function depends upon interpreting it in terms of the autocorrelation of a function made of atoms or. (6. where xj is the coordinate of the jth atom and N is the total number of atoms in the cell. points. The interpretation of this synthesis requires some further derivations. ideally. h h (6. The expression for the electron density based on atomic electron densities can be used to further analyze Eq.6. (6.3.3.11) we obtain which can be rearranged to yield . where F*(h) is the complex conjugate of F(h).1 I).3.2.3.3.3. Substituting F(h)F*(h) = F2(h) in expression (6.9)] 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.7)].8b) and (6. and upon the concept of vectors running between atoms. To be frank. (6. There is an extensive literature discussing this interpretation.10) With expression (6.
. This can be done by dividing the F 2 ( h )by the square of the Fourier transform of the average atomic electron density profile.xj simplifies the above to N N p(u> = j=1 k = l 1 JJJpj(x’)pk(x’ + u . for example.13) we obtain N N (6. + ” M.ujk) d v . The peaks Pjk are the convolution of the individual atomic profiles.3. the atomic electron density profiles of the kth andjth atoms are Gaussians of variances 07 and a the peak centered at ujk will also be a Gaussian of variance fl.13) where ujk = xj . j 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. Pjk is the convolution of the electron density profiles of the ith andjth atoms. A very detailed analysis of the Patterson function and of different deconvolution methods can be found in BuergerI2 (and references therein). translated to the Patterson origin. Buerger. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES The change of variables x’ = x .ujk.k = : . “Vector Space and Its Application in Crystalstructure Investigation.242 6. 1959. the function can be partially deconvoluted in structures which contain a few heavy atoms that dominate the diffraction pattern. If. New York. Substituting (6. In addition. J. u ” 6.14) in (6. There are N 2 . the height of the peaks is approximately proportional to the product of the atomic numbers of the relevant atoms. .3. 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. In addition. xj .xk. Define the integral in the above expression as (6. If all atoms in the structure have similar radial electron density profiles.N peaks for the vectors ujk with k # j. For a crystal structure containing a few atoms in the unit cell the Patterson function can be “solved (deconvoluted).3.14) where u’ = u .3.” Wiley.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.3. 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. (6.
4. This assignment can be used to obtain the coordinates of the heavyatom sites. The interpretation ofthis Patterson function involves two major (A and B) and one minor (C) heavyatom sites. The crystal in this projection contains twofold axes of symmetry perpendicular to the plane of the drawing.lox The main use of the Patterson function in protein crystallography is for the determination of the positions of heavy atoms (Section 6.4.2. (6. Projcction along thc b axis of the diffcrence Patterson function of a gold heavyatom 4. The zero contour is dotted.) .4). (Courtesy of Dr. projections of the Patterson function are used [Eq. Heavyatom derivatives are usually screened and the heavy atoms located using these projections (Section 6. (6. For this purpose. and the negative contours are omitted.4. The vectors between site A and the minor site C (AC and AC') are also present.3. Eduardo Padlan. derivative (AuCIi) or glycera hemoglobin crystals. The contour lines are drawn at equal but arbitrary intervals. The analysis is systematic .6.3) can be used for this purpose.16) Data for such a synthesis can be collected very easily. in many cases. for example. 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').10)]. A single precession photograph or a short diffractomer run (Section 6.4). W ___c CRYSTALLOGRAPHY 243 C FIG.3. These functions can be calculated using data from one section of the reciprocal lattice.
The crystals often have a very beautiful external morphology. Prot ei n Crysta Ilography 6. Bernal and D. C. The symmetryequivalent points are calculated. l4 l5 Ib . Chem. which contain thousands of atoms per unit cell. Nature (London) 133.3. 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 6.244 6. Wiss. J . The latter are collectively called direct methods (a selection ofpapers on this topic appears in AhmedI6). E. 6. Reichert. D.1. “Crystallographic Computing. Muellers Arch. The synthesis of the electron density function [Eq. They realized that protein crystals contain a large percentage of mother liquor filling the space between molecules. The computer then checks to see if these vectors correspond to peaks in the Patterson function.4. R.3.4.5)] requires the amplitude and phase of each structure factor. An example is shown in Fig.. 198 (1849). a computer search assumes in turn that each grid point in the unit cell is a possible heavyatom location. In one version. It was not until the middle of the K. Introduction The oxygen transport protein hemoglobin was crystallized in the latter part of the nineteenth c e n t ~ r y . ed. The problems to be overcome were formidable. Med. the grid point is flagged as a possible site.14 Since then a great variety of proteins have been crystallized. The intensity of the individual Bragg reflections is proportional to the squared modulus of the structure factors. Figure 4 shows an example of how the positions of a heavyatom derivative can be located using a projection of the Patterson function. Crawfoot.. (6. J.435 (1926). Copenhagen. with welldeveloped faces and edges. ’ ~ the enzyme (protein with catalytic and activity) urease was crystallized by Sumner in 1926. Physiol. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES trial and error. 69. Summer. Ahmed. making careful use of the symmetry of the crystal. B. 5. 1970. F. and that crystals become disordered as they dry out.” Munksgaard.2) or upon various algebraic relations between the structure factors. J. In 1934 Bernal and C r o ~ f o o t showed that ’~ these crystals produce a rich and complex xray diffraction pattern. for example. Biol. but all phase information is lost. If so. and the putative interatomic vectors generated. Anat.794 (1934). For the first time there was a tantalizing possibility that the molecular structure of the proteins within the crystal might be determined. p. Their photographs were obtained by enclosing the crystals in glass capillaries. All of these procedures fail when extrapolated to protein crystals. Most challenging of all was the difficulty in measuring the phase of all the protein structure factors.
4. Further. PROTEIN CRYSTALLOGRAPHY 245 FIG.2)typically in the range 1. 287 (1954). Perutz. Proc. The number of Bragg reflections within a reciprocal space sphere of fixed radius is proportional to the volume of the unit cell. To provide accurate models of protein molecules these images have to be interpreted. A 225. l7 D. Ingram. and M. Many other difficulties also obtruded.4. and individual atoms are not visible as they are in the smallmolecule case.5 . Thus protein crystals give rise to hundreds or thousands as many Bragg reflections as smallmolecule crystals.0 A.6. Photograph o f a crystal of the F1 sector of mitochondria1 ATPase. Automated procedures to measure these reflections awaited development (Section 6.4. These are also discussed below (Section 6.4.4). Soc. W. V. the diffraction patterns from even the best protein crystals fade out at much lower resolution than the patterns from small molecules (Chapter 6. Green. to a large extent. which has proved astonishingly successful in the intervening years. Ser. usually in light of a known sequence of amino acids within the protein (Section 6.4. 1950s that the laboratory of Max Perutz” introduced the method of multiple isomorphous replacement (Section 6. The bar represents I mm in the scale of the micrograph. The images synthesized from such patterns are deficient in detail. M . . been overcome by a combination of manual and computerbased model fitting and refinement techniques.53.5). London. F. Difficulties associated with the interpretation of electron density maps have.3).5). R .
7. pH. " ) . 4.'* who appears to have a green thumb in this regard. Calculate an electron density synthesis of the crystal. 6." 8. Usually about 50% of the crystal volume is occupied by solvent. Measure structure factor amplitudes for native and heavyatom derivatives. combine model and data.2. 23.4. however.or C a 2 +could be required in low concentration for crystals to form. Make a new synthesis using calculated phases dC observed amplitudes F. Mol. and rate of precipitant addition. The cycle is abandoned when improvement ceases. ~~ 1. McPherson. Fit a molecular model to this synthesis. are almost universally met. and the literature is filled with examples of apparently random procedures that allow the crystallization of previously resistant molecules. In most cases such precipitates are amorphous or microcrystalline. i. Variables explored are precipitant type and concentration. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES TABLE Steps in a Protein Crystal Structure Determination I.33. One begins with a concentrated protein solution often in the range 1 % by weight of protein. 9.. 249 (1976) B.246 6.e.'' and therefore the crystals are destroyed A.J.. which causes the protein to come out of solution. one of several methods of improving the initial model. hut . Crystallize protein. The final criterion of adequacy is their ability to diffract to the desired resolution. 6.2 mm in all dimensions. We then deal with examples of protein structure determination. such as salt or organic solvent. The crystallization of proteins has been reviewed by McPherson. Anal. In addition. Matthews. Protein crystals suitable for xray diffraction studies are usually larger than 0. The repetition of steps 7. In what follows we discuss each of these steps in more detail. A variety of microtechniques have been developed to allow the systematic investigation of many conditions using a minimum of material. Using above information calculate phases for structure factors. but occasionally single crystals result. Crystallization The crystallization of proteins retains large elements of black magic. temperature. Biol. certain ions such as C1. To this is added either gradually or suddenly a precipitant. Methods Biochem. Once obtained protein crystals must be kept in the presence of mother liquor. Make heavyatom derivatives. Certain conditions. and 4cfrom the model. 2.491 (1968). Calculak structure factors F . Determine position of heavy atoms. 5. Table I is a flowchart of the major steps in the determination of a protein crystal structure. W. and 9 illustrates Fourier refinement. 3. 8.
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). Radiation from storage rings of highenergy accelerators is also used. (6. When Cu xray tubes are employed the K.54 A) is selected by means of one of three techniques.” Hilger & Watts. 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. The measurement of the intensities that provide the desired structure factor amplitudes is often a long and tedious process. 1. eds.” SSRL Rep. etc. 6.1)] one can obtain the unit cell parameters from measurements of the difffraction angle 8. 1978. A. the sensitivity to damage by radiation. how well it diffracts.. The second method involves the use of crystal monochromators.6. The condition in 2o H. Connecticut. The method of choice depends on the particular problem under consideration. which has a sharp absorption increase for wavelengths just shorter than 1. London.~ the measurement of the diffracted intensity of all observable reflections. The first type involves the determination of the unit cell parameters and the symmetry of the c r y ~ t a lThe second consists of . For diffraction experiments the crystals are sealed in glass or quartz capillaries in the presence of mother liquor. when deciding how to collect the data.. the size of the crystals. Therefore the strategy of data collection is of great concern to protein crystallographers. “Workshop on XRay Instrumentation for Synchrotron Radiation Research.3. Winick and G . line (8. Stanford Synchrotron Radiation Laboratory.3 keV.2. Under these conditions they are in general very stable and can be used for data collection. Some of the advantages of the individual methods are discussed in more detail this section. a filter of Ni. PROTEIN CRYSTALLOGRAPHY 247 when allowed to dry. Using Bragg’s law [Eq. They consist essentially in recording (usually photographically) several selected regions of the reciprocal lattice. Stanford. Experiments for the determination of the crystal lattice and its symmetry are simple to perform and easy to interpret. The symmetry of the crystal can be determined by careful visual inspection of the recorded regions of the reciprocal lattice.4. Guinier. No. “XRay Crystallographic Technology.’l In the first. Brown. 78/04. 1952.” In this case the xray beam is reflected by means of an intense Bragg reflection of the monochromator crystal. One should take into account the cell dimensions of the crystal.54& is used to reduce the amount of contaminating radiation.4. X rays for protein crystallography are usually obtained from copper anodes.
Appl. thus providing a high degree of monochromatization.842 (1970). 6).2. 3. Harrison. New York. All reflections have the same size and shape and fall in positions on the film that can be simply and accurately predicted.1) will permit only a single wavelength to be reflected. W. The precession camera permits the undistorted recording of a section of the reciprocal lattice of a crystal on a photograph. A precession angle ji is introduced and the normal to the plane precessed around the xray beam with semiangle i . 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. Press.26 Precession photographs are particularly easy to deal with. eds. T. The choice of a particular configuration for data collection involves a variety of considerations. W. Crystals of quartz. Amsterdam. Willis. namely photog r a p h i ~ ’ ~ . 1977. The mirrors can be slightly bent to focus the xray beam for higher luminosity. 1.” Cambridge Univ. These photographs are ideally suited for the measurements of unit cell parameters. Nockolds and R. Wonocott. H.22 For a given grazing angle there is a strong dependence ofthe reflected intensity on wavelength. J . Two general methods exist to record diffracted intensities. S .4. Instrum. 7.. 26 C. LiF.” NorthHolland Publ. and graphite are the most widely used for this purpose.The film is always maintained parallel to the plane being rei corded to assure that the image is undistorted. E. Arndt and B.” Wiley. Sci. 6) is located at a predetermined distance from the crystal and moved so as to isolate reflections from a single plane of reciprocal space. Crystallogr. the precession camera has become a favorite among protein crystallographer^.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). 2 5 U . Reflection angles can be chosen such that the shorter wavelengths (the most common contamination) can be eliminated. 1966. (6.3..^^ We discuss briefly below the most and ~ common implementations of these methods. Films can be digitized using automatic microdensitometers and the data further processed to give structure factor amplitudes. 1964. J . Intensity data can also be obtained from precession photographs. 22 23 . The third method consists of using grazingincidence reflection from metallized glass flats (mirrors). Ariidt and A. London and New York. 84 (1968). An example of a precession photograph is shown in Fig. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Equation. J . Buerger. A layer line screen of the appropriate radius (Fig. M. 6. ~ counter technique^. U. 2 4 M. “The Rotation Method in Crystallography.^^ All preliminary photographs and initial determinations of cell dimensions and symmetry are done with this camera.248 6. These will emerge as we discuss particular devices. “Single Crystal Diffractometry. J. Kretsinger.1 The Precession Camera. Since its introduction by Martin Buerger in the 1940s. “The Precession Method in XRay Crystallography.
Ewald’s sphere construction for the precession camera: (a) the incident xray beam (arrow) intersects a rcciprocal plane at a point A .4. . the number of longexposure photographs needed to collect the data is unreasonably large. The plane is oriented perpendicular to the beam. 6. PROTEIN CRYSTALLOGRAPHY 249 FIG. the normal is precessed around the xray beam. (6.4.16)].Srri.” However. other problems intrinsic to this method of data collection make it less convenient than those using other geometries. Not all the intensity diffracted at a given time is recorded. This is the general ’’ N. 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). An undistorted image of the reciprocal plane is recorded in the film within a circle of radius (see Fig.2 Oscillation Camera. Freer. The reason for this disadvantage can be traced in part to the use of layer line screens. However. forms an angle p with the xray beam.6 . This allows all reflections of the reciprocal plane within a circle of radius AD to pass through the diffraction condition.3. A film is placed at point Band is oriented parallel to the reciprocal plane. Arta Crystallo.6.qr. A layer line screen is introduced to isolate a cone of semiangle p. the normal to the reciprocal plane (and to the film).2380 (1971). Screenless precession photographs have been used successfully in protein structure determinations. and therefore extended exposure times are needed to collect a complete data set. for crystals with large cell dimensions (> 50A) and for highresoluGZ tion data 1/smax 2 A. Xuongand S. The simplest geometry for recording The screenless photographs consists of rotating the crystal in the xray beam while recording the diffracted beams on a stationary film. 7). To record the photograph. (b) A precession angle fi is introduced such that C E . B B27. Small precession angles have to be used to avoid spot overlapping.. Screenless precession photographs can be used to avoid this problem. Screened precession photographs are ideal for recording one section of the reciprocal lattice to use for the calculation of projection Pattersons [Eq.H.3.
to facilitate the identification of the reflections. the crystal is oriented with a unit cell axis parallel to the rotation axis and perpendicular to the xray beam. to avoid overlap of reflections. Also. For long exposures. Speical precautions are taken to avoid backlash and overexposure of the reflections that are in the diffraction condition when the movement is reversed. (Courtesy of Michael Rossmann and John Johnson. .5 A.23 Usually. corresponding to a resolution cutoff of approximately 7. Cell dimensions simply related to the distance between spots. The photograph was taken using a precession angle of 6". the crystals are oscillated back and forth through this angle. Note also the very large number of reflection present. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES FIG. each photograph is obtained while the crystal rotates over a very small angle (from a few minutes to a few degrees).7.250 6.) principle of the oscillation camera. Symmetry is casily recognized in the photograph. Precession photograph of a uranyl derivative of crystalline southern bean mosaic virus.
The FourCircle Diffractometer. PROTEIN CRYSTALLOGRAPHY 25 1 FIG.) A complete data set is obtained by recording smallangle oscillation photographs for all the necessary different orientations of the crystal around the rotation axis. 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. The oscillation angle is I . sufficient to note that there are several good It ) computer programs available for processing oscillation photographs.6. Since their introduction.The edge of the photograph shows reflections at 2A resolution. (For a complete discussion see Arndt and W o n ~ c o t t ~ ~ is. computercontrolled fourcircle diffractometers have become the . 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.3. 2) from the intersection of Ewald's sphere with reciprocal lattice planes. However.8. (Courtesy of Paula Fitzgerald.3. several simplified geometries have been used successfully for this purpose.5". such as the one shown in Fig. 6.4. The curved rows of spots arise (see Fig.4. Oscillation photograph of crystal porcine pancreatic aamylase. 8.
Hamlin.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).qr. Dickerson.4. x. 289 (1978). 6. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES method of choice. E.H. Nielsen. bidimensional. A A34. The counter can then be set at an angle 20 from the xray beam to record the intensity of the diffracted beam. R. This problem is especially serious for crystals that are very sensitive to radiation. 4 ) which allows the crystal to be set at any desired orientation. Strandberg. Using the diffraction patterns from the native and modified crystals one determines the positions of heavy atoms bound to the protein. Phase Determination Using lsomorphous Replacement As stated before (Section 6. The process whereby these modified crystals can lead to the determination of phase is quite simple. Xuong. the heavy atoms can be located using the Patterson function or other methods from smallmolecule crystallography.4. and because their high electron density makes them stand out above the protein. C. Once the positions of the heavy atoms are known. '' . At least one such machine has been functional for several years. Vernon. Kendrew. I188 (1961).8b)l and can serve as a reference to measure the phase of the protein structure factors.stallo. Srcr. equipment incorporating these detectors is still in the developmental stage. The development of large. the present cost of these instruments does not put them in the category of equipment that can be acquired by a single laboratory. the phase of the scattering from them can be calculated [Eq. C . and W . The crystal is mounted at the center of rotation of an Eulerian cradle (with angles w. With this arrangement any reflection can be made to form in the equatorial plane. S. Fourcircle diffractometers are very convenient to use and are usually the method of choice for proteins with cell dimensions smaller than approximately 100 A.. Freer. 14. Acta c'rJ3.28 In any case. Arta Crj'. The method most commonly used for solving the phase problem for crystalline proteins is called multiple isomorphous replacement or the heavyatom method. and H.yta//ogr.25In this geometry the counter can be rotated in the equatorial plane that contains the xray beam. 2 9 R.28 Unfortunately. The major drawback of diffractometers is that they measure one reflection at a time and miss all the others that are formed simultaneously. Computer control of the circles permits the collection of a large number of reflections with very limited operator intervention. E.3.252 6. (6. T.29 In this method protein crystals are modified by the introduction of atoms of high atomic number.4. J. positionsensitive detectors has resolved this difficulty. Because there are few of these. Introduction of heavy atoms is usually accomplished by soaking preformed N.
4. This means.(h) and FpH(h) are Fdh) = j= 1 C fj exp(2nih . or Sm). Borders. M .g. while causing no other changes. Mu/. For a heavyatom derivative to be useful it must be isomorphous to the native crystal.2) yields F P m = FP(h) + fH(h). which is exposed to solvent. The structure factors of the native and the heavyatom derivative F. E. Heavy atoms normally bind to the surface of the molecule.exp(2nih .VtI * N (6. are their fractional coordinates. in addition to mother liquor.1)and (6. and E. Weinzierl. E.4. FpH(h)= C 1:exp(2. C.9) illustrate many of the facets of this method which we now describe. PROTEIN CRYSTALLOGRAPHY 253 crystals in solutions containing.4.xj). that the heavy atoms have replaced an equivalent volume of solvent in the crystal. 29. However. J .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 . L.4.1) where xj are the coordinates of the N protein atoms.2) here NH is the number of heavy atoms in the unit cell and x. Jr. However.4. Dickerson.. Most heavyatom derivatives are found by trial and error. ffg.x j ) + 1 f.4. r (6. . Bid. L. .. Varnum. Kopka. Pt. it can be used to determine the phases of the native crystals.4. The structure determination of cytochrome C30 and rubredoxin (Section 6. exp(2nih xr). (6. such as the binding of Hg to SH groups are generally useful. varying concentrations of salts of highatomicnumber elements (e. J . Margoliash.4.4. specific modification of the protein using heavyatomcontaining groups has also been used. 77 (1967).6. Substituting (6. Once a suitable heavyatom derivative has been prepared and its intensity data collected. Heavyatom substitution and isomorphism can be monitored by comparing intensities and lattice constants and derivative crystals in precession photographs. For the most part they make interactions with amino acid side chains in ways that cannot be predicted before the structure is known.3) which represents the diffraction that would arise from the heavy atoms alone.3) into (6. Au. certain reactions. J . (6. ideally.nih . j= 1 r= 1 N XJ. It is convenient to define the heavyatom structure factor fH(h) = C S.
A . AF(h) is normally used as the estimate of the heavyatom structure factor amplitude for all reflections contributing to a centrosymmetric projections. SWI. A “good” heavyatom derivative should contain only a few major heavyatom sites.(h) < fH(h) and F . Weinzierl. 997 (1968). The structure factors that contribute to this projection are real. AF is an underestimate OffH. Most space groups have at least one centrosymmetric projection. These parameters can be used to calculate the heavyatom structure factors f H . Ac.254 6.6) (6. have opposite sign (the socalled crossover condition). If moduli are taken on both sides of this equation we obtain IFPHI’ = IFP f fH12. . R €324. 4). sin + b. E.4. Sygusch.)’.5) For most reflections F. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES the cell. and their temperature factors can be refined using leastsquares procedures (Section 6. see also Dickerson et aL3’ and Sygusch3’).4. atid R.4. Palmer. ~1 3 ’ R. However. since in this case fH(h) = FPH(h) + FP(h). The preferred method for locating the heavy atoms involves the use of a Patterson function in suitable centrosymmetric projections with coefficients AF(h)’. The positional parameters of the heavy atoms. When F. (6. (6. 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. and therefore FPH(h) = FP(h) kfH(h).(h) and FPH(h)..4. A A33. J.4. This approximation does not introduce serious difficulties and can be fully corrected once the coordinates of the heavy atoms are known. (6. 512 (1977). and its Patterson function can in general be unscrambled.(h) 2 fH(h) and thusf. their percent substitution (occupancy).. Dickerson.(h) can be calculated from the structure factor amplitudes using AF(h) E FpH(h)  Fp(h). These structure factors can be used to determine native phases through Eq.7) This can be shown to yield F& = ( F cos C( ~ + u ~ ) ’ + ( F .4). Sect. In crystals without suitable centrosymmetric projections a threedimensional Patterson synthesis with coefficients AF(h)’ is usually calculated. E. the heavyatom structure factors f H cannot be directly obtained from the experimentally accessible magnitudes F. Actcr C‘rysfulloy. andf.8. J.qr.ru Crsstallo. 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.
(6. Ac.3.(h) exp(ia) exp( .2nih x). The correct value of the phase will then correspond to the common roots of Eq. 12. addition or deletion of sites.” m(h). The most commonly used weight is called the “figure of merit. 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. (6.7).4. The estimated phases and the values of F .8) This expression is identical to Eq.e. The synthesis using m(h) weighting is often called the “best Fourier”33 and is given by p(x) = 1 v m(h)F. etc.^^*^^ The “quality” of the phases can be estimated from the statistics of the minimization procedure. All magnitudes in Eq.9) The average value of the figure of merit over all reflections. This equation can be solved to obtain the value of a. Crick. For each reflection all heavyatom derivatives should give a common value of a . are then combined to calculate an electron density map using the synthesis p(x) = 7 1 F.4.6. 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. (6. It is roughly true that m(h) = cosrerror in ~(h)]. all the obtained pairs will not have a clear common value. are affected by errors in the heavyatom model (i.2nih * x).4.7) for all heavyatom derivatives. H . There will be in general two values of a that satisfy Eq.4.4. PROTEIN CRYSTALLOGRAPHY 255 In this expression M is the phase of the native structure factor and uHand b. Therefore.4. errors in the atomic parameters. (6..7) are subject to errors. In an actual structure determination the situation is more complicated. provides a useful indication of the overall quality of the phase determination.tu Crystnlkogr. are the real and imaginary components of fH. and F. 794 (1959) . C. (6. Several formulations have been used which differ mainly in the residual chosen and in the detailed form of the observational equation^.. This ambiguity can be eliminated by using several heavyatom derivatives.5) with F(h) = F(h)exp(ia). are experimentally determined magnitudes and a. Usually. exp(ia) exp( . each reflection in this synthesis is weighted according to the estimated accuracy of its phase. (6. Blow and F. 33 D. and b.). (m(h)). even though Eq. M.7) gives two possible values of a for each individual heavyatom derivative.4. 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. F . (6.
This map represents the distribution of diffracting matter in the crystal. The maps are usually contoured on lines of constant electron density and plotted on transparent sheets. The transparent sections are stacked with the proper spacings and illuminated for visual inspection (Fig. 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.4. the electron density belonging to an isolated molecule cannot always be identified immediately. However. Lowresolution maps (I/smax= 6 A). The atomic model derived from this density is shown superimposed. a model of the protein molecule can be built.256 6. when interpreted. Computer displays can also be used. In many cases molecular boundaries can be located as regions of low electron density separating continuous globular regions of high density. . 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. can indicate the general shape of the molecule. Because the crystals are periodic.9. 9). but they are in general not very helpful for the interpretation of lowresolution maps. The calculation of p ( x ) provides an estimate of the electron density in the unit cell of the crystal. in protein crystallography. Suck of contoured electron density sections used t o visualize the electron density function of thc immunoglobulin fragment Fab New. each containing the electron density corresponding to one section through the unit cell of the crystal.5. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Interpretation of Electron Density Maps 6. and other gross structural features. we want to know the structure of the protein molecules forming the crystal. subunit organization. When this separation is unambiguous. presence of helices.
Individual pieces fill the regions within contour lines drawn at some constant level ofelectron dcnsity. E. l l . G . An equivalent procedure can be followed using computer display^.6. The highresolution maps can also be plotted on transparent sheets. Science (Washington. and this makes it possible to adjust the model to fit the density. J . The illusion that the brass model is floating in the contour density is often quite powerful. (Courtesy of E. Balsa wood model of the hemoglobin molecule from the bloodworm. McQueen. M. Tsernoglou.) 197. 10. Mol. Petsko. Love. The procedure is continued until all the density in the crystal is used and the complete amino acid sequence built.) assembled to represent the molecule (Fig.C. E. The rodlike features of the model are CI helices. D. Richards. Hermans. To aid in the interpretation of the maps they are displayed in optical comparators like those shown in Fig. 37. A.. Jr. For this procedure a few (510) sections of electron density are displayed at a time and viewed through a halfsilvered mirror. and D. One then builds a brass model while looking at the reflected image. which can be superimposed over the density being interpreted. A . and the electron density features interpreted using this information. Knowledge z of the amino acid sequence of the protein (primary structure) greatly simplifies this task.4. 3 . 3 4The comparators allow one to fit skeletal models of the amino acids to the electron density. J . 1378 (1977). 10).^' In both cases the final product is a threedimensional atomic model of the protein molecule. A model of this F. Bid.225 (1968). 34 35 . GIyrera clibrcmchiatu at 6A resolution. PROTEIN CRYSTALLOGRAPHY 257 FIG. Padlan and W. With higherresolution maps ( 1/smax 2 A) the polypeptide chain of the protein can be traced.
Secttons 0 1 electron density map o n transoorent sheets H a ll. The geometrical and chemical properties of the active site(s) and other functional regions can be analyzed. Solution and crystallographic experiments can be designed to test or exclude proposed modes of action. (b) Mirror parallel to sections of map. Physical. and physiological properties of the protein can be correlated with structural characteristics.I I . .') kind contains a remarkable amount of information. Prolein model FIG. The procedure just described for the determination of the threedimensional structure of a protein molecule using singlecrystal xray diffraction is long and involved. (From Blundell and Johnson. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES LNqht ba: . sdvered b P r o t e i n model (bl r Lighl box Sectjonr of eleclfm nallsdvcred de L mirror . Opticalcomparator or ' Richard'sbox'whichI:dcilitatescomparisonofthemolecular model with the clec(ron density map.258 6. Important insight can be gained into the mechanism of action of the molecule. chemical. but experience has shown that the wealth of information that can be obtained from such a determination is always worth the effort. (a) Mirror tilted 45" toward sections of map.
6.) represent other examples of proteins for which the interaction with other molecules (which we shall call ligands) can be studied. The complex of the protein and its ligand must be crystallized and the structure determined using the usual techniques. structural correlation of functional properties can be made using the threedimensional structure of the native protein. (6. receptors.e. When enough biochemical and physical chemical information is available. When proteinligand crystals are isomorphous to the parent crystals.4. hemoglobin).C F. In principle these studies involve a completely new structure determination.6. can be used for these experiments.4.2nih * x). repressors. The usual way to overcome this problem is to use nonhydrolyzable substrate analogs.e. Lowtemperature techniques have also been able to “freeze” substrates so they cannot react. In the cases of enzymes these molecules are the substrates.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. etc. and effectors of the catalyzed reactions. just as if the proteins were in solution. For many proteins.(h) exp(ia.. Not surprisingly. products. most of the structure of the proteinligand complex is known (i. It must be realized that in most cases enzymes also retain their activity in the crystals. antibodies. Therefore in the case of hydrolytic enzymes if substrates are diffused into the crystal they will be hydrolyzed before xray data can be collected.271ih * x)... If we use .) exp( . Difference Fourier Syntheses One of the goals of the determination of a protein structure is the characterization of its functional and physiological properties. and binding proteins (i.e. V (6. Carrier proteins (i.4. the protein part. most functions are dependent on interactions with other molecules. however.10a) ppL(x)= 1 V FPL(h) exp(icrpL) exp( . This problem can be simplified enormously if a suitably small ligand such as a substrate analog. Sometimes these small molecules can diffuse into preformed protein crystals (as happens in making heavyatom derivatives) and bind isomorphously to their specific binding site.4. PROTEIN CRYSTALLOGRAPHY 259 6. the binding constants are generally lower in the crystallized protein.) A simple technique termed the differenceFourier synthesis can then be used to localize the ligand. The electron density functions of the protein and proteinligand crystals are given by 1 pp(x) = .
F. Phillips. C. G . Pro(. and F.7. the electron density corresponding to the ligand. ligand difference maps are interpreted by copying the contoured density into transparent sheets which are then displayed in an optical comparator (Section 6. C . Sec. D. Mair.260 6. The proteinligand complexes obtained in this manner can be studied to characterize the interactions between residues of the protein and ligand. to locate the residues involved in the catalytic steps of the enzymatic r e a ~ t i o n .) exp( . L o n r h . in principle. B 167.c. ~ ’ etc.5). C . If a group of protein atoms moves upon ligand binding its original position will appear as negative density in the difference map. T. Usually. 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. L. Sarma. These maps are subject to large errors due to the approximation clpL = and the need to measure small amplitude differences between F. The new position of the group will appear as positive electron density.. K . Ser. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES the approximation clpL = clp we can calculate the difference ppL . and V . R .. (6. Experiments using differenceFourier maps provide invaluable information about the biochemical and physiological properties of protein molecules. Soc.4.pp (difference map).qv. A m Crjwdlo. The perversity of nature R.. North.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.4. Moffat. 378 (1967). however. 6.. Blake.B B27. A . 1414 (1971). .. which is given by 1 ppL . difference maps of ligand are easy to interpret. In practice. N .FP)exp(icc. Movements of side chains of the protein that sometimes occur upon binding can also be detected in differenceFourier maps.11) This map can be calculated from the structure factor amplitudes of the native and proteinligand crystals using the native phases and provides.4. 37 C. Johnson. Henderson and J. especially if the general location of the binding site is known from sequence and chemical data. R. 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.pp = (FpL. A .
protease. E.4. Colman. Another is the study of the same molecule crystallized under different conditionsfrom organic solvent and from salt perhapsto check the constancy of conformation. P. 84. Biol. Further. and J . E. Rossmann and D. Blow. Lattman. I . E. that such crystals are not always isomorphous to native ones. Herriott. unbiased by the original search molecule structure. Wishner. Epp.8) to give accurate representations of the new molecules.97 (1974). Love. 0. K.4. 4 1 M.6. P. Mol. B i d . 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. in principle. and some simplification must be found. 139 (1975). thus generating the desired preliminary model for the crystal structure. Such sixdimensional searches. and W. Struct. R. E. M. inspired by Rossmann and tures (search molecules) to be used as building blocks to construct models of crystals of similar or related molecules. J .J. Ward. Schiffer. E.7. 15. Biophy. A particular parameter set may provide a clear best fit with experiment. and W. B. 4n H. Schmid. Steigemann. PROTEIN CRYSTALLOGRAPHY 26 1 ensures. and other molecules have been successfully determined in this A collection of papers about molecular replacement appears in Rossmann. A third is the study of functional conformational changes which occur upon ligand or substrate binding. The rotational search or rotation function is based upon certain simple properties of the Patterson function. The molecular replacement allows known molecular strucmethod. Schwager. E. 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. in the usual case. These models can then be refined (Section 6. To find the correct model one could. Lattman. 38 3y . 179(1975). are far beyond the scope of even the fastest computers. but it is very efficient compared with isomorphous replacement.8 To construct a model of a crystal using a search molecule requires. Structures of hemoglobin. immunoglobulin. The known symmetry operations of the crystal allow the other molecules in the unit cell to be generated. Lattman. Rossmann and realized that it was possible to separate the rotational and translational parts of the search. Fehlhammer. M. This method is neither as straightforward nor as foolproof as the differenceFourier method. B. however. reducing a sixdimensional problem to a sequence of two threedimensional ones. Mech. M. 24 (1962). 98. only six parameters. The study of the evolutionary relationship of molecules performing identical functions in different species is one example. Mol. 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. Acta Crystallogr. G. three to specify the position of the search molecule in the unit cell and three its orientation. however. M. E. C .
262 6.4.13) where the F s are the structure factor amplitudes of the model and crystal. (6. is the Patterson function of a single. showing that R ( C ) = C Fh(Ch)F2(h). upon a selfvector family in P. All selfvectors are contained within a sphere of radius D about the origin of the Patterson function. depending upon convenience. thereby providing as estimate of the molecular orientation in the crystal. and vectors running between molecules (cross vectors). 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). Indeed.(u). and valleys in P . In practice the success of the method varies widely. The volume of integration is a sphere of radius D. P . one need only seek the orientation for which P . where D is the maximum dimension of the molecule. The integral above is evaluated numerically for each value of C. in others several peaks of nearly equal height are found. In principle the absolute maximum of R occurs for a rotation C which superimposes the whole set of vector peaks in P .12). since P .4. The families of selfvectors are identical. but may be written as an integral over all space. it is best thought of as the set of intcratomic vectors within one test molecule.12) The matrix C is a rotation matrix. isolated molecule and is therefore not periodic.and a maximum in R is sought. To find the orientation of the search molecule in the crystal.4. The correlation function most frequently used for this is R(C) = JJJPM(Cu)P(u) dV. The latter bit of formalism allows one to apply Parseval’s theorem6 to (6. vanishes outside the sphere in any case. coincide with valleys in P. Both the direct and reciprocal space formulations have been used. highly significant peaks in R ( C ) are seen. one can readily calculate its Patterson function P. It is small when peaks do not coincide. . R ( C ) is large when peaks in P . corresponding to the orientations of the molecules within the crystal. plus whatever cross vectors happen to be of length less than D. and considerable difficulty may arise in attempting to pick the correct one. This sphere of radius D will contain one family of selfvectors for each molecule in the unit cell. (6. but appear in various orientations about the origin. If one knows the structure of the search molecule. In some cases isolated. 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). often based upon Eulerian angles. superimposes upon one of these. coincide with peaks in P.
1854(1970).tu Crysrallogr.5) provide coordinates for all atoms in the structure. ed. An extensive literature exists describing variations on T ( x ) and discussing the most efficient ways of evaluating it. In an important advance C r ~ w t h e has ~ r ~ expanded the functions of the righthand side of Eq.8.12) in spherical harmonics and has shown that two out of the three integrals can thereby be reduced to fast Fourier transforms.. However. in “The Molecular 42 . these coordinates are subject to considerable error.14): (6.’ Again.4. 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. A. thus completing description of the model structure.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. of course. (6.14) J J J unit cell Here P. Such a synthesis is. who put it forward for slightly different purposes. Gordon and Breach.4. G . thus speeding up the calculation enormously. This is usually accomplished by the use of a ‘translation function.4. Love.). At the resolution usually attainable in protein crystals (1/slnax 2 A) the maps do not have enough z z detail to visualize individual atoms. only its position remains to be found.. ‘’ R. Replacement Method” (M.’ Full details of the translation function are outside the scope of this chapter.6.4. S w i . 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. 6. is the Patterson function of the model with the properly oriented search molecule at position x. BB26. Rossmann. Ac. the success of T(x)is quite variable. Once the orientation of the molecule is known. 1972. but in favorable cases it gives a clear and unambiguous indication of the molecular position. E. Refinement of Protein Structures Protein models obtained from the interpretation electron density maps (Section 6. Lattman and W. Also. errors in the estimation of the phases introduce errors and distortions into the electron density and into E. PROTEIN CRYSTALLOGRAPHY 263 The above formulation of the rotation function42 is a simplified version of the original function of Rossmann and Blow. E. Crowther.4. a hybrid of the search and correct structures. New York. and it is the function of the refinement process to convert it into an accurate image.
(6.The most commonly used index of agreement between observed and calculated magnitudes F.qr. are used to calculate po. 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 .16) is an observational relation which can be used to refine the atomic parameters. 253 (1966). McQueen. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES the model.^^.4. Acru (’rj. J . ed.264 6.730 (1974). from a physical point of view the models are highly inaccurate.15) with At. 1975. Diamond. Actir C‘rjxtui/o. (6. namely the structure factor amplitudes of the native crystals. 4h R. 21.). Both kinds of methods have been used for the refinement of proteins. In addition.(h) = F.Diamond.(h)  F. Copenhagen. An estimation of the values of this set of observed magnitudes can be calculated from the parameters of the model using Eqs. A A30.16) Equation (6. The correct stereochemistry is usually imposed upon the measured coordinates using “model building” program^. where W(h) is a properly chosen weight. E.U J 2 nv if phases a. R. 292.^^ These models are in general good enough to study most of the relevant biochemical properties of the protein. Ahmed.Sect. p. (For a complete evaluation of some of the methods.(h).(h) and F. Munksgaard. Usually leastsquares procedures are used to minimize 1 W(h)AF2(h).18). see Blundell and Johnson’ and Diarn~nd.s/uIIoqr. This clearly indicates that the minimization procedure can be performed both in direct (real) or reciprocal space.(h) is called the “ R factor” and is defined as (6.4. However. 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.3. 44 45 .. Hermans and J . There is in principle one set of observed magnitudes that can be used to evaluate the accuracy of the parameters.4.~”) K. Hence the models obtained in this manner represent only the best efforts of the builders. bi “Crystallographic Computing” (F.
can then be combined with the observed amplitudes F .N =Al. Mol. The electron density pmode. to produce coefficients for an improved electron density map.).46(1975). (6. J. For a problem with M observed structure factor amplitudes and N parameters the linearized observational equations can be written in matrix representation 1 AN. Therefore all methods of solution necessarily involve iterative procedures. J . which serves to begin a new cycle of refinement. which are then introduced after each Recently Diamond developed a method49 in which the residual J~llo(x) . 509 (1979). The independent variables of the refinement are then rotations around single bonds in the structure. and J . Biol. The electron density p o is pbes.4. Instead they are modified by a leastsquares fit so as to preserve the stereochemistry of the model. the problem has to be overdetermined to provide for a stable refinement. The traditional realspace methods use either electron density or difference electron density maps. Also. The matrix elements are derived from minimizing W(h)AF2(h) 1 47 4R S . The atomic positions are refined by shifting atoms up gradients to the summit of electron density peaks. the resulting system of equations is nonlinear.M6x1. C. 131. . L. This information can be used in two different ways: (i) to reduce the number of parameters. Jensen. R. H.4.P m o d e ~ X ) 1 2d v is minimized. Carter.17) where the subindices represent the number of columns and rows of the matrices.436 (1971). and L.calculated based on is the atomic coordinates of the model using a spherical Gaussian to represent the individual atoms. 49 K. C. Bid. W. can be obtained through a structure factor calculation [Eqs. PROTEIN CRYSTALLOGRAPHY 265 Independent of the particular residual minimized. For convenience we summarize some of the possible approaches in Table 11.3. The known stereochemistry of the amino acids is normally used to overcome this problem. Chem. Jr. Diamond. Reciprocal space methods refine directly the residual W(h)AF2(h). thus reducing the number of model parameters by a factor of approximately three. R .8)]. since the number of observations per atomic parameter is usually quite low (from less than 1 to 2 or 3). T. Kraut.M~ (6. obtained from multiple isomorphous replacement (Section 6. Watenpaugh. A. Freer. After shifts have been imposed a new set of calculated phases a.(h)exp(ia. A A21..4. These procedures are usually performed without stereochemical constraints. These a. and (ii) to increase the number of observations. For a typical protein this condition is not easily met.6.qr. Sect. Acta Crystallo.. D . Alden.4) or from a map calculated using F. 250. The shifts calculated in each minimization cycle are not used directly. Sieker.
etc. All structure factor calculations are done with atomic scattering factors.Pidexl)'P 1 O(P.y.Pideal)' l. but they range from 3 to 50 or more. point. conformational angles: atomic radii ideal. . optimized nN. planarity condition. .  Pmodeb' dV.occupancy.z)*8. 1orz> 1WAzF(h).TABLE Summary of Refinement Options 11. (observational or approximate) 6 ' Gaussian. bond length. 1 WAFz(h) + C W@. are the values of a stereochemical parameter (i.) obtained as an average of a series of very accurate smallmolecule observations (pideal) as calculated from the atomic coordinates of the protein being refined @ and ) . transform of atomic scattering factor (x. ' The values of n and m vary for different implementation of these methods.. . (mlv)" pideal andp.e.rl. . bond angle. 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. This is the implicit or explicit atom shape at the minimization step.
and S . Section 6.. in principle.NAN. Sussman.and firstorder terms in a Taylor expansion. Secl A A33. a very expensive task.5 0 5 0 J . They can be shown to be where F. In this case. Second.M and Ci. In a typical case there will be more than 3000 parameters.4. Acta Crystallogr. R. large blocks of 200 parameters were refined to avoid working with the full matrix. = Defining BN.4. M . PROTEIN CRYSTALLOGRAPHY 267 with AF(h) made linear on the shifts by using the zeroth. (6. L.4. ax These normal equations can be solved for the shifts 6x by multiplying (6. we obtain B6x = C.N = AL. N A 1 . Another method used to diminish the number of parameters consists of refining the positions and orientation of rigid groups. Kim. .(inverse of B). so that 6~ = B'C. the number of parameters is in general very large. M is usually not much larger than N .4. 800 (1977).18) by B. there are two problems in applying this method directly to the refinement of proteins. This expression can be solved for the shifts 6x in the parameters by transforming to the normal equations.(h) and AF(h) are calculated with the present atomic parameters. M . used for a small protein (rubredoxin. and the normal equations will thus require the generation and inversion of a 3000 x 3000 matrix for a large number of cycles.Holbrook.19) As mentioned above.18) The elements of B and C are and Cj = h W(h)aFc(h) AF(h). Church.6. This is. S. The method was. Multiplying on the left by AT (transpose of A) we obtain A~AGX A ~ A . however. (6.9). G .N = A&. H.4. This method has the additional advantage that the stereochemistry of the molecule is automatically preserved. First.
Refinement has led to greatly increased rigor and quality in the protein models obtained by XRDA. Wilf. eds. etc. They differ in function./dxi)(dF. Otherwise they are very small. Ralston and H. (C/. Beckman. ( P ~. Konnert.268 6. 13. 62. S. Hcndrickson and J. This implies that the matrix elements of B and C will now include terms from the stereochemical equations. 1960.). Konnert and Hendrickson found that the system of normal equations is very stable even if the matrix elements not containing stereochemical constraints are omitted. providing further savings in computing time. These terms can be very large if parameters are related by a stereochemical restraint (for example. Rainaseshan. Wiley. R. in a typical case only 1 of the matrix elements have to be calculated and used.9.Pideal)’ k h (see Table I1 for definitions). Case History: Rubredoxin A To illustrate the different aspects of protein structure determination we next analyze the process as applied to a specific case. and refinement. complexity.409 (1952).). These approximations lead to very substantial savings in computing time for the generation of the sparse matrix. Venkatsan./dxj) is small compared to the terms arising from stereochemical constraints when x i and xj are not coordinates of the same atom. A. thus using the stereochemical information as additional observations. H. Nut). They were solved using different methods for data collection. eds. Ncw York. Bur. Such models have become of interest to a wide range of investigators.S. size. S. In addition.01. in “Mathematical Methods for Digital Computers” (A. S. Indian Academy of Sciences. J . M . in “Computing in Crystallography” (R. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In a recently developed method Hendrickson and Konnert5’ proposed refining the residual C W(h)AFZ(h) + C W . if they are coordinates of two atoms that are bonded or belong to a rigid group). Bangalore. we have settled on a 5 1 W. Stiefel. Rrs. the method of conjugate gradients5’ used for solving the system of equations takes full advantage of the sparseness of the matrix. 1980. There are probably about 100 proteins whose structures have been determined (to some resolution) using singlecrystal xray diffraction techniques. for this 1 % of the matrix elements x h W(dF. Hestenes and E.)49. leading to the establishment of a Protein Data Bank (Brookhaven National Laboratory. Sfand. New York 11973) from which atomic coordinates from a rapidly expanding number of molecules can be obtained. phase determination. Upton. p. It is therefore very difficult to choose a “typical” case. Diamond. biological relevance. 6. ’’ . In addition. With this approximation.4. F. and K . p.
Sieker. The final map calculated with phases obtained from multiple isomorphous replacement included data to a resolution of 2.9 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. H. 9. Sect.40’. L. The rubredoxin literature uses a hexagonal unit cell (as is usually done). Acra Crystallogr. The crystal is then said to be rhombohedral and to belong to space group R3. namely. 12. Herriott. Herriott. but the rhombohedral cell is more appropriate for the scope of this book. 943 (1 973).derivative and a multiplesite UO. C. Watenpaugh. ironsulfur protein that has been found in a number of anaerobic bacteria.54 protein was crystallized by The precipitation with nearsaturated ( N H 4 ) . The cell parameters of these crystals are a = 38. Several electron density maps were calculated and interpreted. R. 36. D. 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). The tracing of the polypeptide chain was unambiguous and the iron atom and the four coordinated cysteinesulfurs were located. pasterianum was not known at this time.0 A. The structure of the rubredoxin of Clostridium pasterianum was determined to 1. Watenpaugh. 39. C.6. and L. L. Heavy atoms were prepared by the usual soaking technique. The sequence of the rubredoxin from C . Bid. A singlesite HgIi.” The four cysteines were assigned as amino acids 6. Cold Spring Harbor Symp. 54 K. Examples of the electron density corresponding to several side chains are shown in Fig. PROTEIN CRYSTALLOGRAPHY 269 protein that is small. and L. Most rubredoxins have an iron atom and four cysteines per molecule of approximately 6000 daltons. Jensen. The positions of the heavy atoms were determined using AF2(h) Patterson syntheses. D.~ but its discussion is outside the scope of this book. easy to describe.’ were used for phase determination. Sicker. J. B B29. J. R. and 42 in agreement 5 3 K. H. rubredoxin. and for which more than one refinement method has been used..47. . At least 25 investigator years have been invested in this project. The crystals are flattened rhombs and can be grown to more than 1 mm per side in a few weeks.’. Using sequence data from other rubredoxins.53. Rubredoxin is a nonheme.4.77 A and a = 112. Data were collected using a fourcircle diffractometer. Jensen. S 0 4 at pH 4. Quant. 359 (1971). The structure was solved by the multiple isomorphous replacement method.5A resolution by Watenpaugh et a1. has been solved to high resolution. Xray diffraction experiments indicated that the only symmetry of the crystal is the presence of a threefold axis at every lattice point. the authors built 54 amino acids and were able to identify about 40 of them with “reasonable certainty.
5 Ib) 30 25 2. and 1. s 3 with permission. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES 3.7 A. Electron density through aromatic side chains in rubredoxin. Copyright 1971 Cold Spring Harbor Laboratory. (b) tyrosine.) . 2.7 ICl FIG. The side chains reprcscnted are (a) phenylalanine.270 6.0. Note how the rendition of the groups improves with resolution.0 2. 12. 2.0.5. and ( c ) tryptophan. (Reproduced from Watenpaugh et a / . at resolutions of 3.0 1.
6. 23 peaks in the electron density map were interpreted as water molecules and their oxygen atoms assigned coordinates.5 < < < < d d < 5. The value obtained (0. Copyright 1971 Cold Spring Harbor Laboratory. PROTEIN CRYSTALLOGRAPHY 27 1 FIG 13. The large circle represents iron.0 and 1.15)] was calculated.6 ('x) 5.4. (Reproduced from Watenpaugh P Z u I .7A resolution was calculated and showed the expected improved resolution (Fig. Quant. only 45 % of the reflections between 2.4. Biol. about 95% of the nonhydrogen atoms in the structure were assigned atomic Coordinates. A temperature factor B (Section 6. An electron density map including data to 1. L.3.1) of 15 A2 was assigned to all atoms in the structure and an R factor [Eq. Although a chemical sequence was still not available. Polypeptidechain backbone of rubredoxin.0 d < 3. (6. H.0 2.7 86. 12). It was found that the average intensity of these reflections decreased rapidly with decreasing l/s (Table 111). represented by line segmentsconnecting rcarbon centers.5 A as measured by the authors had intensities larger than two standard deviations above background.9 44. J. 36. Siekcr. Cold Spring Harbor Symp. and L. Watenpaugh.) ~ ~ with the sequence data of the rubredoxin from Micrococcus aerogenese. Herriott. For example. D.0 d < 2. . C.0 3.0 224 816 2478 4254 165 2151 1899 '' From K. and the small ones carbon. Also.0 1. Summary of Intensity Data for Rubredoxin"** Possible number of reflections Number of reflections exceeding 20 218 Range of d (4 Reflections exceeding 2u 91. This gave a grand total of 424 atoms. Figure 13 is a stereo pair drawing of the FeS complex and the path of the polypeptide chain.389) indicated that the quality of the atomic parameters TABLE 111. The ironsulphur complex involved in the redox activity of the protein is also shown. with permission. R.5 A were then collected in order to improve the resolution of the model. Intensity data for reflections with l/s 2 1. thc intermediate circles sulphur. Jensen.3 93. 359 (1971).
only two observations per parameter were available.272 6. With this addition.12 (Table IV) is remarkably low.e.2 A.06 were found to have very large errors and were eliminated from the calculation. As the refinement proceeded the authors introduced additional water molecules to explain properly located positive peaks in their difference maps. In most of these cases the atomic parameters stabilized with subsequent refinement. The authors found no general tendency for the parameters to diverge or oscillate.10. low overdeterminancy and use of diagonal blocks) werc soon dissipated. The value of these factors was restricted to the range 0. occupancy factors proportional to the peak height were introduced for all atoms. The final R factor of 0. The course of the refinement through this and the subsequent steps is shown in Table IV. Also.372. The few cases where it did occur werc corrected using differenceFourier maps between cycles.4. In order to restrict the number of parameters only seven B values ranging from 9 to 25 A’ were included. detailed analysis of the maps indicated that the temperature factor used (12 A’) was not suitable for all atoms in the structure.It is clear from Fig. leaving only a few atoms in the structure with uncertain atomic parameters. N. The standard deviations of the interatomic distances for C. Problems intrinsic to this low overdeterminancy were overcome. giving an R factor of 0. Furthermore. while the atoms at the outside required larger values.. The authors chose the simplest possible approach. and 0 atoms were found 10 be in the range 0. The apprehensions derived from the imposed limitations (i. 14 that the reflections in the intermediate ranges have the best agreement between observed and calculated structure factor amplitudes. In this refinement individual temperature factors were used. Six reflections with s I 0.O. The course of the refinement was also analyzed by computing the R factor for reflections grouped in ranges of i. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES was reasonably high for an initial model. only the protein molecule and the tightly bound water molecules are included in the calculation. Even for this extremely small protein. probably because of the high quality of the diffraction data.8) was initiated. This distribution reflects several of the intrinsic problems of calculating structure factors for protein crystals. First. The leastsquares refinement behaved surprisingly well.3. The assignment was made depending roughly on the distance of the atoms to the center of the molecule.1. Atoms at the inside of the molecule seemed to require lower B values. At this stage a leastsquares refinement minimizing w(h)AF2(h) (Section 6. The refinement was then carried out using differenceFourier maps. They partitioned the matrix in 1012 blocks involving 200 parameters each. The temperature factor was then adjusted in two steps t o a value of 12 A’. the refinement involves a 2000 x 2000 normal matrix. The rest of the unit cell is filled with randomly .
132* 0.312 0.242 0.289 0.346 0.141 0.179 0. < 0.154 0.550 350 2107 2107 286 1 1 1 1 1 100 0.118 0.301 0. including 256 H atoms 4th L.100.225 0. cycle F. D.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. Cold Spring Harbor Symp.394 0. and L. 28 reflections with sin @/A < 0.03 weighted 0.312 0.131 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 2151 0. B i d .31 0. L.389 0. 36. Fo map From 2 A res.372 0.373 0.092 0.260 0. I67 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. C. R.188 0. 359 (1971).328 0.306 0.31 0. H.392 0. cycle 3rd L.O and occupancy 23 23 23 22 22 22 23 106 127 130 130 130 1 1 R 0.209 0.461 0. Jensen.307 0.222 0.05 weighted 0. 6 reflections with sin Qji. cycle 2nd L.123 0.397 0. Quant. including 256 H Number of H.389 0. Watenpaugh.263 0.224 0. Fo map From 2 A res.241 0.389 0. Copyright 1971 Cold Spring Harbor Laboratory.5 12 12 920 730 640 640 .31 ' From K.145 0.080 0.181 0.262 0.271 0.31 0.390 0.392 0. S.10 0.31 0.192 0.376" 0.239 0.261 0.153 0.1670. ' .663 0.385 0. S.192 0.092 0. S.126 00.TABLE IV.292 0.25 0. Course of Refinement for Rubredoxin" R and number of reflections as function of sin O / i Overall Coordinates From 2 A res. J.250.321 0.239 0. Herriott.31 0.389 0.078 765 165 765 765 765 765 765 765 765 765 765 765 765 0.288 0. Sieker.159 0.333 0.112 218 212" 212 212 212 212 212 212 212 212 190b 190 190 0.139 0. S.192 0.170 0.504 0.148 0.
The number of water molecules included is also shown. represents leastsquares refinement and AF denotes difference Fourier refinement.30 R 0. This omission introduces errors in the reflections with low s (lowfrequency components. .2).3 FIG.S. Other factors affect mainly the highresolution reflections (i..3.1 sin @ / A 0 2 0. R factor as a function of sin O/l.2c 0. large s). On the individual curves L.214 6. (=is) for different stages of refinement.14.IC 4 t h L S 130H20 2 5 6 H atoms 1 1 1 I 1 L 0 0. and to the use of isotropic temperature factors (Section 6. Overall.40 0. Chapter 6. determination of the rubredoxin structure represents a highly successful attempt to obtain a very accurate model of a protein molecule.e.s4 with permission. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES 0.) distributed solvent molecules that are usually ignored.1). (Reprinted from Watenpaugh er a/. They are related to the approximations made during the leastsquares refinement which lead to false minima.
Frequently.6. FIBERS 275 In this case it is probable that the extra effort will pay off. . 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. whole globular proteins. Amsterdam. Biological fibers can be divided into classes depending on the nature of the units that build up the fiber.. rather than small monomers. K. In any structure determination of this type side chains can only be visualized as average masses. The fibrous proteins actin and myosin. 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. are polymerized to form the unit. Fibers are also classified by the ways in which the threads are packed side by side to form the specimen. where Bessel functions play a prominent role. and also on the way the units pack together to form the fiber. “Diffraction of XRays by Chain Molecules. They are bundles of threadlike or rodlike objects. also belong to this class. and amino acid side chains in the case of fibrous proteins. 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.Fibers 6. The side chains normally occur in a random sequence and do not obey the symmetry of the unit. and are nonperiodic in the other two dimensions.5. The selfassembly of these units provides interesting systems for physicalchemical study.5. These groups are bases in the case of nucleic acids. Perhaps the most familiar unit is a polymer chain. Monomermonomer interactions are usually noncovalent. For these reasons the theory of fiber diffraction is most conveniently expressed in cylindrical coordinates. The double helical structure of DNA is perhaps the most familiar example of this. Definition and Description Fibers form the other major class of biological specimens studied by XRD. as in most polynucleotides and fibrous proteins.1. called the fiber or z axis. rodlike object.5. The broad definition of fibers given above conceals a number of distinct types.” NorthHolland Publ.55They contain periodicities along one axis. 6. which we now discuss. The packing schemes have great variety 5 5 B. 1966. Vainshtein. which form the basis of many contractile systems. In the other major class of fibers. arranged in an extended or helical conformation. several chains are wound around one another in the unit.
setting limits on the resolution of the useful data that can be collected. In the oriented gel. 273 (1978). J . truly crystalline bundles. 9. " L. . and V. Certain preparations of DNA are of this type. XRAY DIFFRACTIONANALYSIS OF MACROMOLECULES and complexity. s9 A.56 Frequently. Crick. The effects of arcing become more serious as one looks farther from the center of the photograph until. fiber diffraction photographs are usually characterized by arcing. A variety of other specimen types exist. or in which disorder such as dislocations or irregular alternation of the sense of rods is present. Crystaiiogr. 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. and we mention only a few prototypical cases. The canonical specimen in this group is TMV.2. H. 6. Kluy. Such a specimen yields a diffraction pattern composed of discrete spots confined to layer lines normal to the fiber axis.581 (1952). 199 (1958).. Makowski. Crick. WyckoR. the unit rods have random azimuths and diffract independently of one another.5. Fiber Diffraction Theory et ~ Fiber diffraction theory was first presented in seminal papers by Cochran 1 and Klug et aLS9It does not offer any tool as simple as diffraction . which we discuss in detail in Section 6. great experimental art is required for preparation of fibers suitable for xray diffraction study. Attempts to deconvolute the smearing from the diffraction photographs have had very promising initial appli~ation. Acta Crystallo. such as polyethylene. in which the averaged pattern of a unit rod is observed. Arnott. Am.4. Nevertheless. The same pattern would be given by an individual bundle if it were rotated through 360" about z during photography. 5 8 W. a smearing of the pattern arising from lack of parallelism among threads. on the other hand. and a wide variety of techniques. C. in which various stages of semicrystallinity are present. 11. such as extrusion and quick stretching. F. layer lines may merge into one another. 31 (1973). W. Vand. C. H. ~ ~ " S. 5.~' It should be reemphasized that all fibers give rise to azimuthally averaged diffraction patterns. These bundles are then packed with random azimuths (rotations about z ) into the fibrous specimen. have been used. finally.5. Acta Crystailogr. In crystalline fibers unit rods pack side by side to form small. F. H . The information contained in these patterns is much more confused than that in the diffraction by single crystals. Appl. 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.276 6. Crystailogr. 11. Cochran.qr. The problem is especially severe for oriented gels. Assoc. as are specimens of some simple polymers.
is the Bessel function of order n. $. Z (6. so that F(R. Z)in exp(in$).5. z ) exp[2ni(xX + y Y + zZ)] dV. X Then XX Thus = R cos $.4) The factor i" remains by convention.in+) exp(2nizZ)r dr d 4 dz.5.3) Note that F has merely been expanded in a Fourier series in the periodic angular variable. k: Z ) = JJ/p(x. Z). (6. FIBERS 277 from a spatial plane wave in the crystallographic case.(R. z. Z) = c G. We define polar coordinates by x = r cos y. We begin by writing down the diffraction pattern of a general object in cylindrical coordinates.5.6.5. we find x exp( . where J .in$) exp(2nizZ)r dr d 4 dz (6. (6.1) = + y Y + ZZ = rR(cos 4 cos $ + sin 4 sin $) + zZ = rR cos($ . $. Z. but useful and conceptually simple special cases do exist. (6. Y R sin $.2) Using the identity expC2nirR cos($  4)] = c Jn(2nrR)inexp[in($ 'x m  4)]. To emphasize this the triple integral is often denoted G.2.7) F(X.4) + zZ. F(R. Z ) = l//p(r.5. The Fourier coefficients are the triple integral which multiplies exp(in$). $. y = = r sin 4. z = 4.5) is a type of FourierBessel transform. The relation x exp( . . z ) expC2nirR cos($ .5. (6.$)] exp(2nizZ)r dr d$ dz. From Eq.(R.
10) and that g n .11) This shows directly that if one can determine the individual G n . Thus exp( .11).5. . of the electron density are directly available through Eq. except when Z = I/cwhere 1 is any integer.(r)Jfl(2nrR)r = dr (6..5. It is useful and insightful to make an analogous expansion of the electron density function: p is now periodic in d. however. The period of the fiber can be readily introduced...5. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Thus far F is the diffraction pattern of a perfectly general object in cylindrical coordinates.(2nrR)R d R .278 6.... by extension of the previous notation. [which are . .(R) 271 j). = (6. This is a slight generalization of discrete Fourier coefficients for a onedimensional. m = 2x JornGn. will play a central role in the description of the rotationally averaged pattern. If p has period c along z .in$)r d r dd. and z . or. the Fourier coefficients gn. (6.(R)J. periodic function.5. 1 (6.5. (6.7) The coefficients G n . are slight generalizations of the usual Fourier coefficients and are given by It can be shown that Gn.(R)i" exp(in$).. then F will be identically zero except on layer lines of spacing l/cthat is. so that it can be expanded in a twodimensional Fourier series The gn.. d z . complex.6) G.
O). giving F R.dzd4. 0. (6.5.6. 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. First we analyze the diffraction pattern from a wire bent in the shape of a helix of radius r o and repeat (pitch) c.14) ('"f") To integrate over 4 and z a change of variable is helpful: define Then 4 = n(u + u).6) for the diffraction pattern of a periodic object we find x Jn(2nrR)exp( . (6.15) .13) The integral over r can be done by inspection. $. and make possible the generation of an image from a rotationally averaged diffraction pattern.in$) exp ('Y'")r __ dr d 4 dz.1 2) where we assume that the helix passes through the point ( r o .5. R)i" x Jozn J)(& . (6.5. $. Inserting this function into Eq.  ( 1) c = n exp(in$)Jn(2nr.5.5.  ( '1 = nc x c Jn(2nr0R) exp(inrl/)i' n Io2 jo*6(u) exp[ . F R . Substituting I? = ). (6.i m ( u + v)] exp[xil(u  u)] du du.5. We approach diffraction by helical objects in stages.5) exp(in4) exp .(u  0). 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. Such an object can be formally represented as the product of two b functions (6.
shown in this figure. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES simplifying.17) I. 16. one arrives at im(Z + n)] exp[inu(l .) . F R . Thus the main bars of the x are formed by the first maximum ridge of the J . (Reproduced from Vainshteinss. produce fringes outside the main x . (6. First. with the order of the Bessel function being equal to the layer line index 1.5. 15. I).n)] du. 15.16) J. move farther out from the origin as n increases in magnitude. This pattern is greatly simplified compared with the pattern from a general fiber.R) exp(inI))i" Jo Jo6(u) exp[ and integrating over v. with permission. 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.18) F(R. when (6. ~ ( I) = nc n J. the first maximum of J . R The factor 271c is normally omitted.5. The x shaped form of the pattern. I).(Znr.(2nro ) exp(il$). and a similar set of line segments FIG.(2nroR) exp(in$)i" This integral vanishes unless n = exp[inu(l . although not its exact equation. only a single Bessel function occurs on any layer line. can be deduced by a very simple argument from the general shape of the helix. Because of the form of the Bessel functions J 1 this gives the diffraction pattern an x shaped appearance.l/c) = (2n/c)i'J. Subsidiary maxima of the J . As shown in Fig. is at the origin. and the first maxima of the J .5.n)] du dv. (6.280 6. Bessel functions as a function of order (n) and argument (u). This is illustrated in Fig.
.8).) forming the back. The existence of a strong reflection therefore implies the existence of a correspondingly strong Fourier component in the object. forming the arms of the x .(2nrR)R dR. We will shortly generalize this for a helix made of atoms. one Bragg reflection. . (6.5. Each set of lines gives rise to a diffraction pattern of a row of points normal to the stack of line segments.16. A simple choice for the helical case is suggested by the expansion for p(r. Substituting which represents one term in the expansion. In projection. Note the ‘’ x ” drawn through the first maxima of the Js. z/c) given in Eq.. 4. that determine the intensity of the Ith layer line in the diffraction pattern of a continuous helix. we find that . where we showed that by generalizing the usual stack of Bragg planes to a spatial. A uniform helical wire is not a very useful model for most systems. The two rows of points comprise the two arms of the x . (b) the x shaped diffraction pattern is related to structural features in the helix. G 1 .5. 4. We discuss here a generalization of the helical wire whose diffraction pattern is particularly simple.10). with permission. by computing the diffraction pattern of a set of beads (atoms) spaced uniformly along a helix. for G.2niz/c). (Reprinted from Vainshtein”. in Eq. (6.. ( R ) = ~~~J1(2nrR’)J.20) . sinusoidal density fluctuation we could produce an object that could be used to synthesize any specimen.5. so that crude ideas about important periodicities can be quickly obtained. (6. = (6. with g. We choose p ( r .5.. the helix is approximated by the sum of two stacks of sloping line segments.6.19) J . FIBERS 28 1 (a I (b) FIG. z/c) = Jl(2nrR’)exp(id . and which also had a very simple diffraction pattern. This parallels the case of crystals. Dill’raction from these stacks occurs along directions S perpendicular to the stacks.5. Diffraction by helices: (a) values ofthe Bessel function J.
F(R.5. by convolving the Fourier transform of the stack of planes with the Fourier transform (diffraction pattern) of the continuous helix. (6. Harburn. This is illustrated in Figs. The diffraction pattern of this structure can be generated by use of the convolution theorem. and value zero elsewhere. for example. In DNA. Welberry. 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. For other examples see Harburn et ~ 1 .282 6. This convolution amounts to reproducing the x diagram at each meridional point.5. Using this result. all the phosphates on one strand would form one such helix. These helices will all have the same pitch. A. R . New York.. For the case of a helical wire the pitch P was also the true repeat. and is often termed the rise per residue. We now proceed to the example of point masses strung on a massless helical wire.. Note also that the intensity of the diffraction pattern. one view of a helix can be used to generate all others. "An Atlas of Optical Transforms. FF*. vanish. Thus (6. This implies that the whole diffraction pattern is . but their radii and azimuths will vary. all the 3' oxygens another. is independent of azimuth in these two cases." Cornell Univ. Only the phase factor exp(i@)varies. 1975. and T. Press. .2 This model (the discrete helix) is of great general utility. Therefore in this special case the rotationally averaged diffraction pattern from a specimen is identical to the unaveraged one. ~ ' To express this result formally some relation between p and P must be assumed. 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. l/c) = 6 ( R . and so on. 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. 17 and 18. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES Using the orthogonality conditions for Bessel functions we realize that this integral vanishes unless R' = R . $.21) while all other G. Taylor. the diffraction pattern from any view of the object is sufficient to reconstruct an image.')in exp(i@). because of helical symmetry.22) which is a ring with periodically varying phase on the first layer. Ithaca. C. This is consistent with the idea that. "" G . The spacing p between planes becomes the z distance between atoms.
which is a system of planes of repeat distance C* defining the position of the layer lines. DiKraction pattern. from a continuous helix and from helices having closely or broadly spaced atoms. 18.FIG. (d) distribution of the first peaks i n J. permission. (Reproduced from V a i n ~ h t c i n ~ ~ . The example drawn is for seven residues in two turns.. being the transform of a system of planes in real space. over the layer planes. Formulatlon of thc diffraction pattern of a discontinuous helix: (a) system of points with a repeat distance C'*.17.) with FIG. (b) system of planes of repeat distance C*. This illustrates the convolution of the x pattern with rows of points in reciprocal space. (c) convolution of the previous two systems. .
284 6.5.(2xr0 R) exp(in$).25) where the sum is over those values of y1 consistent with the selection rule (6.28) where again the sum is taken only over those values of n that satisfy the selection rule. In general. there need not be a whole number of atoms per turn. we can directly write the diffraction pattern as F R. because many of the J . exist on all layer lines. n (6. in practice only small values of rn generate an n for which J .23) where t and u are integers. however. On layer line lthat is. is nonzero within the resolution of the photograph.26) where the atomic scattering factor4 has been introduced to give shape to our points. Thus helical structures in general have diffraction patterns much simplified compared with a general fiber. can appear.27) then (6. $.24). The full formula for a family of helices with atoms at ( r j .5.5.5. and the phase shifts involving 4 j and z j reflect the positions of the atoms along their individual helices. (6. It is often convenient to separate the angular and spatial variables in this equation. We therefore write the true repeat c c = tP = up. If we write'by analogy with (6. z = 0. and amounts to the structure factor equation for a helical object instead of a crystal. for which all J . only one or a few terms contribute significantly to F.in$j ( + 2xilzj C ).5. 4 = 0.5.  ( 1) = 1 i"J. (6.24) where rn is in principle any integer. are forbidden by the selection rule. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES In a discrete helix. Given this analysis. We can then write h i " J. at Z = l/conly certain J . This expression serves to set the relation between the interleaved x s in the diffraction pattern.5) C ) (6. those which fall on this layer line through the convolution process. (6.5. The helix in this example was initialized by assuming one atom at the point r = r o .z j ) is given below. .5. The allowed values of n are then given by 1 = tn + urn.(2xrj R ) exp in$ .4 j .
b'. b. Inc. (6..(W. Reprinted by permission of John Wiley & Sons. rather than to guess a model.5.. one must know the phase and amplitude of i n meridian xrays 4 .) .' Copyright @ 1966. Fiber diffraction geometry: the shade' qks represent the Fourier transform of a fiber. FIBERS 285 As mentioned before. The observed intensity I is I = (FF*) dl ..5. and c) give a'. (From Holmes and Blow./ exp(in$)i")(x G$ exp( . The points a'. the observed diffraction pattern is rotationally averaged. . / (a) FIG. on the film.in$)( 2)") n. b'..30) If one wishes to reconstruct an object directly from the diffraction pattern. The points of intersection of the disk C with the sphere (a.= ( 91 Gn. Lines drawn through the intersection of the disks with the Ewald sphere from the center 0 of the sphere represent the scattered ray directions. and c' form a layer line. Inc. and c'.29) The integral vanishes unless rn = I R./(W2. respectively. Therefore d$. (6. . Note that point B does not give rise to any point on the tilm.6. / equator .19a.5. John Wiley & Sons.27c (1 I' G.
. there is no variation in intensity as one moves azimuthally in any plane. Since each G . Same as (a) but the fiber axis has been tilted so a& bring the point B into the reflectto ing position. l/c) via Eq. the problem of recovering the Gs from the Is is sometimes referred to as separating the Bessel functions. Remarkably enough.1%. complex F(R. and use these to generate p from Eq.8). (From Holmesand Blow. .is generated from only one J. Alternatively.5. Note that the sphere intersects each reciprocal space .286 6. diffraction from fibers is confined to a set of planes in reciprocal space.) each G.. Since the data are rotationally averaged. .4).5. Reprinted by pcrmission of John Wilcy & Sons.* Copyright @ 1966.. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES (b) FIG. A word about how the intensity data are measured is appropriate here. this process has actually been realized in the case of TMV (Section 6. $. Inc. From these one can calculate the full.5. one can use Eq. As we have seen. John Wiley& Sons. Thus all the available data are contained in radial scans running from the meridian to the limits of resolution in each of these planes. Inc.28) and then synthesize the object by Fourier transformation of F... (6. Now that poini B projects onto the meridian at B’ and that thc layer line is no longerstraight. (6. (6.5./.11) to generate the g. Figure 19 shows the Ewald sphere construction for a fiber.
Arnott. “The Double Helix. CampbellSmith and S.4 examine here the simpler and problems that are more characteristic of the field. The geometry and chemistry of the monomer often provide severe constraints on the model. 61 62 J. the “linkedatom least squares” (LALS) method.g.5. and. Problems can be simple because the fiber structure itself is simple (e. P. Polypeptides and polynucleotides may fall into this category.. Later. so that lowresolution images upon which a model can be based are frequently available. This was accomplished by using rotations about single bonds as the independent variables. has been successful in providing accurate atomic models for a variety of polynucleotides and polysaccharides. Only a region near the meridian is missingthe blind region. Joint use of electron microscopy is very common for these specimens. modeling techniques have also provided images of specimens in which the repeating unit is a whole protein.3.5. D.5. The required photographs are stillsneither the fiber nor the film moves.3 (1978). such models were adjusted by trial and error to give improved agreement with the observed data.. In the case of polymers whose elements are fairly simple monomer units.” Atheneum. J. atomic models are possible. They can also be simple because the diffraction patterns are poor and contain so little information that only a crude structure can be determined.6. 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. FIBERS 287 plane along an arc which provides almost all of the desired radial scan. A AM. when the specimen is a polymer having only a small number of atoms in the monomeric unit). similar to the process described earlier for crystals. Arnott and and others6’ made use of the structure factor formula as an observational equation for leastsquares refinement. To deal with the relatively small number of data. in a sense. which in any case. only a very small number of the first models may be possible. A c f a Crystallogr. Watson. 6. is often modeled as an ellipsoid or a cylinder. If data from the blind region can be ignored. Techniques for modeling various types of disorder have also been incorporated into the analysis. a single photograph provides a complete data set. On a much larger scale. In these cases no internal detail is seen in the subunit. . it was necessary to reduce the number of parameters in the model as much as possible. This. 1968. The second panel shows how tilting the fiber can help fill in the missing data. Sect. while constraining the lengths of bonds and the angles between them to canonical values. Solution of Simple Fiber Structures We defer discussion of TMV to Section 6. New York.
Three nucleotides are bound per protein subunit. so that the rise per subunit is only 23 A x & = 1. TMV is a rodlike virus. drawing of the virus. 216 (1977). Caspar. the . as its structure was understood in A the early 1 9 6 0 ~is shown in Fig. 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. Virus Res. with a central hole of radius 20 A. Note the Dutchshoeshaped protein subunits on the outside. Adu. Tobacco Mosaic Virus Structure As mentioned earlier. and the R N A wound in the lumen of the central. Warren. Caspar. Drawing of tobacco mosaic virus. The pitch of the helix is 23 A. . (From Ca~par. Stubbs.225 (1960). an atomic model of TMV has been obtained from fiber diffraction studies. or whether it only appeared to be so because of arcing.5. 7. Holmes. A ~ PProtein Chew.65 Because the helix is so shallow. Nuture (London)267. Klugand D. 20. It represents by far the most complex fiber analyzed in this way. and K.20. The RNA follows the same helical path and is positioned at the edge of the central hole.~~ opening angle of the x in the diffraction pattern is very small. D. D. and was only made certain "'G . L. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES FIG.4A. long and 90 A in radius. L. .~') 6. S . and it was experimentally difficult to decide whether a reflection was actually meridional. Identical protein subunits build a helix with 49 subunits in 3 turns. 37 (1963). cylindrical cavity.4. Thus the value of 49 residues in 3 turns was in dispute for some time. 64 A.288 6. 18. D. some 3000 8.
The selection rule is I = 3n + 49m.) by Franklin and Home@ with the use of a heavyatom derivative. 21.5. FIBERS 289 FIG.at the repeat is 3 turns. Photograph of the dill'raction pattern of a fibcr of tobacco mosaic virus containing data to 3w resolution. C. When I = 0. (Courtesy of Gerald Stubbs. Fr. 213 (1958). E. An example of such a pattern is shown in Fig.inklin and K . The rods form an oriented gel which gives diffraction patterns of very high quality. 11. on the hh R.6. so that the pattern is essentially the rotational average of the diffraction intensity from a single rod. . It is immediately clear tl.21. consisting of the loworder Bessel functions from the central x in the diffraction pattern. Acfa Crjstallogr. since every third layer line has intensity near the meridian. Holmes. All the native data nccded to reconstruct the fiber are contained In this photograph. The effects of interparticle interference are very small.
(6. (6. N .290 6. Imagine now that a heavy atom is bound. to a specific site in TMV. We pass over for the moment how this might be accomplished. . + ... and write as well G =A + iB. Barrett.433 (197 I). . . (6.5. Cold Spring Horhor Svnip. W .28) and (6.which are simply related to the native G. and P. This heavyatom derivative will give rise to a diffraction pattern built from the Gb. + a 1 ) 2 + (B. since these lead directly to either F [Eq.8)]. /= G". K. which becomes nonzero at about 10A resolution. von Sengbush. G .1.5.+ h .35) 6 7 A . We recall that from Eqs./+ Yn.. C. . + u2). Holmes in Heidelberg.. and mixes hardly at all with J. = ( A . B i d . and the next is J . Leberman. . Barrington Leigh. . Mandelkow.5. Let . y = u + ib.5. Holmes. and generated the original hope that the atomic structure of TMV might be revealed by it.33) Then I' + B: + A: + B: + . E..28)] or p [Eq. = 1G " . . where m runs only over all heavy atoms." where a generalization of the isomorphous replacement method was devised to deal with this problem. .34) (6. (6. C. . Qinmt. .5. However. J. . as in a protein crystal.27) that while the observed intensity is 1 = (FF*). The final stages of this project were carried out in the laboratory of K. .32) (6. + b 2 ) . 1 = A: (6. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES equator.5. R .. n It is sufficient to find the values of the individual G . ) 2 + ( A .. which we can determine. the zerothorder Bessel function appears. which is only a small fraction of its peak value at this point.5.5. . 36.. + (B. only the sum of G 2 is observed. . The great wealth of detail in this photograph is readily apparent.
G. that the azimuthal angle 4 for a single heavy atom cannot be determined from a rotationally averaged diffraction pattern.5. say 1/(4 A).34) has only six terms in itthree A s and three Bs.34) is therefore the most accurate of the set. Bessel functions occurring on the same layer line differ in order by integer multiples of 49. Second. 15) that they remain zero until their argument is about equal to their order. Stubbs and R. The determination of heavyatom positions was fraught with diffic~lty. This means that Eq. so that the as and bs are subject to considerable error. J. Holmes. within a given radius of reciprocal space. because of the selection rule for the TMV helix.68 First. 192 . Only when cross derivatives involving two different heavy atoms are examined can a A 4 between the heavy atoms be calculated. The polynucleotide chain with its electrondense phosphorus atoms is the most prominent feature in the maps. Further. the additional constraint inherent in the smooth variation of the individual Gs was taken into account in a global way.~’ Consider. ‘’K. since they can be calculated from the known heavyatom positions.34) and (6. it must be emphasized that Eqs. In the actual TMV case seven heavyatom derivatives having five independent sites were used. The proof of all these elaborate methods lies in their ability to produce an interpretable and plausible electron density map. (1975). Acta Crystallogr.5.6. First. A minimum of six heavyatom derivatives.5. Sect. Thus. Given the radius of the virus rod. and U. (6. one can readily show that out to 4A resolution at most three Bessel functions contribute at any point along a layer line.35) are for the I s and Gs at one point in the diffraction pattern.35) as a constraint. In the technique actually used the heavyatom equations are solved using (6. (6. Further. Gallwitz. the others will all be very small.5. two convenient circumstances make this possible. and required great insight and care. All the as and bs are assumed to be known. one for each term. The 4Aresolution structure of TMV published in 197769makes clear the fundamental soundness of the method. Diamond. A A31. and proved very powerful in eliminating false solutions at certain points. it is characteristic of Bessel functions (Fig. They must be solved at a large set of finely spaced sample points to provide the desired information. C . is required to solve these equations. for example. Mandelkow. the difficulties in assigning accurate parameters to the heavy atoms are even greater in fiber diffraction than in protein crystallography. Solution of these equations is not trivial. E. only a limited number of Bessel functions can contribute to the diffraction pattern. Equation (6. What remains is to calculate the As and Bs. FIBERS 29 1 An additional equation like that for I’ arises for each heavyatom derivative. Stubbs.5. 709 (1975).. so that very few loworder J s can appear. J . Nuture (London) 254.5. The coat protein subunits ’’ G.
23. and in this area chain tracing was not possible. Schematic drawing of two adjacent TMV coat protein subunits. 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. (Reproduced from Stubbs P I dh3 with permission. R a n d 2 represent the radial and fiber axis directions in the structure. Fortunately.292 6. and these made possible the tracing of the protein chain through most of the subunit. near the outer 808. this portion of the molecule can be visualized in a closely related structure. The cylinders represent helical segments. termed the TMV disk.63with permission. The region shown contains RNA.) . For technical reasons the effective resolution of the map drops to about 5. has been FIG. (Reproduced from Stubbs Pt al.5 8. radius of the virus. Perspective drawing of sections of contoured electron density function of TMV. 22.) contain extensive stretches of ahelix. The interaction of the RNA with the protein is also shown. An intermediate in the assembly of TMV.
A schematic of this arrangement is shown in Fig. Erickson. In many 7 0 J. Butler. A section of the RNA binding site of TMV and a schematic drawing of the structure are shown in Figs. E.A. New York. The diffraction patterns they produce are also the rotational average of the onerod pattern. provides an interesting example of the f” amount of information that can be gained from a lowresolution study.5. and J. 14. ’These groups showed that the exterior of the tubules was char~ acterized by an array of 13 socalled protofilaments running parallel to the microtubule axis. Cell Sci. Klug. 6.6. Rosenbaum. 22 and 23.. 1976. but are much less rich than the pattern from TMV. J. ” R. An xray diffraction study of brain microtubules by Mandelkow et a. G .” Cold Spring Harbor Lab. Amos and A L. Sci. which readily form an up heterodimer. Pror. Additional refinement is now being carried out at 3. A. Cohen.3370 (1977). each staggered axially with respect to its neighbor by about 9 A. Stubbs.74. For the first time considerable detail of proteinnucleic acid interaction can be made out. Pollard. aand Ptubulin. transport of materials from one part of the cell to another.. 153 (1974).71Like TMV they are hollow rods. 24. and an improved version of the TMV model is expected shortly. Champness. eds. Looking at Fig.5.4 resolution (G. ’* . C. Cold Spring Harbor. Bloomer. personal communication). and C. 7 3 L. Cell Biol. P. The dimers readily polymerize into microtubules with increasing temperature. G. 60. Mandelkow.S. ~The outer ’ portion of the subunits are clearly seen in this structure. and A. “Cell Motility. T. Book C : Microtubules and Related Proteins. Acad. Microtubules have been studied by electron microscopy in the laboratories of K l ~ and ~ ~ g E r i ~ k s o n . We now consider in a crude and simple way how a microtubule diffracts x rays. The spacing between tubulin molecules within a protofilament is about 40 A. In the virus the R N A chain follows a complex path ranging from 35 to 50 A from the virus axis. U. Microtubules are built from two closely related monomers. and these all wrap around one segment of helix (termed the left radial) like a roll enclosing a hot dog. It also illustrates very useful interplay between electron microscopy and xray diffraction. and other motile properties of the cell. Goldman. J . P. Nature (London) 259. A . N. 74 H. 523 (1974). For microtubules this occurs at the inner and outer surface of the hollow rod. 24 one can see that there are many different helices which might be drawn to connect the tubulin monomers. Bricogne. Diffraction takes place where there are large fluctuations in electron density. Thomas. J.5. J. Klug. FIBERS 293 crystallized and analyzed in the laboratory of Aaron K l ~ g . Natl. Structure of Microtubules Microtubules are cellular organelles involved with cell division.20 ( 1976). J . There are three bases associated with each protein subunit.
The vcrtical protofilaments ( I 3start helices) and the 3start helices are drawn in. (Reproduced from Mandelkow Z I r d 7 ’ with permission.24.FIG. Each lattice point is represented by a black disk.) 294 . Surface lattice of the microbule.
~~ 295 . Slyrofoam modcl of reconstruction of microtubule. (b) view from outside of tubule. (a) View along tubule axis. (c) vicw froin insidc of tubule. Note that the highrelief helices are differenton theouterand inncrsurfaces. 25.) . (Reproduced from M a n d e l k o w ~ f n lwith permission.FIG.
For lack of a direct way to phase the reflections.lreduces approximately to two terms: a J . Such bimodal G. Reflections arising from the inner and outer walls of the microtubule were identified by their distance from the meridian. Inside. The investigators concluded that the inner and outer radii of the tubule were 70 and 150 A. 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. A reflection corresponding to the spacing between the 13 protofilaments was also observed. are indeed observed. XRAY DIFFRACTION ANALYSIS OF MACROMOLECULES cases several helical lines have to be drawn if all the subunits are to be included. since each problem in fiber diffraction is unique. have the same arrangement of subunits. Further. Because there is very little density fluctuation within the rod wall. electron microscopy. at least according to this preliminary model. or other arcane methods. respectively. it is not necessary that the same families be prominent both on the inner and outer surfaces. It was found that on the outside (Fig. 25) running at about 45” to the tubule axis. However.. additional progress on this structure can only come with the use of heavyatom derivatives. most problems are solved by resorting to specialcase reasoning. In some sense this is a hopeless task. The inside and the outside of the microtubule. 24 is a threestart helix.296 6. Next. using the ideas that went into the derivation of Eq.5..5. Clearly. It is these particular helices that will contribute most to the diffraction pattern.27) for the G. (6. so that the same families of helices can be drawn. which are themselves clearly divided into subunits with a strong modulation from a tenstart helix (Fig. for the inner tubule radius and a J . Also..22). 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. (with the same order) for the outer tubule radius. (6. the tenstart helices on the inside and outside are not in register. . 24) the predominant feature was the separation of protofilaments. which contains information about the projection of the tubule down its axis. of course.. Each line is said to be a “start. Rigorous demonstration of this would require a knowledge of the phase of the relevant reflections. the major feature is the tenstart helix: the protofilament separation and subdivision are very weak. each new problem illuminates and extends some part of helical diffraction theory. Eq. The analysis began by examining the equator. Extremely clever deductions from the diffraction patterns are a . Conclusion With these two examples we have tried to illustrate some of the scope and limitations of the fiber diffraction method.5.” so that the slanted family of lines in Fig. strong reflections on the first layer line were examined. 6.6.
it is actually possible to get the wrong answer in some cases. We find this area of XRD continually entertaining and intriguing. grant GM25432 (to L. lack of good specimens is a limiting factor in many cases. atomic information is difficult to obtain. Except for quite small structures.I.6. research career development award AI00271 and N. In addition. Acknowledgments This work was supported by N. and N.H. FIBERS 297 common feature of the literature.I.5.I. A few generalizations do hold. however. .H. Lattman). research grant AI14820 (to Eaton E. Mario Amzel).H. when very indirect reasoning is used.Furthermore.
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1). Our purpose is not to provide an exhaustive review of biomedical uses of laser light scattering but.2 we outline the basic theory by which laser inelastic light scattering phenomena are understood.3 contains a discussion of typical instrumentation and certain common experimental procedures. Not only do lasers provide intense. to familiarize the reader with various classes of applications. such as measurement of the diffusion coefficients of macromolecules. detection of changes in blood flow. The changes which are measured then can be correlated with molecular reorganizations of the sample. Chapter 7. Emphasis primarily is given to fluctuation spectroscopy.7. rather. Introduction Light is scattered when it passes through an optically heterogeneous material. determination of the electrophoretic mobilities of biological cells. Spatial variations in refractive index result in a diffraction pattern which fluctuates with changes in the structure of the scattering medium. and measurement of the elastic moduli of soft biological materials. Inc All rights 01 reproduLlion in m y form rcsxvrd ISBN 0124759629 . LASER LIGHT SCATTERING By Ralph Nossal 7. VOL. 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. 20 Copyright 0 1982 by Acddeniic Press. at a rate which depends upon the kinematics of the refractive index changes occurring within the sample (see Fig. Recently. assaying for the swimming speeds of motile microorganisms.1. essentially monochromatic sources. The remaining chapters are concerned with specific topics. but we also discuss some kindred aspects of classical light scattering techniques pertaining to the intensity of the scattered signal. some excellent review papers have appeared in which particular biological 299 METHODS OF IXPERIMENTAL PHYSICS. In Chapter 7. The availability of lasers has facilitated measurement and analysis of such fluctuating refractive index distributions. In so doing we hope to communicate some of the spirit of the physical modeling required to interpret measurements. The intensity of the scattered light falling upon a small area varies accordingly.
The latter is defined to lie between the axis of the laser beam and a line drawn ’ H . N. LASER LIGHT SCATTERING applications are described. 10. ‘. Z. 133 (1970). Bloomfield and T. for weak illumination. J. those whose polarization is perpendicular to the incident polarizationarise when the offdiagonal elements of the polarizability tensor are different from zero. 1974. Chen. Annu..300 7.. V.421 (1981). 371 (1977). Z. 2. C h ~ mScr. “Photon Correlation and Light SeatingSpcctroscopy. . Cummins and H . and we say that the scattering is “polarized. MethodsEnzymol. Z. Depolarized scattering is generally very much lower in intensity than polarized scattering. New York. Swinney. Carlson. and R.” “Depolarized” components of scattered lighti. 2. and Physics. R. Unless otherwise stated. we assume the scattering medium to be optically isotropic. A good bibliography is given in S ~ h u r r . 4. B. New York. B. however. 9b S. Pike. R. the electrons and nuclei within the material move oppositely to each other and produce an oscillating dipole field. 1976. M . Moore. New York. ed. New York. in “Chemical and Biological Applications of Lasers” (C.. ’ B.” Plenum.). with a field strength which is proportional to d2P/dt2. Biophys.Laser Light Scattering. t ) . “Scattering Techniques Applied to Supramolecular and Nonequilibrium Systems. 193 (1972). 1974. 4. Academic Press. ~ ’‘ ’ 7.” Academic Press.. Berne and R. F. V. Chu. In this case the plane of polarization of the scattered light is parallel to that of the incident field. H. 1977. Ford. H. Theory When a substance is illuminated by an electromagnetic field.243 (1975). Rei. New York. if the scattering medium is optically isotropic the offdiagonal dements of a are zero valued and the diagonal terms are equal. C R C Cri/. Bioclzem. Schurr. and thus has not been of much concern in biological applications of quasielastic light scattering. H. is proportional to the incident field Eo(r. Bioeng. Op/. L.” Wiley (Interscience).” Plenum. Nossdl. D. “Dynamic Light Scattering with Application to Chemistry. ihid. Jr. A. In addition. 48F. Bloomfield. Biology..2. t).e. Pike. Pecora. A n oscillating dipole is itself a source of electromagnetic radiation. Cummins and E. Chu. B. t ). eds. eds. and conference proceeding^''^ contain extensive discussions of some of the topics that follow. New York. 415 (1978). C. K. Reu. Prog.” Plenum. J.8. Ware. Cummins and E. ‘ ’ . several books7. The proportionality in general is described by a tensor polarizability a(r. Chaptcr 5. 1977. Lim. Vol. which. B. “Photon Correlation Spectroscopy and Velocimetry. This field is characterized by the macroscopic polarizability P(r.. R. 1981.eds. The intensity of scattered light is a strong function of the scattering angle 8.
57. reradiate electromagnetic waves which interfere with emissions from other scattering centers.  R .0. exp[ . Dubin. and the output is vertically polarized. (Each dipole in the sample reradiates energy as a spherical wave. but it is assumed that the detector is positioned sufficiently far from the sample that the resultant wave itself is spherical. B. Nu/[.A. In idealizations of the scattering process the incident electromagnetic wave usually is taken to be a plane wave Eo(r. the intensity of which fluctuates when thc scattering centers move. Greytak..g. B. Lunacek. Benedek and T. Dipoles are induced in the vertical plane. there is an additional field component which induces dipoles oriented in the horizontal plane. Tanford. "Physical Chemistry of Macromolecules. C k m . and G . we see that the detected electromagnetic ). is the locus of directions of equal scattering intensity. S. THEORY 30 1 Frc. lo I' .k * r)]. 1623 (1966). and if the detector is in the horizontal plane. t . U. the laser is operated in the TEM.i(u. which is proportional to sin cp.7. G. and the scattered wave is taken to be spherical. mode. 1961. Benedek. which are induced by the incident field. J .S. 1. The phototube views a small part of the resulting diffraction pattern. Dipoles.. Proc. I n this case the angles cp and 0 are related as cp = $7 . the factor sin cp is I for any scattering angle 8. Wiley. 40. 1." Chapter 5. for which sin cp = cos 0. H.) 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. 1604 (1964). ZEEE53. Schematic o f a typical light scal(ering experiment. from the phototube surface to the sample through a set of collimating pinholes in the collection optics (Fig. 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. t ) E. Sci. The inset shows the scattering from an individual dipole: I. T a n f ~ r d ' ~ Referring to Fig. If an unpolarized source is used.2. 1164 (1967). Pecora. Proc. l 3 C. Acud. J. New York. 1). Phys. B. e. Usually..
and t’ = t . P(r.2.) 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). “Electrodynamics of Continuous Media.” where n is the index of refraction of the scattering medium (c/n is the propagation speed of the emitted wave). Landau and E. M.14 this quantity is given as (7.” Academic Press. V is the volume of the effective illuminated region of the sample. Reading. t)E(r.r l/c is the “retarded time.2) where the scalar a(r. we restrict ourselves here to the RayleighGansDebye theory. New York. In other words.7 we discuss scattering from microorganisms and other large particles. Kerker. For a planewave incident field ’*“ ” I J L. 377. P(r. c is the speed of light. Van dc Hulst.” Wiley. 1957. AddisonWesley. Massachusetts. . 1960. According to classical electromagnetic theory. 1969. t ) may become quite complicated because of multiple scattering or absorption.n 1 R . “The Scattering OF Light and Other Electromagnetic Radiation. Because we here restrict ourselves to optically isotropic media.2. Lifshitz. “Light Scattering by Small Particles. D.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. New York. t ) can be written as P(r3t ) = a(r. 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. 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. (In Chapters 7. If the sample is optically dense.’ 3. the determination of E(r.” p. t). l 5 H. and I R 1 is the distance from the detector to the origin of the sample. t) is the polarization at point r. and indicate modifications which are necessary in such cases. C.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. t ) is the local electric field within the sample.5 and 7. ‘I’ M.302 7. (7. t ) is the polarizability and E(r.
ao(t) might be taken as the polarizability of the solvent and C(r. t 1 e i Q .5) and Eq. We also noted that the temporal change of the polarizability is slow in comparison with the oscillation of the electromagnetic field. ( R . (Note that 8 lies in the plane defined by the incident and scattered beamssee Fig. f) as the difference .2). THEORY 303 E = Eo exp[i(k . r d3r 2 M(~>S(Q>. i.r).i .k') r] d3r  (7.2. (7.3) becomes 1 a(r. From geometricarguments onecan show that themagnitude ofthe Braggscattering vector is given as IQI = Ik .2.2. (7.2. t ) FZ . 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. the polarizability can be expressed as a(r.oo ) ] we t find. 1.k' is known as the Bragg scattering vector. of course. t )expii[k * r ~ co ot 'y  '711 d3r N R x J.t ) o I V a(r. The quantity Q = k .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.2.r (. then the integral in Eq.4) where 6' is the scattering angle. (7. (7. exp[i(k' R .k'I = 21klsin)O = (4nn/A)sin)6'.2. so terms of O(d2ct/dt2) could be neglected.2.rl FZ k . 1 V%A3 where S(Q) is the Dirac 6 function. t ) = a(t). where A is the wavelength of the incident radiation.7.e. In this particular case scattering occurs only in the forward direction. (7. ordinary transmission.r E. t). I k' I z I k I = won/c = 2nn/A. t).1) and (7.) If the sample is optically homogeneous so that a(r. In general. from Eqs. t ) = ao(t) + i(r.. that R ):f x Eo sin cp exp( . For a suspension of particles.(R.2.coot)]  t ) exp[i(k .3) To derive the latter we substituted (won/c)lR .
if the particles are much smaller than A.5) that the scattered field is given as Es(R.304 7.Rwot) R c i aieiQ. (7. R . one has i where ri and Ei are.2. we implicitly assumed that the incident field is not appreciably disturbed as it passes through the sample and that multiple scattering can be ignored. the ensemble average E i E j eiQ'crir~)) simply the sum is E'. Also. if they do not interact with one another. If the particles are uniformly distributed throughout the scattering volume and. Therefore the scattering process is sensitive to the optical shape of the scatterers if particle dimensions are comparable to the wavelength of light.7) It is apparent from Eq.ii(r)eiQ'r d3r.2.7). In this case we find from Eqs.2. We then find E. (7. = w0/2nc. (7. (7.2. We find since A = V/L. Classical Light Scattering The intensity .2. also.(.2. 7.6) and (7.w o r ) scatterers c JQ. (7.2.1.r. respectively.3) and (7. LASER LICiHT SCATTERING between the solvent polarizability and that of the particles. (7.2.9) where N is the number of particles in the scattering volume. which.. For identical particles we thus have <xi cj xi I / I o = (167c4 sin' ~ J / A ~ R ~ ) ( E ) ~ N . for sufficiently long measurement times.a of an electromagnetic field is given as the square of the field amplitude9 = E*E.7) that the particles will be invisible if their index of refraction and that of the solvent are identical. t ) = (yr  Eo sin 4) &(k'.7) we considered that scattering caused by local variations in solvent density is insignificant and can be neglected. the position and excess polarizability of the ith scatterer.2.6) where Qi is the spatial extent (volume) of the ith scatterer. can be related to the ensemble average of the spatial distribution of scatterers according to Eq.2. Early light scattering techniques involved measurement of the time average of the intensity I = I E t E . however. To derive Eqs. . (7.(R. t ) = (y)2 f"  ___ e i ( k ' . 1.
2.2. Phys.7. the “ zaveraged ” molecular weightI3 is found. we should start with Eq.pM L4R2d (7. which generally is taken to be proportional to the mass distribution.Thus. the only angle dependence ” Is drp(r)eiQ” l2 (7.10) Since all quantities other than M that appear in Eq.9) we find Because p = N M / d . If the density of scatterers is sufficientlyhigh.6) rather than Eq. p ( r ) is the distribution of inducible dipoles within a particle.2. when particles are large. (7. (7. eiQ’crlrJ)) be expanded in can terms of virial coefficients.2. THEORY 305 Equation (7. (7. . the molecular weight of the scatterers can be ascertained by measuring the total intensity of scattered light.2.’3 First.1 = 4na. first.11) where the structure factor Y ( Q ) .defined as Y ( Q )= is sensitive to the shape of the scatterers. Chem. J . From Eq. we have 1 10 4n2 sin2 rpng(dn/dp):.7).10) in principle can be independently determined. where M is the molecular weight of a scatterer and d is Avogadro’s number.) Two important assumptions have been invoked which.. where p is the mass concentration of particles in grams per cubic centimeter. Zimm. (7. if relaxed. if no is the index of refraction of the solvent.10). For small particles. we note that the index of refraction of a medium n can be related to the volume polarizability a according to n2 . (7. 16.2. (7. for identical noninteracting randomly oriented particles.2.2. the analog of Eq. Consequently. interactions can be probed since the expectation C.2. We recall. imply modifications of Eq.(dn/dp)p = 4nNcl.I7 Second.12) B.9) is (xi (7. we have n ’  n: % 2n. that the particles were assumed to be noninteracting.9) can be put in a somewhat more familiar form by the following manipulations. Here. (If several sizes of particles are present in the sample. 1093 (1948).2. H.2.
2.. quasielastic light scattering is concerned with extracting information from the temporal behavior of the diffraction pattern. Lather. .. we can determine the autocorrelation function (.g. LASER LIGHT SCATTERING of the intensity is through the term sin2 cp.306 7. Classical light scattering from bacteria and other biological cells is discussed in papers by Wyatt * and Cross and Latimer. that the system is in a stationary state. by measuring the angle dependence of the scattered light. Consequently. a measure of the kinetic behavior of the scattering medium can be obtained. J . 183 (1973). 1225 (1972).e. Wyatt.2.000 daltons) can be studied with this technique. These time variations are related to local changes in refractive index of a sample. Assuming that the average properties of the sample remain constant. where those modifications of the basic theory which are peculiar to each case are indicated. 8. D. Appl. 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. l9 7. Intensity Fluctuation Spectroscopy We have just seen that classical light scattering techniques relate the intensity of scattered light to certain molecular properties. 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. and. Opt. repeat the measurement at some other times t’ and t‘ z. A. l 7 1 + cos’ 0. 11. consequently. monochromatic. for large particles additional angle dependence affects the intensity through the Q dependence of the structure factor. i. repeat the measurements at yet later times and add those results to the former. For didactic purposes we now consider how the motion of small particles (e. and add the product thus obtained to the first. By limiting the aperture of the collection optics. and. is given as l 3 . Method. divide by the + + + In l9 P. 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. The recent availability of amplitudestabilized laserssources of intense.7 Microhiol. protein molecules of molecular weight 80. finally. temporally and spatially coherent radiation makes such studies practicable. However. fluctuations in the intensity of the light that falls upon the detector can be discerned. Other applications are discussed below. In contrast. information on the size and structure of a particle can be determined. which. 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. Cross and P. when unpolarized light is used as the incident source. most importantly.
*. A : Gen.16) E.(t + z)EsC(t + T)).2. THEORY 307 number of additions to the sum. Plenum. t + 7)eiQ" d3r)* Jv a(r.. namely. Eq. Analysis of the joint expectation of four parameters generally is a difficult matter. and S. A .(t)E. E. ) (A3A4) + ( A 1 A 4 ) ( A 2 A 3 ) . The more often one repeats the measurement. E. Pike.). we can write for the autocorrelation function of the detected intensity W(Z) = (j(t). Z. Cardoso. t + T)eiQ" d 3 r . x (1" a(r. New York. Jakeman. Nucl. for a scattering process which obeys Gaussian statistics. t)eiQ" d3r li (7.14) ) The complicated expression given in Eq. Gen. it ( A i A 3 ) (AzA4) then follows that (AlAZA3A4) = ( A ./(t + t)) = (E. E. . p. Saleh and M. A .*. Thus.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.2. 4. Fortunately. 6. Phys.2. an important simplification occurs: If A . if the statistical behavior of the scattering assembly can be characterized as a Guussiun random process. A : Math. Pike. 517 (1971). Phys. (7. Phys.15) where io(Q) = O ' Ilv a(r. R. 2 2 B.7. R. J . . (7. the closer will this experimentally determined quantity be to a theoretical function whose properties derive from the stochastic characteristics of a representative sample.2. J . Considerable study has been given to the manner in which finite sampling effects the reliability of deduced kinetic paramet ers. eds.14) becomes + (7. (7.14) must next be related to some interesting material parameter via a physical model for the kinematics of the sample.(t)E.2. A z . Cummins and E. (7. A . 1897 (1973). in "Photon Correlation and Light Beating Spectroscopy" (H. F. Because the instantaneous intensity is the square of the field amplitude.3) and (7.2. 75. Swain. (7.2.2. A4 are Gaussian random variables. 1974.13) and from Eqs. . Jakeman.
t ) . z). 28. one situation for which it most likely holds is the case when refractive index fluctuations derive from movements of Brownian particles. the integral a(r. (7. that the scatterers are indeed undergoing Brownian motion. however. J. we shall see.2. t)eiQ'd3r)* f. z) = [io(Q)] (( Jv a(r. now. Of course.18) is required (see Chapter 7. Berne." Equation (7. 561 (1976). t + z) d3r (7. 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. if these conditions are not satisfied.2.Generally. additional features will be present in the scattered light ~ p e c t r u m .2. from Eq. is (7. + (aa/aC)aC(r. A : Math.17) The physical properties of a particular sample are related to acquired data through the quantity I l(Q. (7. Phys. t)eiQ"d 3 r can be expressed as t eiQ'ri(f). a generalization of the expression given by Eq. that for large scatterers. . Schaefer and B.308 7. Lett. when the scatterers are identical particles much smaller than the wavelength of the incident radiation. Gen.15) will hold. then by the law of large numbers the sum will be Gaussian distributed and the factorization indicated by Eq. Voss and J.1 ( E ) 2 ( x eiQ""" 1eiQ'rJct+'r) j \ (7. z) (the "intermediate scattering function") is defined as I l(Q. however. Rev. .a(r. However. t ) = a.2. Eq.7)]. thus.17)by assuming that the local polarizability a@.18') also could have been derived from Eq.2. W. + 24 D. F. (7. t ) = [io(Q)]." Suppose. The assumption of Gaussian statistics is oftentimes difficult to substantiate. . As before [cf.5). . (7. if scattering arises from ~~*~~ a large number of statistically independent random events.475 (1972) R. such as macromolecules in solution.2. 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. t ) is the "density fluctuation" and the expression within the angular brackets is the "densitydensity correlation function. Clarke. LASER LIGHT SCATTERING and I l(Q.2. J . note that the quantity i We eiQ"i(t) the Fourier transform of the density operator. for which intraparticle scattering must be considered.17) one has ) ci xi i I Z1(Q. 9.18) where hC(Q.2. Phys.
(7.2. (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. However. (b) inverse relaxation time r. 2a.. ( Q . Consider. 2. sin $0. (7. It has the form of a constant back. the diffusion coefficient would be determined from a representation of data such as that shown in Fig. I . plotted versus the square of the amplitude of the Bragg scattering vector Q = 4nni.19) where D is the translational diffusion coefficient. In such cases the ’.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.4. 2b.15) is often called homodyne scattering. The experimental situation depicted in Eqs. (7. 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.19) is shown in Fig. certain kinds of particle motion cannot be detected by this technique.13)(7. (7.2.. by measuring the autocorrelation at several scattering angles. for example.7.2. T) = e’Q2‘.18’) thus becomes I . FIG. As shown in Chapter 7. Eq.15) and (7. The photon autocorrelation function derived from Eqs.2.2.2. movements of particles with constant velocity V.2. ground term plus a simple exponential whose decay is given as z = (2DQ’)Thus.19). THEORY 309 The motions of noninteracting Brownian particles can be described by Fick’s diffusionequation.
15) may be invoked to simplify data analysis.as indicated by Eq.  + 7.(t).’ From Eq.so that Eq. then the analog of Eq.18) becomes I. (7. the autocorrelation function would not contain any information about the drift velocity of the scatterers. we note that heterodyne scattering can always be used to test whether the “Gaussian approximation” given by Eq. Indeed. Finally. t)I2 = Z:Zl = ( t i ) 4 N 2 / [ i O ( Q ) ] 2 .2.3. ..*.20) Consequently.*. z) = + + T) [io(Q)]’(E)2N ei(Q’v)r.13) is g(z) x ( ( E : o E ~ o ) ~ + 2(E&JL0) > Re(E.15).2.1.(t)E..3. 3. (7.. .20) we see that Re<E. I Z 1 ( Q ..2. (7. Suppose we designate this unscattered field as ELO(t) = ELOei(mot+q). .(t)E.(t z)) cos(Q * VT).2.”) If we assume that an experiment is designed so the amplitude of ELO much greater than that of is the scattered field E. Instrumentation and General Techniques 7. so W(T) in this case does indeed provide information on the drift of the scatterers. 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.(t + z)) + . (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.t ) as 1 rw (7. (7.3. since the information contained in the frequency distribution of a timevarying signal is equivalent to that which can be extracted from autocorrelation. the power spectrum of the scattered light S(Q. o)is related to the autocorrelation function V(Q.2. However. that the total field at the so phototube surface is E(t) = ELO(t)+ E. by the WienerKhintchine theorem.(Q.310 7.2. (7. we could as well have considered spectral analysis of that signal.21) where the righthand side of the equation also contains a negligible “homodyne” component.. Relationship between Photon Autocorrelation and Spectral Analysis The previous section emphasized autocorrelation techniques for analyzing the fluctuating intensity of scattered light.1) The equivalent nature of these functions is illustrated in Fig. (7. (7.2. However. 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.
2.3. I).3.3. (7.3. The average number of detected photons is proportional to the intensity of light falling upon the photodeteclor. e. If the photon autocorrelation function is exponentially decaying. (7.g.4.. 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(01.15)].1) are shown in Fig.2) The frequency width of this function contains the same information as does the decay of the autocorrelation function. the related spectral power [see density is Lorentzian. (b) subcutaneous blood flow or randomly swimming microorganisms. and (c) particles drifting with uniform velocity. 4.3.2. INSTRUMENTATION AND GENERAL TECHNIQUES 31 1 ? FIG. t ) e2DQ2z Eqs. Processes whose autocorrelations (a) (b) (C) FIG.7. I f ( Q . Examples of the transformation effected by Eq. (7. .  (7. Typical results for (a) diffusing macromolecules. Correlation functions [Il(r)'] and spectra [S(w)] are related according to Eq.19) and (7.
the signal in the first (zeroth) channel of an autocorrelator is blocked or neglected and the shotnoise term e( i)6(z) ignored. 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.(T)= e(i)h(z) (i)2gz(z).2. an instrumental autocorrelator necessarily samples the scattered photon field over discrete intervals of time. One really does not directly analyze the field intensity when an autocorrelation analysis is performed on the scattered light.1 In practice however.. Similarly. manipulations are made on the photocurrent emitted by the detector (Fig.3. 7.2.Ar/Z + z + s’) ds’ ) + z + s’)ds’ ). [gz(T) is related to the autocorrelation of photopulses n(z) as + S A T ) = <n(t)n(t + ~)>l(n(t>>2.312 7. .(T) is given as1 C. 3).2.6) and standingwave oscillations of soft gels (Chapter 7. Rather. if the scattered signal is very weak or the time scale of the fluctuations very fast. Characteristics of Measured Autocorrelation Functions Although theoretical analyses based on Eq.13) pertain to continuous signals. i. lo)].e. where (i)’g2(z) is proportional to the quantity given by Eq. the correlated components really are integrated functions.9)]. when spectral analysis is performed the shotnoise term provides a constant background which is subtracted from the remaining signal. (7. %&) =(A212 1 (J Ar/Z n(t Ar/z + s) ds J Ar/Z n(t Ar/Z . (7. Therefore. 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.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.3.14).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. also.7) and subcutaneous blood flow (Chapter 7. efficient collection and processing of primary data presently require photon autocorrelation techniques. the expression given in Eq. Also. (7.2. Thus the photocurrent autocorrelation function C.3) 1 ‘v (1 AriZ Z(t Ar/Z + s)ds J Z(t . (7.
Applications involving low count rates and short correlation times.e. if the pinholes through which the sample is viewed by the phototube are very large.2. An exhaustive analysis of the ways in which p depends on various instrumental parameters has been performed by Jakeman and associates. The longer one accumulates a signal.3 below) and coherence “losses” at the photodetector surface. The correlated signal actually appears as U(Q. some of the information contained in that pattern is lost. Oliver. theoreticai and experimental analysesz5 indicate that an accuracy of approximately 1 % can be achieved for quite reasonable experimental conditions (e.. L45 (1970)..15) also is but an idealization. ’’ E.. however. In the case of diffusion coefficient measurements of monodisperse samples.6).3.4) where p < 1.3. even though the diffraction pattern of the scattered light fluctuates. seem to be most successful when spectrum analyzers are employed (see Chapter 7. Phys. the greater will be the accuracy of the parameters that can be extracted from the data. using a 50mW HeHe laser).3. . the fidelity of g(Q. Indeed. where scatterers move in preferred directions. Jakeman.3. A : Gcn.e. p w 0. The latter arise because the scattering volume “seen” by the photodetector is of finite size so that.2 but such considerations are beyond the scope of this introductory article. 3. measurement times less than 2 min for a sample composed of 5 mg/ml haemocyanin.3. The choice of instrumentation largely depends on the uses anticipated for the spectrometer. z) also depends on the count rate (i. (7. . R Pike. such as measurements of the diffusion coefficients of small proteins. N (1 + BCIi(Q. Of course. i. 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. Equipment Data can be processed either with a spectrum analyzer or with a pulse autocorrelator. J . and E. C.I. and sample concentration are changed.g. (7. INSTRUMENTATION AND GENERAL TECHNIQUES 313 The factorization gz = 1 + f: as shown by Eq. the total number of scattering events). channel width. scattering angle. 7. 2)12).20. Phys. The proportionality term fi accounts for statistical errors introduced by clipping (see Section 7. are best done in a photon counting mode utilizing autocorrelation analysis. Velocimetry experiments. 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 .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.
Jakeman. and C . 44. One.3.. Instrum. 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. and theory of such devices has been described by Pike and his associatesz6 and Chen et al.27 Later. if the average count rate is low (e. Oliver. the ability to simultaneously accumulate correlation functions at different sample times. Rer. R . R . and an option for up to 1096 channels for resolution of the correlation function. a clipping level of 2 would produce a “ 1” if the number of counts in the interval is 3 or greater). J . *’ S. R E P .Sci.g. Appl. 506 (1973). that of a “singleclipped’’ autocorrelator. 51. including a builtin display. the actual number of delayed counts n(t . operation. 3197 (1978). Several firms manufacture excellent realtime spectrum analyzers. Foord. Opr. of clipped autocorrelators. R . 1356 (1975). Instrum. C. Wood. In some laboratory installations the correlator output is fed directly to an electronic calculator for immediate manipulation and storage. depending on whether a preset level is exceeded (e. essentially performs the operation indicated by Eq. California) is producing a spectrometer incorporating a clipped autocorrelator which contains a microcomputer dedicated to diffusion coefficient determinations. Clearly.g.Sci. it has many of the attributes. Veldkamp. Ref. Nicomp Instruments (Santa Barbara. Malvern Scientific Corporation (Ronkonkoma. Yangosand S. B. J . several highquality commercial instruments now are available. P. less than 0. 2 8 R. LangleyFord Instruments (Amherst. and A. Chen. Blagrove. E. i. C. Ford.j AT)is replaced by either 0 or 1. 242 (1970). 46. LASER LIGHT SCATTERING Two kinds of correlators have been used for photon counting applications. the autocorrelations formed by a clipped autocorrelator operating at zero clipping level will be virtually identical with those formed by a full autocorrelator. H.3). it has many desirable features.e. Asch and N. E. Holz and S. J . Pike. Peacocke. Chen. it repeatedly performs multiplications of the actual number of counts appearing in the various time intervals. H. Sci. such as short sampling times and moderate cost. 17.. C.314 7. New York) markets similar signal correlators. Chen.. (7. Ntrtitre (London) 227.5 pulses per interval AT). H. z9 M. a multiplicity of interfacing options. The construction. In the other case. Early R. 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. a “full” autocorrelator..29 Although an investigator previously had to build his own correlator. as well as a complete laser photon counting spectrometer. Instrum. W. 344(1980). E.. Lai. 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. *’ . Jr.
in many instances high power can be a nuisance.g.67.586 (1975) .7. Argon ion sources..9). (7. The laser must have good amplitude and beam direction stability. however. INSTRUMENTATION AND GENERAL TECHNIQUES 315 efforts to use laser light beating spectroscopy relied upon sweptfrequency analyzers which were slow and cumbersome. Flynn.7). Nicolet Scientific Corporation (Northvale. and should have a polarized output. Instrum. Sci.4 Good temperature control for the sample is usually advisable.2. various pulse shapers and/or amplifiers are used to interface the photodetector and analyzer (see. New Jersey) and Princeton Applied Research (Princeton. However. Rev.3. The design of the sample holder or cuvette generally depends on the anticipated application. The situation is now greatly improved. emits narrow pulses. 3o Inexpensive photodiodes can be used instead of phototubes for certain applications involving high signal levels and slow fluctuations (see Chapter 7.10). e. Depending on the particular instrumental configuration. Ware4).10)] seem to be favored for research applications where low concentrations of small macromolecules are being probed. and for many applications spectrum analyzers are at least as convenient to use as are correlators (see Chapter 7. the detected signal must arise from only a few “coherence areas”’).e. ” J . New Jersey) currently offer suitable instruments. A variety of HeNe and argon ion gas lasers have been used. S. Electrophoresis experiments (see Chapter 7. Lowlightlevel photon counting applications require a phototube which has fast response time. Gethner and G . Lowpower HeNe lasers are quite adequate for scattering from structures such as biological cells and tissue (Chapters 7. Several tubes are available which have such characteristics.6) require particularly sophisticated engineering skills. causing heating artifacts or providing signals which overload the detectors and analyzers unless attenuated with filters.. 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. 46. because of their generally higher power and shorter emission wavelengths [scattering intensity varies as see Eq. W. the performance of which has been reviewed in a paper by Gethner and Flynn. and has a low probability of producing afterpulses. as is vibration isolation. 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. The detector optics must be designed so that adjustments can be made to delineate the region of the sample which is being observed.
2) where k is Boltzmann’s constant. Westhead. information about the size and shape of a particle can be deduced. if we subtract a constant term Co and define 6C = C . B. The results of such a measurement can then be used to obtain other information. such as indications of changes in molecular conformation or of modified interactions between various constituent subunits. 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. t)/& = DV2C(r.4. t ) / & = DQ26C(Q. Once D has been determined. The derivation of that expression follows directly from Eq. For example.2) would be a proper expression for analyzing an experiment even if the bare particle were not spherically symmetric. and N . However. so that Eq. Benedek.2..1) is the translational diffusion coefficient.18’) and the diffusion equation for the time dependence of the concentration of particles C(r. O)SC(Q. ’~ 3’ 32 R. if the scatterers are grossly asymmetric a different model is necessary. O)e’Q2‘.2. in particular. (7. (7. we find. t). LASER LIGHT SCATTERING 7. 327). R. and a the effective particle radius. C.g.1) The coefficient D which appears in the exponent of Eq. Ford. (7. JsC(Q. t). the diffusion coefficient for ellipsoidal particles is given by the Perrin equations (see e. The effective particle size may be somewhat larger than that of a “bare” molecule if solvent is dragged along with the moving scatterer. Immunochemi. (7. X ( r .4. T is the temperature.Ptrj7 12. 14. t ) = SC(Q.4. T a n f ~ r d .19). Gabler. J . .316 7.32 ~ If a sample contains identical (monodisperse) Brownian particles. D = kT/6nqa. (7.4. 349 (1975). if the scatterers are spherically symmetric the particle radius can be determined from Stokes’s law. Cohen and G. t). The solution is SC(Q.namely. B. t ) is given by the expression (7. from which an expression for the densitydensity correlation function then follows as (SC*(Q. after taking spatial Fourier transforms. E. ie. p. Thus.. Biophys. 528 (1974).Co.4. W. the intermediate scattering function ll(Q. y~ the viscosity. 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. t ) ) = ((SC)2)e’Q2‘. Another effect of solvent association might be to smooth out the shape of a nonspherical particle.
Because the intensity varies as the square of the mass. 13. 48 14 (1972). One procedure which can be used to analyze data from polydisperse samples is the “method of cumulants. (7. Biophy. G(T) dT = 1.4.s/r~~ 960 (1974).’ J P‘(u)[ii(a)]’ eD(a)Q2r da. ribosomes. and phospholipid vesicles.3) is derived in the Chapter 7. d dz r)2G(r)dT = d2 dz (7.7. P/iys. =  I I(r . Biop/iI. a practical lower limit on the size of the scatterers is presently 20003000 daltons.s. M.. 57. 29. Koppel.hrwi. J 3Sa J.34 and small bacteria have been studied. ~If ~ I . the radii of which fall within the range 15 A 5 a 5 lo3 A. the analog of Eq.3).(t) = f G ( r ) K r r d r with l.33 vesicles of sarcoplasmic reticulum. then : the moments of G ( r ) are related to the time derivatives of Z. ( T ) ] ~ = ~etc. 505 (1978). ’’ ’’ . D. 337 (1976). and R. if the particles are small and. [A generalization of Eq.4.3) where io(Q) z . J . J . Bioph.t ) for a mixed collection of particles.2. D. (7. CC can be taken to vary directly with the mass of a particle (frequently a good approximation). Yeh.1 If.(z) as f = M . N. (7. such as micro tubule^^^ and actin filament^.19) is I1(Q. Carlson. J . 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). Franklin.4’P(a>[E(u)]’ and b ( u ) is the relative density of scatterers du having radius a. CameriniOtero. C. if it can be assumed that the density of scatterers is so low that interactions between particles are rare. . J .2. 16. Rioc. 37 (1980). i. J. E.z) = [ i o ( Q ) ] . DIFFUSION COEFFICIENTS 317 Particles of many differing types can be studied.[ln I . However.^^" Complexities occur if the scatterers are not all of the same size. Measurements are frequently performed on particles such as proteins. J . and R. (7. To compute the intermediate scattering function I1(Q.4. the size of small peptides. D.4. S. Pusey. Newman and F.ys.. also. The situation for large scatterers is complicated by the fact that intraparticle interference requires integration over the contours of the particles.e. = rc(r)dr= M. R. . (7. Gaskin. Y. ( T ) ] ~ = ~ .~. E. D. Schaefer. Uicm. Baskin.. P. 24. Gcthner and F.4. W. let us again refer to Eq. D. Selser.17).5.4) [ln I . Koppel. then a scatterer contributes to the scattering intensity in proportion to the square of its molecular weight. Viruses. Considerable attention has recently been given to studying large spatially anisotropic molecular assemblies. in Eq.” first described by K 0 p p e 1 . t) is represented as I. ( Q .
if intensity fluctuation measurements are combined with conventional light scattering measurements. (7. it is often more effective to fit the data to a weighted sum of exponentials. lrnmunoassay in the agglutination inhibition mode. 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 ~ / . where pi is the weight concentration of scatterers.) Ab free Ag FIG. parameter determinations may be facilitated. Equation (7. ~ * ) . if the particles scatter light in proportion to their molecular weight. the first derivative of Il(Q. Generally no more than a few (two or three) exponentials can be used to fit data if parameters are to be unambiguously determined. D could be represented as B = piMiDi& p i M i . in which case data have to be fit to a weighted set of Lorentzians. Specific antibody is used to "crosslink" antigencoated ( A g ) spheres. particularly if the time scales for the exponential decays pertaining to sample and dust are widely separated. z) in the limit z 0 is proportional to an average diffusion coefficient 6. The ratio M . An analysis of errors expected in values of D is given in Koppel's paper.4.5) If the molecular weights of the species are known. which.4. In this case Eq. since r = DQ2. 5. such as protein dimerizations or polypeptide helixcoil transitions. LASER LIGHT SCATTERING f Thus.318 7. that is.4. . however. / M : is a measure of the relative width of the distribution. (Similar considerations arise when frequency analysis is used.3) becomes xi (7.36 If only a few particle species are present in the sample. is a zaveraged quantity. the relative concentration of particles can be determined and one can deduce the equilibrium constant for simple reactions.5) is also quite useful for analyzing data from samples which contain a small amount of residual dust or other largesized contaminant.
. K.963 (1976). First. ” G. by which we mean correlated scattering from several points within a particle. K. 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. R. 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. B.5. and G. and in certain instances can approach the sensitivity of radioimmune assays. Second. i f I 5 2(n .g. Beiiedek.. Cohen. 5).5. if the particles are not spherical the Q dependence of the relaxation times of the autocorrelation function may differ from that of spherical particles.nO)AK1L < 1.” In recent years attention has increasingly been given to similar inelastic light scattering situations.e. Intraparticle interference. The effects of size have been of concern ever since light scattering was proposed as a spectroscopic technique almost 100 years ago.7. but only a few special cases have thus far been analyzed in detail. Y55 (1976). i.’6 is permissible. N. Benedek. B. and G . even when all scatterers are of identical size and shape. R.37The assay can detect very small amounts of antigen. LARGE PARTICLES 319 An interesting and potentially important application of this technique is a laser light scattering immunoassay for detecting antigenantibody interactions. J .. i. . fmmunoc~humisrry 13.32.e. Changes in B are used as an indication of the agglutination of antibodycoated latex microspheres by polyvalent antigen. Excellent discussions of classical light scattering from large particles may be found in the wellknown books written by KerkerI6 and Van de Hulst. voii Schulthess. This condition is satisfied if the phase shift experienced by a plane wave transversing the scatterer is small. 7. 3 8 G. 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. 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. J. e. that the presence of other scattering centers can be neglected in considering scattering from any particular center. of the order of a few microns or less (10°K) is easily effected if the socalled RayleighGansDebye approximation’s. can have two effects. von Schulthess. A generalization to the case of particles whose sizes are. 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. Cohen. fmniunochemisfry 13. Sakato.
t ) can be rewritten as E. A useful transformation to a bodyfixed coordinate system can be accomplished by writing r = R(t) + p.(R. LASER LIGHT SCATTERING where FI . 6). (7. .1) where Q ( t ) indicates that the integration is to be performed over the volume and orientation of the particles. (7. t )  eiQ'R(*)d 3 p ~ ( p t)e'Q'P.6 .2) where P(p.' If the RayleighGansDebye approximation holds. / FIG. 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.6) that the scattered field may be written as E.2.3 20 7. L is the maximum dimension of the particle. Thus the expression for E. t ) is a weighted distribution of scattering centers.5.(R. Transformation to a bodyfixed coordinate system.no is the difference between the refractive index of the particle and that of the solvent.(R. we see from Eq. J (7. In general. . which for optically homogeneous scatterers is simply proportional to the mass distribution within a particle. however.5. t )  1 particles i(r)eiQ'r d3r. scattering from a large complex particle can be evaluated only by resorting to the solution of Maxwell's equations. and A is the wavelength of light.
however. and Z1(Q. LARGE PARTICLES 32 1 Let us here assume that the scatterers are identical and noninteracting. Orientation and translation are strongly correlated when flagellated bacteria such as E .(Q.t ) Y(Q)(eiQ’ARc‘)). t)eiQ’P implicitly depends on the spatial orientation of a particle because of the term eiQ‘Pin the integrand. t)eiQ’P’ eiQ‘P. (7. p.3).5.Qa cos Qa)}’ Qa (spheres). we now consider the diffusion of an assembly of large solid spheres. Then the intermediate scattering function Z1(Q.5. (7. If.5. if its translational motion is correlated with its spatial orientation. t ) [cf. i.5.5) .2. the particles have spherical symmetry.) is the “dynamical structure pol factor. Eqs. p t ) + Y(Q). A similar conclusion holds whatever scheme is used to compute the electromagnetic fields within the scatterer. the time dependence of axial reorientation will affect the photon autocorrelation function.4) where pLtis the cosine of the angle between the scattering vector Q and the internal reference axis of a particle. In a heterogeneous assembly the Q dependence of the structure factor can affect the form of I1(Q. 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 7.5. the dynamical structure factor depends only on the magnitude of Q and not on particle orientation.5. if a particle is nonspherical and. (7.5. These last comments hold only if all particles are the same size.e.7. This perhaps is clearer if we write Eq.” given by the integrals within expression (7. (7.4) is difficult. . Note that Q generally is fixed by the geometrical relationship between the source and the detector. Y(Q. so the computation of the expression given in Eq. so d3p P(p. These organisms are cigar shaped and have internal structure. For example. coli locomote.7). (7. Y ( Qp o . It is easy to show” that in the RayleighDebyeGans limit. Hence the particle structure in this instance would affect the intensity of the light scattered at a particular angle.3) as (7. t) ( eiQ’AR(t) p’ d3 / 1 d3 p P(p’..2. 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. Thus. but not the Q dependence of the decay.3) ) where AR(t) is the displacement of the center of mass at time t. Y(Q) is given as  Y(Q) = {[3/(Q~)~](sin .18)] can be written as I. further. O)P(p. t).15)(7.
34 Chen et ~ 1 have derived a similar expression for coated . T. the relative intensity of the scattering from a 0. Chrzeszczyk. Jr.6) In the limit that a is small. Y ( Q a )decreases markedly for large values of Qa. 503 (1977). t + + (7.5. . consequently.) = Bo(QL)eQZD' B1(QL)e(Q'D+6err . Thus. P e ~ o r a ~ ~ was the first to recognize that.(Q.5. and S. 48. 187 (1977). Chum. the intensity varies by only 15 % over that range.1) are carried out. Lipids 13. Chen. ~ ~ ellipsoids and have investigated relationships for dynamic light scattering which arise when the integrations indicated by Eq. Chu. Kunze. The structure factor for spherical shells has been derived by Tinker3' and by Pecora and Aragon4': Y(Q> {[3/Q3(a3 a:)] (sin Qa . artifactual scattering arising from dust contaminants in a sample can in part be eliminated. thin molecules would contribute features to the scattered light spectrum from which information about the rotational diffusion coefficients of the scatterers could be extracted. Phys. R. "R .3) is Z. K. J (7. . 4 I26 (1 968). 1 (1974). 4" 4' 42 R . since large particles usually move more slowly than small particles. P h p .4.05pm particle. Opt. 64. Lipids 8. Day. Y ( Q a ) + 1 and the expression given in Eq. Binelrim. the signal from larger particles will be relatively less significant as the scattering angle is increased .3) is recovered. J . For example. in contrast. T) = [io(Q)]' [ 9 ( a ) Y ( Q ~ ) [ t i ( a ) eDca)Q2r ]~ da. Thus the analog of Eq. C h m .5pm particle decreases by a factor of almost 300 as the scattering angle is increased from 20" to 90". for a heterogeneously sized collection of scatterers.5. 0.322 7. Appl. R. (7. however. Pecora and S.8) '' D. for a 0. 4 J S. Tartaglia. Phys. The situation for rodlike scatterers is more complicated. (7.Qa cos Qa =  + Qai cos Q U ~ ) } ~ (shells). F. and B. which was derived for rigid. C. Acta 470. J.sin Qai . Chem.Tinker. (7. Holz. A. Churn. Ardgon. . LASER LIGHT SCATTERlNG where a is the radius.5. reorientations of long. Chen. ~ ' and membraneous structures such as particles formed from sarcoplasmic reticulum. E.4. H . Sun. is Zl(Q. Phys.3403 (1976). 16. . Ho. P. 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.. (7.7) where a is the outside radius of the shell and ai is its inside radius. M . under certain circumstances. This expression is important in analysis of the scattering from phospholipid ~ e s i c l e s ~ ~ . when a sample is illuminated with light from a HeNe laser (red). T. Pecora's expression. and P. Bioplys. J . 230 (1972). Pecora. vanishingly thin rods of length L.
4s 46 . flexible molecules are e ~ a r n i n e d . Caloin. Mol. 68. If the correlation times for translation and rotation are comparable. However. 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. ~ scattering techniques to measure the rotational diffusion coefficient of tobacco mosaic virus. ~ ~ H.5. K. I. Macromolecules 6. Eiopolymers 16. Herbert. 18. : In addition to the requirement that the scatterers be rigid. Carlson. LARGE PARTICLES 323 where B.9) and (7. sizes of the order of several microns). B i d . S. the length of which is approximately 3000& and for which diffusion coefficients are found to be4’ D N 2. S. Schmitz. and S. 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. 4 8 S. Z. Fujime. Schurr. Lee. 9. 2091 (1977).The . Fujime and M. and S. in which case a single exponential characterizes the autocorrelation function. Murayama. Biophys. B. Additional features in the autocorrelation function may arise from molecular configurational fluctuations if the scatterers are not rigid. 4 7 S. information about 0 can be obtained.5. 49 M . 237 (1973). Cummins et ~ 1 used light ~ .g. Second. C. D. First. 347 (1972). Opt. the intensities associated with higherorder terms in the expansion are quite significant.518 (1969) M .(x) and B . is the spherical Bessel function (of the second kind) of order 1. H. Wilhelm. M. Murayama. F. term if QL is less than 3. 583 (1976). Kotlarchyk.5.7. Woods.8). Asakura.8 x cm2 sec’ and 0 1 320 sec’. J. T. Asano. for large scattering angles and particle lengths (e. J . J .. Chen. if correlated with particle orientation. Appl. S. and G . Internal modes of motion such a stretching and bending may thus be discernible when long. Biopolymrrs 16.8). (7.. such that QL > 5). and M. several other assumptions were made to obtain expression (7.g. Cummins. Daune. and J. 2470 (1979). M. Lin. ( x ) are defined as (7.5. ‘I’ W. leads to deviations from the simple exponential decay given in the first term of Eq. it was assumed that particle orientation and direction of translational diffusion are uncorrelated.10) where j . Almost all of the scattered intensity is represented by the B.5.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 .
the measured ) photon autocorrelation function V ( T is related to the square of the densitydensity correlation function I SC*(Q.324 7.6.2. order to extract information In 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.V * J.1) When the effects of boundaries are neglected.2).2. We thus have aclat = D V ~ C # E . Q ) * . In a homodyne experiment. is the fluctuation of scatterers about an equilibrium value C.18‘)].6. where E is the electric field acting upon the particles and u is the electrophoretic mobility. A kinetic equation to describe movement of particles in an electric field can be obtained from the continuity equation aC/& = . with the current J now taken as the sum of a “diffusion current” J./at = 0. O)SC(Q. t ) will have special characteristics. 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. as shown in Eq. namely. were an electrophoresis experiment to be performed in this configuration.(Q.Re (SC*(Q. the scattering function Z.( D Q 2 + i u E .6.C. the solution of the above equation is (jC(Q. In this situation the spectrometer is operated in a heterodyne mode and.6. the useful signal is then given as )’  Il(Q. (7.2) where 6C = C . t ) = SC(Q.  (7. If. in which only components of scattered light are mixed on the surface of the photodetector (see Chapter 7. Thus.3) where vE is defined as VE = U E .4) Analogous expressions are realized for other processes in which drift is .2. = uEC. (7. O ) e . we would obtain the result normally seen for diffusing particles. (7. O)SC(Q.Determination of Electrophoretic MobiIit ies In the preceding chapters we have assumed that the translational movements of the scatterers were spatially isotropic. the scatterers move preferentially in certain directions.21).DVC and a drift term J. V(T) 1 + fle2DQ2r. however. LASER LIGHT SCATTERING 7.t ) )  eDQ2r cos VF * Qt. t ) [see Eqs.15) and (7. (7.vc. which satisfies aC.6.t ) . (7. = .
5. Weiner. A(. In fact. Furthermore. Op/.5) Note that as the diffusion coefficient decreases so does the spectral width.s 53 R.53 Recent applications of electrophoretic light scattering include an assay for antigenantibody reactions. Ph. were the scatterers to have identical surface charge the spectrum would appear as a sharp spike (cf. Fig. However. I<. It also might cause electrode polarizations. the chemotactic response of ba~teria~lsee Chapter 7. a constant applied electric field can cause a sample to overheat. In this case the relative height of the spectrum at a given frequency o.3). '' D. 304a (1979). ~ w m w dMrt/iods 10. P w c Ntrtl. B ~ ( J ~ / ~JJ. although we implicitly assumed that the electric field is time independent.= Q * vE.6. Cambridge. The spectrum of scattered light would be given as a Lorentzian shifted from the origin by an amount o.'^ 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 . Uzgiris. i. Smith.. 2388 (1976). Cheii. Siege1 and B. 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. A .e. Several other studies of cell surface charge are discussed in Smith. U .D. Cornrnuri. R. Smith. Sci. for large particles such as biological cells. a n d K. the particles in a sample generally do not all have identical charge on their surfaces. . '25.. the scattering cell and electrodes must be carefully designed in order to obtain a determinable electric field in the scattering region. E.7. For example. the autocorrelation function would be given by Eq. DETERMINATIONOF ELECTROPHORETIC MOBILITIES 325 superimposed upon spatially isotropic motion ( e g . A. Massachusetts (1977). 85 (1976). Harvard University. Nossal and S.54 and a study of the fusion of secretory vesicles with membrane receptor^. (7.= Q vE. Thesis.7). J . H . ~ Certain difficulties not present in other applications must be resolved when engineering an electrophoretic light scattering experiment. a specially designed pulse generator must be used to cycle the electric field in coordination with data accumulation by the spectrum analyzer.6.73. I17 (1972). . A . E. If a collection of diffusing particles all were to have the same drift velocity. nor are they all of the same size. . though. and the sample holder 5' '' B.is directly proportional to the intensity of light scattered by particles whose electrophoretic mobilities are consistent with o. resulting in convection. R . B. Thus. 4c).6. Warc.[ild.S. (7. 7). P. Extraction of electrophoretic parameters from the data can be somewhat complicated if several Lorentzians overlap. S .  S(O)  1/[(0  Q * v E ) ~+ ( D Q 2 ) 2 ] . Ware. if diffusion can be ignored the entire distribution of electrophoretic mobilities of a sample can in principle be determined quite easily. Consequently.
t). and the organisms' velocities seem to be essentially constant over similarly long distances (see Fig. the entire distribution of swimming speeds of a sample can be deduced. if measurements are made on protein solutions. d o not interact. We first assume that the scatterers are homogeneous in size. and. the diffraction pattern that results fluctuates at a rate dependent upon the swimming speeds of the scatterers. Finally. Motility Measurements When a suspension of motile microorganisms is illuminated by laser light. the implication of which is that the rootmeansquare (rms) speed or. at which times changes in specd or direction take place. in which case one runs the risk that particles will be leached off the surfaces of the electrodes and contribute an artificial signal.7. 8).7. and are spherically symmetric insofar as their light scattering properties . Electrophoretic light scattering spectrum of mononitclear cells obtained lrotii the blood of lcukcniic patients.53 7. In many cases the tracks of motile microorganisms can be represented as essentially straight lines with intermittent stops. wave front matching to accomplish adequate signal heterodyning must be done carefully. very small ( 5 5") scattering angles must be employed in order to reduce spectral broadening relative to the frequency shifts arising from electrophoretic drift. (Adapted from Stnitti should be cooled to effect adequate temperature control. These straightline trajectories occur over distances generally much greater than a length which is the inverse of the Bragg scattering vector Q . LASER LIGHT SCATTERING FIG. These considerations are discussed at length in Smith's thesis. in some circumstances.326 7. Certain applications require high field strengths. In this instance a relatively simple model can be used to compute I1(Q.
(7. For motile bacteria. 1 . 15 (1978). are concerned. For the model illustrated in Fig. where V is constant. MOTILITY MEASUREMENTS 327 FIG. movements during the rest period are in fact generated by the microorganism itself.2) results from an assumption that during locomotion the trajectories are given simply as r(t + z) = r(t) Vz.8. H.7. 8 it follows from Eq.7. (7. however. Chen. an analysis of the twiddle movements predicts functions which are similar and in good agreement with data. 500 (1972). The second term follows if we assume that movement occurs only by diffusion during the resting phase.t ) is given as (7. The first term in expression Eq.7. Brown.a)eD"*'. Approximate pattern oi' the trajectories of motile microorganisms. 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. Berg and D.57 + '' M . (7.7.2) where LY is the fraction of the population of microorganisms locomoting at the instant that the sample is probed.7. Biophys.1) hi LY(eiQ'vr ) + (1 .c( therefore represents the resting fraction.7. (7.(Q.2. A. Certain strains of flagellated bacteria stop and "twiddle" between successive straightline portions of their tracks (see Berg and Browti'"). C. J . '' H. . Nature (Loiidoii) 239.18) that Z.2). Holz and S. 23.
unless data for I ' ( X ) can be fitted by an analytic function. the procedure indicated by Eq.3) is the Q dependence of the predicted autocorrelation function : if data taken at different scattering angles are plotted as a function X = Qt. F.7. the signal arising from diffusing particles scales as Y = Q't.7. Hallett. Chen. B."' 1': 1 P(V) sin q dq do. 21.7. so that the distribution of velocities is spatially isotropic.7. 171 (1972). ~ ' employed to deduce features of W (V ) . Consequently. In contrast. J . Biophys. Fr. (7.3') we see that the swimming speed distribution can in principle be obtained by Fourier transformation as W ( V )=  2v 71 m!onr sin X V [ X l ' ( X ) ] d X ."') will superimpose. Biophys. H. Marsh.328 7. (7.7.) 33CI. 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. [The distribution W (V )is related to the velocity distribution of the microorganisms P(V) by integrations of the latter over dire~tion. the autocorrelation functions associated with locomotion (eiQ. (7. (7. J . Stock. R.865 (1974). LASER LIGHT SCATTERING If no external forces act upon the microorganisms. 1 The most striking property of Eq.4) may be impractical because of uncertainties related to truncating the transformation. ~ ~ been A useful parameter of motion can sometimes be obtained by examining the 5H '" J. J . Berne and R.2) takes the following form: (7. .7. 14.1) 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.g. "I '' G.58 If Eq. Nossdl. Consequently.4) But. R.. Biopltys. (7. W (V ) = 471V' s.7. (Orsay.~' e. (7. the term (eiQ'vt)in Eq. N o s 4 and S.3) where W (V) is the distribution of swimming speeds of the scatterers. a mixed correlation function such as that given in Eq. 203 (1978).7. Phys. 79 (1978). J . 22. Craig and J.3) is written in terms of the scaled variable X = Qt as (7. T.
and they are so large that structural factors must be considered (cf. Generally.s. Chen.5) Many assumptions have been employed in order to derive the relationships given in Eqs. and B. J . Biophys. P. exhibit scaling of the halfdecay point over a wide range of scattering angles. some rotating or twiddling individuals. R. B’ D. . these perturbations are unimportant at very small scattering angles. 35. Perhaps the most severe is that of spherical symmetry. (7.7. Chapter 7. coli bacteria. 164 (1974).7.60 whereas a model of a prolate ellipsoid coated with a thin membrane leads to periodic deviations from Qt scaling. 14.7. t). For solid particles having the size and shape of E. ~ ~ . swimming microorganisms have the shapes of cylinders or oblate spheroids. 244. H. The discrepancy between the autocorrelation functions for spherical and ellipsoidal particles increases with the size of the scatterers or degree of asymmetry. However. (7. by careful experimental design it should be possible to distinguish and quantitate certain complex movements of the scatterers. ~ ~ F~ ’ example. Unfortunately. J .7.5).3) is seen to be related to the rms swimming speed as (V2) = 1 0 00 V 2 W ( V ) d V= 6X’[1 . If it is assumed that the direction of locomotion of a microorganism is strongly correlated with its orientation ( e g . as they generally contain translating but perhaps wobbling scatterers. or . though. b3 64 ” R . where only translational motions are r n e a s ~ r a b l e . ~ when a continuous ellipsoidal mass distribution is assumed for the scatterer. differences by a factor of two or greater might be observed. 847 (1974). Noture (London) N ~ M i d . samples of microorganism such as flagellated bacteria are amenable to almost unlimited possibilities of data fitting. (7. Nossal. 253 (1973). ~ ~ indicate that wobbling motions and rotation should increase the rate of decay of Il(Q. movements other than simple translation should also contribute to the light scattering spectrum. Nuture (London) 248. Nossdl and S. Racey. W. (7. and S. that motion occurs parallel to the major axis of the scatterer). Boon. S .62 which from Eq. G. 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. and S.5).62a Because the dimensions of microorganisms are large compared with the wavelength of light. J. MOTILITY MEASUREMENTS 329 initial decay of the autocorrelation function. then significant deviations from simple Qt scaling as indicated by Eq.~ . Chen. H . Theoretical a n a l y s e ~ ~ ~ . Hallett. Biopl7j. Banks. J. ~ ~ Thus. Various model calculations have been performed in which such spatial asymmetries are taken into account.557 (1981).7.7. a ’”T. Schaefer. Nickel.Z(X)]lx+o. Alpert.7.3) are p r e d i ~ t e d . R.1)(7.43 Recent experimental data on Chlurnydornonas.
108 (1976).. and G. Muslacich and B.8. Biophys. A. Ford.373 (1976).Applications in Cell Biology Laser light scattering will probably receive increased use in studies of the dynamic behavior of biological cells and tissues. Biochim. Thus. J. Hiochirn. GaddumRose. 68 K . Acta 496. A particularly suitable application is the study of protoplasmic streaming in large algae67*68 giant slime molds. Langley. Hallctt. generally the more complex and uncertain will be the physical models needed to explain experimental observations. Biophys. Nuture (London) 252. V. Ware. H. Blandau.254(1977). P.8. Piddington. Ross. Sattelle. Biophys. h9 R. H. Verdugo. LASER LIGHT SCATTERING fraction of resting or diffusing particles. R.229 (1977). Instrum. 893 (1976). though. Craig. '' R. C. 1. P. 17. Ware. and D. Acla 444. and B. several points must still be resolved concerning the interpretation of light scattering data from cells as large as spermatozoa. 457 (1979). R . 'OS. 71 1 (1975).and P.330 7. among which is the importance of head rotations as compared with translational motion.66" 7. '"T. 28.^^ One potentially significant biomedical use of laser light scattering is in studies of spermatozoan motility. J. Inws/. Sattelle.6. W. R . Jouannet. '' W.the entire swimming speed ~~ distribution could be determined from the Dopplershifted components of the spectrum (see Chapter 7. the more ambitious the applications. Berge. B. F. Nickel.6). Biuphj's. V. Langley. However. 47. Unfortunately. 7). Newton. 16. Mustacich and B. one can determine the velocity distributions of cytoplasmic constituents quickly and objectively.^'. M. I. G y n c w l . and members which collide with each other. and D. N. D. P. particularly Fig. 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. Lee. B.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 ~ . David. Rui. R . 2 I2 ( I 977). 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. K. Sci. selfconsistent computerassisted data fitting gives reasonably good agreement with certain simple theoretical model^. using techniques similar to those developed for measuring the speeds of charged particles responding to an electric field (Chapter 7. DuBois. R . However. .69770 and Protoplasm contains inclusions ranging in size from that of mitochondria down to that of submicroscopic particles. O n the other hand.
the theoretical analysis is rather complicated and several approximations have to be made. B i ~ p h j . Potential applications include detecting the onset of shock in patients confined in a criticalcare facility. I I I S (1976). cinematographic methods are expensive and cumbersome. W. R. Industrial and Scicntilic Conference Management. B i o n w l . Bowen.  ’? .~ 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. and A . S. Lee and P. D. Ware.9.Sci. 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. Inc. 248 (1977). Nevertheless.74“ 7. 27 (1981).33. F. 1258 (1980). ’‘ R. B i d . Bonncr. 305 (1978). ‘’ R. Yoshino. P.633 (1975). Ins/rwiz. c‘. D. Even for this simplified model. Lee and V e r d ~ g o ~have ~ l./ i m . Bonner. ~ R .C. 65. Carlson. and R .. in “Proceedings of the ElectroOprics Lascr ‘78 Conference. Verdugo. Carlson. c‘olcl Spring Ilurhor Symp. However. and generally cannot be used to study tissues in situ. Fujinie and S.~ . D. 389 (1972).8. 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”). Carlson. R. R. B / o p / i ~1. I . 1. Lee and P. Blood Flow Instruments based on laser light scattering can be designed to monitor local blood flow in a noninvasive manner. Haskell and F. Pli~siol. Gy. Chicago. determining blood flow in tissues chosen for biopsy.7.72 A theoretical model of ciliary beat phenomena used to explain light scattering data72 trcats the ciliated epithelium as an oscillating rough surface. Illinois. Ann. Grn. and assessing the extent of peripheral vascular disease. K. 37. Folger. . J . B. and D. R . Nossal. 5 . Fraser. R P P . Rowman. . 7Jr ” W.’F. Bonner and F. Verdugo. the theory gives a good representation of the prominent features of the measured autocorrelation function. 51. F.“ p. Compact instruments currently are being d e ~ e l o p e d ~ “ ’ ~ for specialized clinical applications which utilize flexible fiber optic probes ’’ W.9. 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. 53Y. C. L. B. 197X. Giddon. Quunr. Light scattering instruments based on fiber optics techniques can potentially be used for clinical investigation of ciliary activity in the respiratory tract. Biophys. investigating the effectiveness of flow in skin transplants. 1. Wunderlich. ~ 16.
L.. 56 (1974). 7 R d R . although several collisions with static tissue elements might occur. Thus. The motion of red cells in the microvasculature causes Doppler broadening of the incident light.5. (7. and scattering from surrounding tissue. Stein. D. once scattered by a red cell. R.. and J .^^^^^" Assume that. A m . H441 (1977). We also presume that the cells move in random directions in the capillary bed. R. J . A . Chimosky. do not interact with other moving targets (i. Assume further that photons. is given above in Eq. Lappe.332 7. LASER LIGHT SCATTERING to collect light scattered from tissues such as skin or gums. various s t ~ d i e s ~ ~have~ . at the time of the first interaction of a photon with an erythrocyte illumination from all directions is equally probable. We need not consider the details of photon trajectories subsequent to interaction with the erythrocytes because. Parma. 20. Nossal. Kciscr.3). Boiiiier and R.~iol. before interacting with an erythrocyte. D. Osgood.78 process This is difficult to model analytically because of possible multiple scattering from the erythrocytes. other blood cells) before emerging from the tissue. Bowman. Bowen. E.1) where the 0 integration accounts for all possible scattering angles. P. L. we assume that the scatterers are spherically symmetric. photons make many collisions with the surrounding tissue and are randomly diffused. Suppose that laser light is shined on a subject’s extremity (such as a finger) and detected as backscattered radiation. 2097 (1981). G.~ ~ that changes in the width of the * ’ indicated Dopplershifted spectrum can be correlated well with changes in flow. ) The brackets indicate averaging over speeds. P.4) and (7. M. Jr. D. Holloway. 231. 79 M. the intermediate scattering function can be written as (7. D.7. Nonetheless.y. Phl~. Stern. R.77. Nuiurc (London) 254. Photodiodes are used as detectors. in the RayleighGans limit. App/. P/i. even though the precise value of the proportionality coefficient is hard to determine theoretically.5). Opt. Stern. (Lundun)232. assuming a fixed detector and 4n incident illumination (sin 8 is a geometric factor). D. having an effective radius a. F80 (1979). only the net path between a red cell and the fixed detector determines the phase shift of the scattered light. Consequently. with velocities which are constant over distances large compared with the wavelength of light. R. One model which shows how the spectral bandwidth scales with flow rate is as follow^. Stern. H . even though light is seemingly incident upon the tissue from a specific direction.e. uncertain knowledge of the spatial pathways for flow through the vascular bed.5.9.sia/. Bowen. 77 18 . and specially designed lowcost electronic analyzers are used to extract flow parameters from the data. For simplicity. J . W. and ~ ( Q u is the structure factor which. Bowman. L. D. (7. M. J . by Eqs. and R.
+ (7. A m w . T.9.*l Also. 830 (1974) T. I .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 example. Gels and Solutions of Fibrillous Proteins Various biological materials.. BenSira.9. 526 (1978). Riva. I(t) = 2 4 2 ~ 1 T2). either in situ or as reconstituted models. 7. which are. (7.2) and (7. Soc. Feke and C. 189 (197.(Mitrslritrqron. we find the halfdecay of the autocorrelation function to be related to the rms flow speed as ( V2)1'2 = 0. Opt. extracts of cell '" Tanaka. numerically evaluate Eq. T.*2although the necessity for surgical intervention limits the usefulness of this particular procedure. " '* G. The equivalent relationship to the halfdecay of the power spectrum is ( V2)1'2 = 4.1.1) Y(X) = (7. Opt.2) where CI = 0.3). (7. When 2uL2 is large (as for red blood cells) the resulting expression for Z ( t ) effectively becomes a function only of T. Riva. respectively. (7. S(. f1.4).ricc. E.9. .) 186. .5) which facilitates the integration in Eq. blood flow in arteries of experimental animals has been measured with a fiberoptic catheter.5. 68.C.5).1av1. Examples are the lens and oitreous humor of the eye. If the speed distribution is Gaussian. 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. (7.1 and z.10. have the mechanical characteristics of amorphous solids or highly viscous fluids. it then follows6o that the expression in the angular brackets is (sin[Q V t ] / [ QV t ] ) = eQ2(v2)'2i6.3) where ( V2)"' is the rootmeansquare value of the velocity. we can express the result of the integration in terms of reduced variables of length and time. J . (7.9.9. GELS AND SOLUTIONS OF FIBRILLOUS PROTEINS 333 A good analytical approximation to Eq.71atQ:. Using in Eq. and C.ic.10. An instrument based on their prototype studies now is being used for clinical diagnosis. 14.1) the expressions given in Eqs. ' ~have shown that inelastic light scattering can be used to study blood flow noninvasively in human retinal vessels. (7. Benedek. Tanaka et ~ 1 1 .9. Tanaka and G.9.7. if we set a = 0. L = 4nnll'a and T = ( V2)li2t/$a. Appl.
To compute U(r. whereas conventional rheological techniques probe bulk moduli. particularly if a sample is easily perturbed by the stresses imposed by conventional rheometers.) Another advantage of certain light scattering techniques is that they can be used to study local properties of a sample. Hocker. In general. Thus. J . Upon taking spatial Fourier transforms we find 6p(Q. In Chapter 7. = Ar. long molecules tend to align in the stress field between the plates of a viscometer. If the interaction between the polymer chains is sufficiently strong. gels formed of fibrin and other blood constituents. in essence they can be characterized as aqueous solvents containing a relatively high concentration of long polymeric molecules. t).2) where p o is the equilibrium density. cervical mucus. 5151 (1973). It is preferable to consider a gel or dense polymer solution as a continuum. (7. the material is resistant to deformation and has gellike characteristics.1)  where U(r. . Clearly. 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. . and G . t)v U(r. in which case light scattering fluctuations are linked to local displacements of material within the sample. B. In a continuum the deviation of the density p from local equilibrium is given by the continuity equation M. Tanaka et considered the question of light scattering from a gel and took the *3 T. Tanaka.10. Although these substances typically contain many constituents. LASER LIGHT SCATTERING cytoplasm. and it exhibits viscous behavior. We here assume that our sample is optically homogeneous on a length scale comparable to the wavelength of light. L. t). 0. physical models invoked for diffusing macromolecules are unsuitable for analysis of viscoelastic materials whose properties derive mainly from frequent strong interactions between polymer chains.18’) we see that thc autocorrelation of photons would be sensitive to autocorrelations of various components of the Fourier transform of the displacement vector. Laser light scattering can be used as a probe of the viscoelastic properties of such materials. Benedek. It then follows that the diffraction pattern varies because of local changes in density. and lubricants of joints such as sinovial fluid. P h y ~59. t) (7. however. Chew. (For example. In other instances the material can flow if it is subjected to steady stress.4 we discussed procedures for probing the motions of dilute molecules in solution. the more easily it is disturbed if classical rheological techniques are used for measurement. (7. by Eq. t ) .3 34 7.ipoQ U(Q. t ) one first needs to assume a physical model.2.10. the less rigid the material.
4) Thus the photon autocorrelation function would decay with a time constant whose Q dependence is the same as for Brownian motion [cf.) 39.7. D. The basic QZ dependence of the decay time has been verified in several l a b o r a t ~ r i e s . J. (7.10. J . (Orsay. only the moduli appear in the term which multiplies the exponential. Hild.3) and (7.4) is given asE3 (7. Note that.~ derived from Eq. and L the illuminated length of the scattering volume. t ) ) = (WFY) exp{C(K + $p)/flQZt). and f . Eq. (7. J .(Q. and the term ( UZ(Q)’) is evaluated by thermodynamic argum e n t ~ The ~field autocorrelation function for polarized scattering then . Hecht.(Q. 86 . (Orsay.4. S. L. and G .2. M.971 (1977).2. E.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. H e n .10. Geissler and A. The other terms in the expression have the same definitions as in Eqs. 955 (1978). u ) . yielding’ the following expression for the autocorrelation of the component of the displacement which is parallel to Q: (U. P. Thus. Their assumed kinetic equation has the form83 pazu/at2 = p u + (K + $p)v(v. Candau. (7. by measuring the intensity of scattered light and knowing instrumental and material H4 85 J.10).jau/at. Phys. although the parameter which can be obtained from the decay of the autocorrelation is the ratio of the elastic moduli to the friction coefficient. T the temperature. assuming infinite spatial boundaries. Carlson. Munch.10. Solution of this equation. Thus they considered movements of a gel lattice against the fluid. Fr. ~ ~ * ~ ~ although deviation from a single exponential decay also has been reported. Fr. yields waves which propagate or are damped depending on the values of K.10. K .86 In these studies the driving forces for the displacements are thermal excitations. (7. Wunand F. Nonpropagating modes are predicted for soft gels. Phys. 190(1975). (7.1)J but with a proportionality coefficient given as (K + $p)/f. p. O>u.10. GELS AND SOLUTIONS OF FIBRILLOUS PROTEINS 335 polymer lattice to move as an elastic continuum subject to frictional interactions with surrounding fluid. p.5) where k is Boltzmann’s constant.)38. Marromolecuk~s8.
Fraser. LASER LIGHT SCATTERING parameters. fh. + " T. Phv. Sc.) 197.. . one can obtain values of K + $ p andf separately.Lett. Jr.vs. 31 1 (1979). 38. I . Tanaka. Nossal. '" NOSSdi. Macromo/oc.273 (1974). B. .336 7. 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. '' T. Mu/. F. Ishimoto. Ishimoto. R. D. J . and Ishiwata. it closely approximates p for the lattice alone. L." Significant deviations from an exponentially decaying form have been observed for the autocorrelation function. 139 (1975).1) should be used to describe such fluid like. 89. Biol.u/cs 12. 50." 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. to examine the formation of cataracts in excised bovine lens. though highly viscous. This procedure has been used by Tanaka et aL8' to study phase transitions in polyacrylamide gels and. (7. Gelman and R. D. Recently a light scattering scheme has been developed which yields values of the bulk shear modulus G. 3105 (1979).' 9*90 Attempts have been made to use scattering schemes based on thermal excitation to study actin gels. A.10.771 (1977). Carlson and A.66 and it may be that a modification of the basic kinetic equation (7. Chylack. This modulus pertains to the solvent and lattice acting together. 95.A p p / . C.irnce (Wrrrkiny!on. T. Similarly.4) have been observed in studies of the properties of cervical mucus. discrepancies between experimental results and the expression given by Eq. but heterogeneities in the sample have made it difficult to obtain consistent results. 1010 (1977).C. in a somewhat analogous fashion. 8 9 R. RcL'.10. materials. but because of the low shear resistance of the solvent.7. S . Tdnaka. and C.
The technique. Moore 8. In biological experiments the scattering studied is the incremental contribution to the total scatter of a solution due to macromolecular solutes.1. The Nature of the Technique When shortwavelength radiation (A about 1 A) strikes amorphous materials. most of the macromolecular scatter is confined to regions close to the direction of the incident radiation. Second. a few tenths of a milliliter of a 1 or 2 % solution can often produce interesting data. Measurements of this kind have contributed significantly to our understanding of the structures of gases.9 . is the topic of this article. molecular size and shape.” Smallangle scattering commends itself to the biophysicist for three reasons. and amorphous solids. Observed under proper conditions. Analysis of this contribution can lead to accurate estimates of molecular weight and volume. Since macromolecules are large compared to the wavelength of the radiation generally used in such experiments (10100 versus 1 A). therefore. Thus the potential for artifacts is minimal and the 331 METHODS OF EXPERIMENTAL PHYSICS. liquids. First. Introduction 8. diffuse scattering results. In addition. it is easy to prepare samples. is called “smallangle’’ or “lowangle’’ scattering. the elastic component of this diffuse scatter can yield useful information about the structure of materials at the molecular level. the information obtained relates to the molecule as it exists free in solution. and knowledge of some aspects of internal structure. SMALLANGLE SCATTERING TECHNIQUES FOR THE STUDY OF BIOLOGICAL MACROMOLECULES AND MACROMOLECULAR AGGREGATES By Peter B.8. is not a prerequisite. Crystallization. or sometimes simply “solution scattering.1.1. All that is needed is a monodisperse solution of the molecule or macromolecular aggregate in question. 20 Copyright 0 1982 by Acdderni~P r e s . for example. The variant of this experiment of most interest to biologists. sample sizes are small. the study of the diffuse scatter given by solutions of biological macromolecules. VOL. Inc All right5 of reproduction in m y form reserved ISBN 012 415962.
At the time of introduction.3. 161 (1939). the appeal of the micrograph is often overwhelming when compared with a model inferred from scattering data. The interpretation of the molecular images generated by an electron microscope can be difficult. by what is to many a nonintuitive process. Yet. While this radiation is effective.338 8. smallangle techniques were virtually unique in offering a direct means for estimating the size and shape of biological macromolecules. 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. 8. but historical factors have certainly played a role. neutron radiation has many advantages (see below). (Poris) [5] 12. and Brookhaven laboratories. are widely used and become more effective every year. First.1. The Sources of Current Interest Crystallography and microscopy. a few days often suffice. a new kind of radiation has become available for smallangle work. 8. Third. The smallangle field grew up using x radiation as its standard tool. the competition. the smallangle field is as vigorous now as it has ever been. Detectors can be constructed to count ' A. a new kind of radiation detector has been introduced into the field. the positionsensitive detector.1. and its use has significantly broadened the range of problems approachable by solution methods. Phys. but the results cannot be equaled by any other technique. There are undoubtedly many reasons for this. electron microscopy and xray crystallography. The attraction of crystallographic analyses of macromolecular structures lies in their richness of detail and accuracy. it has never become widely practiced despite its virtues. data collection IS fast. The first neutron facilities suitable for biological work came on line in the mid to late 1960s. Ann. SMALLANGLE SCATTERING data correspondingly likely to be relevant physiologically. paradoxically. The effort required is very large. thermal neutrons. Although the method has always attracted adherents. notably at the Julich. two new techniques which could provide similar information had come of age. Harwell. Second. By the mid 1950s. when the scientific world was completing its recovery from World War 11. The completion of the splendid installation at Institut LaueLangevin in France in the early 1970s gave tremendous impetus to the field. Four important innovations have contributed to its current strength. but the pictures it produces are visual and direct.2. . Guinier. However misleading it may be.
stepscan mode of data collection and increase the speed of data acquisition roughly 100fold. INTRODUCTION 339 either x rays or neutrons and to determine where in the sensitive volume counting events take place. As a result the sophistication of data analysis in the smallangle field has improved and along with it the reliability of results obtained.1. Fourth. The widespread availability of computers has encouraged the development of new and powerful algorithms for processing smallangle data. 8. The applications of these methods will be illustrated from the recent literature. Among other distinctive features. The experimental problems faced will be discussed in general terms. its potential is still under investigation. and their use is now routine. including timeresolved experiments. Computers have also materially simplified the task of data collection. These devices replace the traditional singledetector. 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.4. the influence of the computer has been felt in the smallangle field as in so many others. as will the approaches generally taken in data analysis. In addition.and twodimensional detectors are feasible. There are many steps in the analysis of smallangle data which require numerical processing beyond the capabilities of hand computation. There is a definite possibility that the unique properties of synchrotron radiation sources may engender some new applications of smallangle techniques.8. They can be built to count all the scattering angles of interest in a smallangle experiment simultaneously. a new kind of ultrahighflux xray source has become available. Both one. . Third. Purpose The objective of this article is to provide the reader with an introduction to the macromolecular smallangle scattering field. a synchrotron radiation source is a pulsed radiation source rather than a continuous one. in the past few years. There is hardly any physical technique that would not enjoy strong interest in response to stimuli such as these. 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. the terms of trade between experimenter’s time expended and data acquired have improved 1001000fold. as is the case with ordinary xray generators.1. 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.
12 Bacon13 has provided an excellent work for those wishing an introduction to neutron diffraction. Timasheff. 11. P. B. New York. 1955. Brookhuwn Symp. J . Many other reviews can be cited which deal with the xray smallangle In addition.I4 ’ A. Brumberger (ed.). Hundb. Birmingham. N. 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” has . England. Syracuse. 1967.5. 1975. E. London and New York. 1962. Pilz. 27. 32. “ SmallAngle XRay Scattering” (Proceedings of Conference on SmallAngle XRay Scattering.1. H. 0. Press. Q .Krdtky and I . London and New York.Re[). Pilz. Kumosinski. Part C. Bacon. “International Tables for XRay Crystallography. Academic Press. 39. 27 (1976). W.). R. Pilz.g. T. G. James. Lovesey. 5. W.” Oxford Univ.Biophys.Jacrot. ed. Oxford Univ. Rep.340 8. and M.~ reviewed the neutron smallangle field. A rigorous theoretical development of the theory of thermal neutron scattering can be found in Marshall and Lovesey. the book by Guinier and Fournet2 remains invaluable despite the fact that it has reached its 25th anniversary. in “Physical Principles and Techniques of Protein Chemistry” (S. the proceedings of a number of symposia on smallangle topics have been published over the years (e. 9.” Kynoch Press. 1968. For example. ’‘ Neutron Diffraction. “SmallAngle Scattering of XRays. H. Biol. W. B. Phys. J. 1971. I. Background information on xray techniques may be obtained from James’ and the International Tables for XRay Crystallography. Leach. Lonsdale. New York. Guinier and G. 321 (1957).” Wiley. 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. June 1965. F. ‘ ’ ‘” ’ . Gordon & Breach. W. and S. Methods Enzymol. Webb. Brumberger’). (ed. “The Optical Principles of the Diffraction of Xrays. New York). “Theory ofThermal Neutron Scattering. 0.” Cornell Univ. l4 W.. W.911 (1976). Earlier Reviews The smallangle field has been reviewed regularly over the years and a number of useful works are available. Schoenborn. ed. Rev. Prog. Anderegg. Press. Press (Clarendon).” 3rd ed. two symposium proceedings have appeared in the Journal of Applied Crystallography (1974. tthaca. Beeman. 141 243. Biophys. pp. Kaesberg.481 (1972). New York. P. New York. Krdtky and 1. 39 (1978). Marshall and S.. 8. Pessen. Fournet. 1973. SMALLANGLE SCATTERING and sufficient references provided so that an interested reader can complete his study of the field. Q. K. Phys.1978). 151 (1973).).
] In the neutron literature.2. hencefi = h i . Equation (8. The scattering of the macromolecules in solution. (Leipziy) [4] 46. . bi being the scattering length of the ith atom. The Scattering Signal If the scatter given by a single isolated molecule could be observed over a period of time as it diffused rotationally.2. Flux is measured in photons (neutrons) per unit area per unit time. which are measured relative to a vacuum which has an averagefper unit volume equal to zero. which is not zero. where molecules are well separated and behaving in an uncorrelated manner. Equation (8. in Eq. For x rays. small scattering angles) can be regarded as a continuum having an average f per unit volume. 1. The effect of being immersed in this continuum can be taken into account to a good first approximation by replacing each. It can be ignored in many cases or included infi(R) as a small correction factor. fi and fj are the scattering lengths of the two atoms appropriate for the type of radiation used. 1).2.f.8. It is also the correct basis for understanding macromolecular contributions to the scattering of a dilute solution.2.2. a timeaveraged scatter I(R) would be measured which is directly related to its ~ t r u c t u r e: ~ ' (8. THE EXPERIMENTAL PROBLEM 341 8.1) Here R = (2 sin @/A.. 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. where there is no physical equivalent of electron density. atomic scattering properties are tabulated directly as scattering lengths. will not be identical to that which they would give in uucuo. where 26' is the angle between the incident and the scattered radiation and A the wavelength of the radiation (Fig. if the molecule is experiencing an incident radiation flux of one photon (neutron) per unit time." [The polarization correction will be very small for low scattering angles. (8. or scatteringlength density.The Experimental Problem 8.1) by P. 809 (1915).1) describes the scatter given by gases at low pressure. r i j is the distance between the ith and jth atoms in the molecule and both sums are taken over all atoms. at a given R. Ann. Phys.e. where the area is measured in the same units as the scattering lengths.2.Debye. 1 3I(R) is the number of photons (neutrons) scattered by a single molecule per steradian of solid angle per unit time. however.1) usesfis. (e2/mc2)J(R).2. Solvent viewed at low resolution (i.1.
2. Cheni. under conditions in which Eq. reflected in the cylindrical symmetry of I ( R ) about the direction of the direct beam. in order to carry out the subtraction indicated.1).342 8. However. a number of lowerresolution aspects of the molecule's size and structure can be elucidated once Z(R) is known (see Chapter 8. as is made clear below."(R). (8. Cohen. Note that knowledge of 6. 849 (1975). SMALLANGLE SCATTERING j .the macromolecular contribution. 8. Goodisman and H. is required. Ser. 19 A. The experimental objective of virtually all macromolecular smallangle studies is to measure Z(R). Isol(R)and Zs0. Brumberger. leads to the loss of all information about the relative orientations of the interatomic vectors whose lengths rij appear in Eq. 327 (1973). with appropriately evaluated As. Mwrornolrculrs 8. is to be J . of the solution.3).2) where u is the volume of solvent in an aliquot of volume u. averaged over the time of observation. 'I' l7 .2) will hold. Timasheff.2. which are simply the counts registered by the detector at each scattering angle. 125.2. The two measured profiles. 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: (8.2). If solvent molecules bind to the macromolecules or organized regions of solvent exist around them. . The separation of macromolecular contributions from those of the solvent is usually done in a simpleminded manner. 37. Hyman. 355 (1968). Chem. as is knowledge of concentration of the solute. Bio/. A sample of pure solvent and a sample of the solution are measured in the same apparatus under the same conditions. N . This subtraction is justified provided there are no correlations between the positions of the molecules of the solvent and those of the macromolecular solute. 598 (1973) H. Mol. Adv. the scattered intensities due to solute and solvent will simply add to give the total measured signal.2. J . and Eq. Isolation of the Macromolecular Signal All components in a solution contribute to its scattering.'6p19 Equation (8. therefore. the reverse operation is not possible: a knowledge of Z(R)will not give back the structure. 104. Monursh. although knowledge of a molecule's structure at atomic resolution enables one to compute f(R). problems can arise. Eisenberg and H.2.the partial specific volume of the solute. '* S. The reader should recognize that. (8.u i p .'6 If these positions do not correlate. (8. S.2.1) holds. The rotational averaging involved in the production of the signal. where uiis the volume made inacessible to solvent by the ith atom and p the solvent's average scatteringlength density.2.
the macromolecular contribution will be proportional to I(R). Schematic diagram of a lowangle scattering apparatus.3. THE EXPERIMENTAL PROBLEM 343 regarded as a firstorder approximation for estimating I ( R ) from experimental data. (5) plane of registration. (2) collimatian slits.2. and (iii) the type of measurement to be made. Equation (8. hence its name. and ( 6 ) detector. 8. (ii) the kind of radiation source available. In the dilute limit. The radiation scattered by the specimen is measured by the detector at some distance from it. (3) guard slits.2. SmallAngle Apparatus: General Features All smallangle instruments consist of the same set of elements: a radiation source. While it is not appropriate to discuss instrument design in great detail. and a line drawn between the center of the beam’s intersection with the sample and the detector axis. I . a monochromator. appropriately collimated beam of radiation directed at a specimen held in a fixed position.1) be measured is that the sample be thin. and a detector (Figs. . (8. a collimator. Distortions in Z(R) due to these effects can be dealt with by making measurements at several concentrations and extrapolating to zero concentration.2.1)does not hold for multiple scattering (see Beeman et d 3 ) .the profile for a single molecule. The scattering angle 20 is the angle between the optic axis of the instrument. The plane of registration is more properly a surface of registration and is the region of space explored by the detector. The purpose of these elements is to provide a monochromatic. defined by the beam center. Critical components and positions in the instrument are numbered: ( I ) radiation source. Its lowangle region will be well approximated as a plane. 1 and 2). (4)specimen. The macromolecules themselves may aggregate or otherwise correlate in solution. If the sample is thick.8. 4 5 FIG. some general comments are in order.2. The only condition besides diluteness which must be met by a sample in order that the scattering described by Eq. a sample holder. a significant fraction of the radiation scattered by the average macromolecule will be scattered again by others before emerging from the sample. The design of these elements is dictated by three factors: (i) the type of radiation being used.
given by Eq.2.2. The instrument depicted herc is in operation at Yale University. The scattered radiation should then be detected using a device with an infinitesimally small acceptance aperture. and the weighted average of 1(28).1). In order to measure such a profile. 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. 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. It was designed by D. (8. the ideal smallangle instrument should have a sourcemonochromatorcollimator combination which delivers a perfectly monochromatic. The numbered elements are ( I ) xray tube. The SmallAngle Compromise The principal problem in measuring diffuse scatter stems from the fact that the signal being measured is continuous. and (6) positionsensitive detector. Xray smallangle instrument equipped with a linear positionsensitive detector. Johnson. (2) focusingmirror monochromator. 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.344 8. 8. the profile measured . Engelman and constructed with the assistance of J . even an ideal detector will be seeing scatter from particles whose angular relationships to the detector vary. (3) quard slit assembly. The two functions Z(28). 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) sample holder. are different.4. infinitely fine pencil of radiation which is directed through an infinitesimally thin specimen. ( 5 ) flight path chamber.2 . measured by instruments which deviate from the ideal. Failure to achieve perfect monochromatization of the beam has a similar effect. The reason this kind of geometry is desirable is that if the detector aperture is finite. SMALLANGLE SCATTERING FIG. If the beam is a finite size. M .
the detector used must have a finite aperture. (A full discussion of synchrotron sources and their possible application to smallangle work may be found in a recent review.5.19).2. The beam knocks electrons out of the lowlying orbitals of 2o H. 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.Rec. THE EXPERIMENTAL PROBLEM 345 is a distorted version of the profile desired. ensure that there is no single “best” instrument for smallangle purposes. 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. and the monochromatization must deviate from perfection in order to ensure a practical rate of data acquisition. This instrumental distortion effect is called “slit smearing. the smaller the magnitude of the correction which needs to be made.2. the more accurate the final result is. As a general rule. 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.”) These sources work by exciting the characteristic xray emission lines of the metallic elements. the collimated beam must have finite size. Stuhrmann. The practical problem is compounded by the fact that the signal of interest is often quite weak. The larger the physical size of the beam. Since small molecules scatter more weakly than large molecules on a weight for weight basis. This situation is not entirely without hope. While computational correction of measured signals for instrument smearing effects a posteriori is possible (see Section 8. . An electron beam having an energy ofa few tens of kilovolts is directed against a metal target. as has always been the case. B. the smaller the molecule. the larger are the angular regions around zero where no measurements can be made. The deviation of the measured from the ideal signal depends on the properties of the ideal signal. which is to say. it is obviously desirable to minimize smearing as far as possible by careful instrument design. 11. however. 8.2. Biophys. on the structure of the molecule being studied. the larger the smallest angle at which measurement must be made is. as well as on the properties of the instrument making the measurement. Q. 71 (1978).” Given the finite brightness of real radiation sources. The interactions between the characteristics of the instrument being used and the structure of the material being studied.8. X Rays For the foreseeable future. most xray smallangle experiments will be done using conventional laboratory xray generators. as well as the additional constraints imposed by the purposes to which the data will be put.
ed.6. illuminated area.. and its K. SMALLANGLE SCATTERING the atoms. The area of the target illuminated by the electron beam is the xray source. Where generators differ most significantly is in their brilliance. Kynoch Press. radiation.346 8. The refilling of these holes with electrons from higherlying orbitals gives rise to sharp emission lines. The simplest technique for monochromatization takes advantage of the fact that the linear absorption coefficient of metals for x rays is strongly wavelength dependent. The other half shows up as a white background continuum which has a maximum cutoff energy equal to the electron beam energy. W. but strongly absorbs Cu K.). emission line (A = 1. D. There are a variety of generator designs on the market differing in size and shape of the. Monochromatization of X Rays Conventional xray sources have a partially monochromatic character due to the way they generate radiation. It has a high transmittance for Cu K. in “International Tables for XRay Crystallography” (K. Thus near the absorption edge.11 mmz in area and rectangular in shape. In general the source areas are 0. metal foils will act as bandpass filters. lowintensity generator may more than balance the gains to be made in data collection rate by the purchase of a highintensity source. In a typical generator roughly half the emitted xray energy will be contained in these lines. which has a somewhat shorter wavelength and which is the second most important emission line from a copper target. A variety of steps can be taken to “clean up” their output to the extent required for smallangle work. nickel has an appropriately positioned absorption edge. the faster the rate of data acquisition and/or the smaller the beam one can use is. High brilliance is achieved by raising the amperage of the electron beam falling on the source. Vol. 1968. 73. The more brilliant the source.54 A) is the one usually used. which leads in turn to high initial cost and maintenance problems. High intensity requires elaborate expedients to prevent the destruction of the target due to melting.’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. passing radiation of wavelengths longer than the critical value and strongly absorbing radiation of shorter wavelengths. 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. radiation. The favorite target material for biological studies is copper. Parrish. England. the number of photons they emit per area of source. 8. For Cu K.2. Birmingham. . Lonsdale. The radiation emerging from a nickel filter of appropriate thickness will be B. Roberts and W. 111.
22 23 . Kratky. The ultimate expedient involves monochromatization by directing the beam onto a crystal. A. radiation..'^ A useful degree of monochromatization can also be imposed by the detector. one whose edge is at shorter wavelength than Cu K. and the other whose edge is at a wavelength longer than Cu K. The discriminating power of these detectors is not very high. can then be used for scattering purposes. Collirnation Xray smallangle instruments stand or fall on the performance of their collimating systems. shape. The radiation scattered in a strong Bragg reflection. and all longerwavelength radiation from the source. The reflected rays. contributions and also be significantly reduced in shorterwavelength white radiation. A surface placed in a beam at the critical angle for Cu K. THE EXPERIMENTAL PROBLEM 347 effectively free of Cu K.22This technique employs two metals.Sect.7. will not reflect Cu K. 0. contributions to a measured scattering profile can be more rigorously identified using the twofilter technique of Ross. Ross. 325 (1943). Both mirrors and crystals can be bent to make the total reflection or Bragg reflection process focus the beam. can be found el~ewhere. A thorough discussion of the properties of monochromators of both kinds. which include Cu K. and divergence of the beam must be such that at P. or any of the other components in the beam of a shorter wavdength.. 425 (1926). for example.2. The size. Witz. A collimator often consists of a sequence of slits which shape the beam and control its angular divergence.8. Phys. The Cu K.’~ 8. both focusing and otherwise. two further techniques can be employed. but sufficient to contribute to making a scattering experiment effectively monochromatic. Xray beams can be totally reflected when they strike smooth surfaces (“mirrors”) at glancing angles of incidence. For applications where better monochromatization is required.2. Acta CrjJstallogr. The combination of nickel filters and detector energy discrimination often suffices. can provide an excellent source of virtually pure Cu K. 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^. A A25. 28. taken at the correct angle. 30 (1969). Nrr/urwissenrchaficn 31. The purpose of the collimating system is to define the size and shape of the beam incident on the specimen. Reo. 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 critical angle for total reflection is a function of wavelength.. 2 4 J .
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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SMALLANGLE SCATTERING
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
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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 and (vi) c a m e r a ~ , ~ ~ *(v)Opositionsensitive detector ~ ,~’
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). 4 2 .. 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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SMALLANGLE SCATTERING
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 ther’~ mal, 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 probability of a scattering event occurring when a neutron or l~ 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 velopment of multilayer monochromators.49~’0 multilayer monochromator 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).
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. Mol. Almost all the scattering from water is incoherent scatter owing to its 'H content. with the same detector used for measuring scattering profiles. the relationship between total sample cross section and measured intensity follows.367 (1961). h3 h4 D. just as is done with H 2 0 in the neutron field. Reo.1).Instrum. L. absolute scale is reasonably easy to achieve.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. Absolute scale is harder to establish for x rays. Luzzati. A set of attenuators of known thickness can be prepared and beam intensity measured as a function of total attenuator thickness.. the total integrated beam intensity. The second '' V. Lucite is a satisfactory attenuator. G.) In such circumstances attenuation will not be logarithmic and accurate extrapolation to zero attenuation impossible.13 Thus it gives a strong scattering profile which is virtually constant with angle.8. The cross section of water is well known. the simplest being a resort to standards whose scattering can be calculated a priori. A 20A. H.63 Examples of substances useful in this regard include sulfur hexafluoride and octafluorocyclobutane. J. which are gases of known structure25 whose scatter can be calculated using Eq. and once again the absolute scale is set. Mueller. A second method in wide use is to employ a standard scatterer and calibrate in terms of the scatter it gives.O is excellent for these purposes. For neutrons. 3. and A. attenuaor beams ~ ~ ~ not ~ ~ tion with filters is ~ s e f ~ 1 . . Darnaschun and J. most detectors used for measuring scattered intensities would be damaged by exposure to the direct beam. 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. Two other approaches have been used.2. Bid. A semilog plot of the data permits extrapolation to the unattenuated intensity at zero thickness. Foils of Cd can also be used. THE EXPERIMENTAL PROBLEM 359 scale. J . 691 (1963). 34. Indeed. Witz. (Such strong wavelength dependence is not seen with neutron attenuators. hence the observed I ( R ) is the true Z(R). Thicknesses of several centimeters are required so that the preparation of a set of blocks of wellcharacterized thickness is easy. Z. 1274 (1965). F~ ~ * ~ which are ~ . The thickness of the sample.25. For scatterers for which I ( R ) is constant there is no slit smearing effect. For an xray apparatus in which the beam is fully monochromatized (by Bragg reflection). Sci. Nicolaieff. Noturforsch. (8." Provided the volume of the sample in the beam is also known. Weinberg. and the solid angle accepted by the detector suffice to set measurements on an absolute scale.2.
Kratky and his collaborators have used mechanical devices to attenuate the beam in order to estimate its in conjunction with the calibration of standards. Kratky and H . Chipman.3) '' 0. 981 (1963). Hendrichs. h5 (8. Wawra. Schmidt. under conditions of ideal geometry and monochromatization. 1655 (1978). 179.122. 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 .2. Passage through the specimen attenuates both x rays and neutron beams because of absorption and highangle scatter. W. J.94.2. R. Crjstullo. C'rysrulloyr. Slit smearing alters both the shape of a profile. Kratky. R. R') dR'. the beam and the scattered radiation of interest are equally affected by attenuation.21. De Marco. It is easy to show that for low scattering angles. 8. The knowns in this case are used as secondary standards. h7 0.68 (1961).2.R')W(R. . 315 (1972). J .24 (1966). C'hcw~. on which estimates of molecular size and shape depend. and J. Phjs. J . Stahinger and 0. 11. The effect of smearing on the data can be thought of as a convolution. Pilz. Data Correction Before data sets are analyzed consideration must be given to the impact of slit smearing (see Section 8. and its absolute magnitude. Commission on Crystallographic Apparatus. In general it is advisable to attempt to correct for these effects computationally. ~ ' For either xray or neutron experiments. the option is always open of determining molecular weights by comparison of unknowns with molecules of the same chemical type of known molecular weight. and P. from which estimates of molecular weight and volume come. under equivalent experimental conditions. " B. D. the curve actually measured.and a report has recently ~~ appeared on the accuracy with which profiles can be m e a ~ u r e d . I96 (1978). A p p l . 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. 5 . It should be recognized that the beam intensity of interest in any scattering experiment is the intensity ujter passage through the specimen.360 8.4). Batterman. RPP. Hence one always wishes to compare measured scatter with the at tcnuatedbeam intensity.qr. Colloid Iriterfiice S(. O ( R ) . Appl. C h m . I . Monaish. 7" H .19. is given by the following expression: I(R . M n k r o m d . SMALLANGLE SCATTERING alternative is to prepare precalibrated secondary standards.i. J . W. Kratky.
2. An extensive bibliography and discussion of techniques has been prepared by S ~ h m i d t . Clatter.as measured. includes some error. Appl. Crystullogr. Cry. the amount of averaging varies with angle. J . THE EXPERIMENTAL PROBLEM 36 1 where I ( R ) is the ideal profile and W ( R . the beam’s spectrum will usually be known and will be provided to the experimenter by those running the facility. There are a number of techniques for solving such equations to obtain I(R). I975 p. Tech. 362 (1976). R. ’ ~ some practical and comparisons of algorithm performance have been performed.3) is an integral equation in which both W ( R . Summrr Sch. Equation (8. The art of data correction is in a fairly satisfactory state at present. 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. Summer Sch. further progress may be expected in the future. Schelten and F. F.I. The first of these can either be calculated from the geometry of the beam collimator and the detector’s properties. Schmidt. 83 (1977).^^ the spline and the indirect transformation algorithm of Schelten and Ho~sfeld. Suc.yr. Reo. R’) and O(R) are known.’~ method of G l a t t e ~ Deutsch and L ~ b a n ’have recently proposed a new . 7 3 J. Clatter. I n / . For xray experiments wavelength effects are usually small. London. Kranold.~~ ~ method for dealing with slit height correction. I . 87. Comput. W. . 618 (1931). J .23. CrystuIlo. Comput. Appl. 75 J. 11. CrystoNoyr. Hossfeld. In addition O(R). 210 (1971). Actu Phvs. J. Mueller. Proc. Lake.yiulloyr.^^. A. Proc. Proc.R’) has two components: (i) a component due to the size and shape of the beam and the detector aperture. R. W ( R . Jones.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. The problem is to find an estimate of I(R) in the face of these limitations in the information given. Tech. R. Aciu Crystullo.. Austriucu 47. as seen by the detector. Crysiulkqr. Deutsch and M. However. R’) the weighting function representing the averaging effects of the i n ~ t r u m e n t . 4. 1975 p. 7 7 M. 147 ( 1 974). the ideal algorithm is yet to be discovered. and (ii) a component due to the spectrum of the radiation used in the experiment.8. 16 (1938). from counting statistics if nothing else. In the case of neutron experiments. ” 72 . 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. 383 (1976). 7 4 0. 191 (1967). 98 (1978). What is required is the size and shape of the beam at the plane of registration. Luban. Ser. ’’ G. or measured directly. ’’ 0.W. Damaschun. One might single out for mention the iterative algorithm^. A 166. 7 8 P. 38. ’ ~ W is generally a function ”~ of R . Spencer.yr. J. I n t .. Appl. Phys.2.. and G. Waltcr.
(8.. Biophys. I(0)cannot be measured directly.PO)262. Bioph. Ii is the flux in the beam measured ufter its passage through the sample.3.1. 8. (The range of R over which data must be collected in order to permit extrapolation depends on the size of the molecule. (8. . provided data have been collected at R sufficiently close to zero.1) at R = 0 becomes 2 = (Fh . Prn. Data Analysis Suppose that full data have been collected on the scattering of a given macromolecule in solution. = (8. Defining fi = ( x i .3. SMALLANGLE SCATTERING 8.PoU) 3 where u = ui. and li are measured in photons or neutrons per second.3.3. The interest in I(0) lies in its relationship to molecular weight and molecular volume. Chem.ys.is the total volume of the molecule inaccessible to solvent and p o the scatteringlength per unit volume.362 8. will be true that it 4 x p ( R )= N W . Criteria are discussed in Beeman et aL3) Guinier’ demonstrated that the lowestangle portions of the scattering curve of any molecule are Gaussian in shape.q. Because of the direct beam.2. A d is the area of the aperture of the detector and d the distance from the sample to xi *’ 0 . Thus the lowestangle data plotted as ln[lexp(R)] versus R 2 will be linear. 13.1) 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. 107 (1963). extrapolation to R = 0 is straightforward. one obtains (Luzzati31. For such a corrected scattering profile lexp(R). see Kratky and Pilz4) lexp(o) (Ad/d’)ZiAtC(MW/NO)(p . Z(0). Kratky. mental geometry into account. However. 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.1). or scattering length density of then taking the incident flux and experithe solvent.2.3162*80 Equation (8.j&h. 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. and extrapolation to R = 0 follows. The data are then used to construct a scattering profile corrected for concentration effects and slit smearing.2) Here I.
2 Thus the molecular weight value obtained from a lowangle experiment is a thermodynamic quantity.).3. In a neutron experiment po can be altered over a wide range by mixing D.If M W is known. The EinsteinDebye theory developed first for light scattering” is correct in this case as well.3. Pergamon. While Eq. 6 the partial specific volume of the solute. can be added to the solution. (8. The scattering of solutions near R = 0 must be considered in thermodynamic terms. Thus data collected on the same macromolecule at different pos will permit determination of the po for which p = p o .8). N o Avogadro’s number.3) where I = ZiA. and C its concentration (mass/volume). in “Electromagnetic Scattering” (M. When R 0. if 6 is known. p. “Physical Chemistry of Macromolecules.3. V follows. Given the atomic composition of the molecule as obtained..OD. glycerol. for example. (8. it is the fluctuations in solute concentration within these volumes that give rise to the scattering seen. it is necessary to be aware that its theoretical justification requires something more rigorous than Eq. A is the area of the sample illuminated by the beam and t its thickness.’EsZ  Wiley.. Then MW = I. The xray volume will be that inaccessible to the solute used to manipulate po and will be a hydrated volume.p o . Tanford. This procedure can be carried out on data that are not on an absolute scale but simply normalized for differences in transmitted beam. M W is the solute’s molecular weight. ed. [Iex.11). In an xray experiment lowmolecularweight solutes of high electron density. 1961. These values can be used to calculate M W [Eq.O mixtures will be the anhydrous molecular volume. The scatteringlength density of the solute is generally obtained from knowledge of its atomic composition and 13. Oxford.” . volumes much larger than that of a single molecule are being examined.12).O into the solvent (see Section 8.1).3.3)]. Timasheff. N. The scatteringlength density of the solvent can be obtained from its chemical composition and density. 337. 6 follows from p.3. such as salts.3..(0)/C]l’z is proportional to p . po can be varied (see Section 8. (8.2) suggests a method for obtaining V from scattering data. Kerber.3. Note that the molecular volume emerging from a neutron study using H. the total number of neutrons or photons in the beam. M W follows. New York.ztC[p .(0)d~No/A. 1963. with the proviso about hydrogen exchange explained below (see Section 8. its extension to xray or neutron scattering may be found in Eisenberg and Cohen” or Timasheff.3) is often convenient. S. (8.3. from its amino acid or base composition. or sugars.3. DATA ANALYSIS 363 the detector. 82 ’’ C. The partial specific volume of the solute must be measured.8.p0]262. leading to a determination dependent only on scattering and atomic composition data. Equation (8.
Hyman and P. (8. p. In practice the molecular weights obtained from xray or neutron forward scattering have been found to be of excellent accuracy. a correct molecular weight will be found. SMALLANGLE SCATTERING In practice published values for 6 for macromolecules (within a given chemical type variation is small) are often used in estimating ( p . different molecules differ only in the breadth of this Gaussian. 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.*3In principle one should measure Gp/GC. (8. Bruniberger. 1967. they are equally necessary here.3. Under these conditions. scatters almost entirely in the forward direction when observed with radiation of sinall A.2. Dust. 8.2. Molecular Size and Shape: Radius of Gyration As already pointed out. the scattering curve of a macromolecule at low R is Gaussian in shape. It can be shown by expansion of Eq.2)] should be equilibrated by dialysis with the solute solution of interest.2.3). Additivity will not be observed if solute and solvent interact. This usage assumes that the scatteringlength density of the solution is the sum of the scatteringlength densities of both solute and solvent. The advantage of smallangle molecular weight determination over its light scattering analog is that sample preparation is much easier. and use it in place of ( p . The buffer sample whose scattering profile is used for background subtraction [Eq. see Eisenberg and Cohen”). .364 8. in “SmallAngle XRay Scattering” ( H . Recognizing the thermodynamic nature of I(O). ed. 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. Gordon & Breach. 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. can be measured under the same conditions for the solutesolvent combination of interest and 6p/6C estimated from it (for details. as they do with polyelectrolytcs. Vaughan. 8 3 A.po)V for use in Eq.1) as a power series in 2nRr and considering the first two terms not only that the curve is Gaussian. (8. Similar precautions are routine in the determination of molecular weight by other thermodynamic methods.). it does not interfere with this technique. comparable to sedimentation equilibrium except for a few situations involving highly charged polyelectrolytes where the precautions mentioned above were not observed. 477. In practice hd/hC.p0)V. A. where d is the weight density of the solution.3. New York. which is the bane of light scattering studies.
) Radius of gyration is the sole fact about a macromolecule which can be deduced from its solution scattering profile without resort to ancillary data.3. Useful information can be obtained. Because this parameter is extractable from lowangle data in such a direct fashion. where T i is the vector from an arbitrary coordinate origin to the ith atom. Porod. 125. virtually every smallangle paper ever published includes radius of gyration estimates.9. thin objects.8. The scattering radius of gyration gets its name from its analogy with the mechanical radius of gyration. however. 83 (1951). where R. is the radius of gyration of the molecule. 51 (1952). (8. Porod. Furthermore.^^ '' G . The sums in all cases run over all atoms. .' For small R. I ( R ) = I(0) exp( $n2RiR2).3. where y i is the distance from thc ith atom to the center of mass ofthe scattering length of the particle. 124. it will often prove hard or impossible to measure those radii because of the narrowness of the central Gaussian.4) Hence the slope of the In Z(R) versus R 2 plot used for estimating Z(0) will be $rc2Rl. Quantities Related to the Radius of Gyration Long. or o n the transverse distribution in the case of flat plate^.3. i. 8. to the location r. Ko//oidZ. When smallangle data are collected on such objects. on the crosssectional distribution of scattering length in the case of long.O solution will be close to the mechanical value for the molecule in question and provides a useful indication of the molecule's size. thin particles or thin plates will often have radii of gyration which are very large indeed.. The value found by xray methods for a protein in water solution or determined by neutron scattering for a protein in D. H4 G . DATA ANALYSIS 365 but that the breadth of the Gaussian reports the molecule's radius of gyration.. (The relationship between molecular structure and radius of gyration is considered at greater length in Section 8. radii can be obtained from data which are not on an absolute scale.e.^^. Ko//oi&%.3.3.
Madison (1968). where R. and B.. Evaluation of M I L and the radius of gyration of the cross section for chromatinrelated structures has been reported by several g r o ~ p s . 491 (1967). V.89 (1976). the analogous plot is ln[R2Z(R)] versus RZ. SMALLANGLE SCATTERING For long. A . R . We shall call this value “f(0)” for this purpose. Tardieu. Saludgian.. R. M I L and diameter information are valuable parameters since they can constrain modelbuilding studies. University of Wisconsin. is the result. I. and P. chromatin). Bran]. have often been measured for elongated structures. S. Bram. F E B S Lett. J .D. ~ ~ . Biochimiu57. For a uniform plate. they materially assisted V. P. 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. Mathis. Biqiolymrrs 5. For platelike objects. 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. similar use of 27tZ(O) will give an estimate of the molecular weight per unit area. thin objects it will be observed that plots of ln[Rf(R)] versus R2 are linear over an appreciable range of R. and they are hard to obtain by other means. 64. For the thin plate. 51. Aleksanyan. The possibility of measurement of membrane thicknesses and mass per unit area by the method just suggested has seldom been taken advantage of. Biopo/lwrrs 14. (8. These same techniques are useful for the analysis of far less elongated molecules. 1301 (1975). is the radius of gyration of the cross section of the object. R7 88 8y ’’ ’‘I . For indefinitely long structures like these. The classic material for studies of this kind is DNA and its various complexes with histones and other protein (i. I133 (1975). A. Sperling and A. Ph. f.e.3) in place of IexP(O)and an estimate of the molecular weight per unit length of the object will result. L. 3 (1975). Mass per unit length ( M I L ) and radius of gyration of the cross section. Thesis. Once again a linear region will be discerned whose slope is 47t2R:. Zipper and H. for the long rod 21(0)can be substituted into Eq. T 2 is the thickness of the plate. Biochem.366 8. 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. If measurements have been made on an absolute scale. I.~ ’ and Bunemann9’ have recently described the alterations Zipper in DNA structure due to drug binding in a paper which includes a careful explanation of the data analysis used in MIL and R. V. Masson. Bunemann. The slope of this linear region will be 27c2RI where R. however. is now the radius of gyration of the density of the plate projected onto a line normal to its surface. Luzzati.3. investigations. thin shape or a thin plate shape as the case may be. Federov. Eur. Vorobev. 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. S.
P. H .3. and E.=r c fifj sin 2nrR 2nrR and I ( R) = allr c c fifj sin 2nrR 2nrR rij=r 0 ’ Let us define a new function p ( r ) : where. Length Distributions Equation (8. = (1i ) / N and N is the number of atoms in the molecule. Schlimme. von der Haar. Phys.4. Thus the total information contained in a solution scattering 92 I. the expression for Z(R). Pirenne. then (8. DATA ANALYSIS 367 Pilz et al. 401 (1970).2. Kratky.one could equally well make a list of all atomic distances and collect these distances into groups of identical value.1). Pilz. 8. (8. P.92in their recent analysis of the tRNA structure from smallangle data. It follows from Eq. 0. Cramer. (Lripziy) [ 5 ] 33. Bueche. Often it is more convenient to consider p(r) a continuous function. Debye and M. 15.f.3.3. 20. Eur. then f where the sum is over all distances r. 617 (1938). Ann. Biochem. 93 94 .8.5) where p is the average scatteringlength density of the object.3. F. Phju. J .3. M.6) Note that knowledge of p ( r ) is equivalent to knowledge of Z(R). J . is written as a double sum over all atoms of terms of the form (sin x ) / x . The contribution of the group to Z(R) is simply all pairs r.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. the two are Fourier mates. In computing I(R). F. 518 (1949).. p ( r ) dr is the (weighted) number of vectors in the structure having lengths between Y and r + dr. Appl.. Debye and A.
For structurcs of uniform internal scatteringlength density. when the molecule is observed at low rcsolution such that R . A function closely related to p ( r ) is P o r o d ’ ~characteristic function” ~ ~ ~ ~ ~ yo(r).3. 8. which is. thc probability that a point a distance r from a given point in the structure will also be within the particle. However. not true of a real moleculc. For real molecules. First. 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. this l/R4 dependence of Z(R) holds rigorously. The radius of gyration of the object can be obtained from the length distribution2: Ri = Jomr2p(r) dr/Jomp(r) d r .3. The intercept of the trend line . can also be estimated. where a is the average distance between nearestneighbor atoms. the trend may be significantly obscured. (8.$ a. I(R)e.3. In fact it should be flat. 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. The largeR data should show a linear trend. (8. of course. but one in which all volume elements have the same scatteringlength density.p will approximate the profile predicted by the characteristic function abstraction of the true molecule.. SMALLANGLE SCATTERING prqfile is the distrihirtion of interatomic vector lengths in the structure under study. where S is thc surface area of the molecular volume.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 .368 8. In practice this relationship is used by plotting the data as R41(R) versus R4. Several intcrcsting aspects of yo(r) can be readily demonstrated. rmaxrthe largest vector in the structure or its longest chord or maximum linear dimension. provided background subtraction has been done perfectly and internal fluctuation contributions are not too severe. nucleoproteins).g.8) For volumes of uniform density.7) where v is the volume of the structure. “ p ( r ) = 4nr2vy0(r). d yo (r )/d r = S/4v at r = 0. averaged over all points. For structurcs with gross fluctuations in scatteringlength density (e.5.
B. Model building is. ~ ~ . 101. ~ ~ : I(O)/Q = U.8. This being the case.9) where ti is the molecular volume and Ap the difference in scatteringlength density between solvent and solute. I15 (1976) . The simplest kind of model building is equivalent ellipsoid determination. one is at the mercy of their reliability. Unhappily. of course. Mateu. Procedures exist for comparing measured data to the profiles of ellipsoids of the same radius of gyration which differ in axial ratio. 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. the trend of the highangle data arc established. J . Tardieu. Without knowlege of the trend of the data this integration would be impossible since data are never taken at large R . Once S and. L. where choices between alternatives are made on the basis of other data.3. and H. no hard rules can be given for how well a model must match the data to be useful. Stuhrmann. the analysis of scattering data can lead to an estimate of the shape of the molecule which gives rise to it. Porod’s invariant Q can be evaluated : Q = 4n /omRzI(R) R d = (AP)~u.p)’u’. T h ~ s ~ ~ . on average.6. models differing significantly in morphology can give rather similar scattering curves. Note that I ( 0 ) = (A. Mol. A . Model building is not straightforward.3. B i d . correspondingly. Luzzati.3. DATA ANALYSIS 369 at R = 0 yields an estimate of S. 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.3. guided by the numerical information whose derivation is described abovc as well as by whatever information may be available from other sources. 8. Finally. 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.3 The “best ellipsoid” 95 V. This shape is then taken as a description of the molecule.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. (8. especially when R is large. (8. Molecular Shape In addition to permitting one to evaluate the parameters just described.
Quigley. and A. Men. however. Press. is a view of the Same molecule as deduced from xray crystallographic data. G . Rev. Hill.” Q. Published by Cambridge Univ. “’ G. M u / . Acra Bid. H.92. .370 8. The equivalent ellipsoid models for E. Two other recent modelbuilding studies by the same group were guided electron microscopic results. Anderegg. a finding recent crystallographic W. Studies on Gtype immunoglobulins in solution demonstrated a T shape for the molecule in solution. Suddath. J . Sneden. Comparison of the smallangle model of yeast tRNAPheand its crystallographic structure. F.)179.C. D.~ crystallographically several years afterwards (see Kratky and P i l ~ and The Figure 3). J. with its principal dimensions indicated. Science ( Washin. S.96“ Pilz model was derived entirely from solution scattering data. J. 3.7. rather than a Y shape as indicated by electron microscopy. Rich. McPherson. J. 44.] so found constitutes an unsophisticated model for a macromolecule whose main virtue is that its limitations are well understood by physical chemists. L. 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 . H. J. SMALLANGLE SCATTERING FIG. 11. London and New York. [Rcproduced with permission from Kratky and Pilz. Such models can be useful. J.39( 1978). coli ribosomes arrived at by Hill et dgSa smallangle data have from 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 .89(1969). A number of studies of this kind have been reported in the past few years. Bielka. On the right is the ellipsoid model of Pilz rr a/. Kim. Ger.D. Kim. W. and M . Mueller. D. “ A comparison of xray smallangle scattering results t o crystal structure analysis and other physical techniques in the field of biological macromolecules. On the left. 817 95a (1974). A. Damaschun.285 (1973). 33. Biophys.qton. E. J . Thompson. J. Bid. Weinzierl. and J . drawn to the same scale.96”The trace of the polynucleotide backbone is shown. ~ ~ More elaborate efforts involve modeling in terms of assemblies of spheres and/or ellipsoids and explorations of more complex shapes. Bottger.9Z arrived at from smallangle analysis.
Not only will the ellipsoid of axial ratio unity fit its lowangle data best. Ostanevich. Spherical Objects If an object is spherical or nearly so. Cser. the model building may have been biased by spurious electron microscopic results. Mol. P. Harrison. Pilz.g.8. the lowresolution data from viruses can be analyzed in a more sophisticated manner than is normal in the lowangle field. 0. M. its radial density distribution. 13. J. Harrison. and B. but at low resolution they are effectively spherical. Nature (London) 266.3. J . A. 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. M. Bid. 28. and Yu. '" I A. 417 (1977). Fishbach. Mol. Witz. IS (1975). Eur. C. In addition. DATA ANALYSIS 371 results have tended to A less useful effort was that made on . Jack and S. Bid. A.16). L. Some of the more complex modelbuilding studies in macromolecular aggregates are discussed below (Section 8. Z. M. c ~ l iIn ~ ~ case.4998 (1973). I . see Kratky and Pilz4).Krdtky. Biol. Mol. 124. but also there will be secondary maxima at higher angles at predictable positions. In most cases phase assignments can be made. S. and D.e.. Both of ~ ' ~ xray l o o and neutron e x p e r i r n e n t ~ ' ~ ~this~kind have been carried out 97 97n I. and M . this fact will be readily apparent from its solution scattering profile. J . Kozlov. C. A. 99 F. 457 (1969).. R. discussed in the next sections. Contrast variation methods. Biochem. The reader should appreciate that direct statements about the distribution of material in an object result from smallangle data in no other case. 42. B i d . Jacrot. J . and J .638 (1965). Sela. it will be true that the maxima in the profile have real phases and alternate in sign. W. . 0. and J. where I F(R)I is the modulus of the Fourier transform of the object's scatteringlength density distribution. Biochemistry 12. i. Rubassay. Gladkikh. Nezlin. Pilz. The classic biological spherical scatters are the small isometric viruses (e. The problems of extending such an analysis to resolutions higher than those where strict spherical symmetry hold have been discussed by Jack and Harrison."' Because of their quasispherical symmetry. 8.3. Harrison. this the DNAdependent RNA polymerase from E . Chauvin. Mol. Jacrot.3. C. J. Chauvin. 68. Witz.205 (1972). I.7. l o ' B . Thus I(R)"* = I F ( R )1. 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. Kratky. Licht. 99.'"' Viruses are complexes of protein and nucleic acid which sometimes include lipid. Anderegg. M . 283 (1976). These viruses are in fact icosahedral in symmetry. FEES Lett. J . "I3 C. can be used to decide where the different chemical components of the virus reside. Ogievetskaya. Fishbach et for review. 641 (1978). A. l o o S.
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. These developments have led in two directions: (i) toward better solutions of the classical problems of molecular size and shape. i.e. this prediction does not hold. Contrast is a concept used abovc without calling it by name. 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. not in shape. is determined.. from which R .g. thc argument being that for small enough R . Contrast may also exist between different regions within the same molecule. and (ii) toward a characterization of the internal structure of macromolecules and macromolecular aggregates. 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 . p o . the intensity of the scattering signal they produce.. 8. however.e. is proportional to Ap2.p o .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. Appl.via/ln~r. and of the effect of variation in solvent scatteringlength density on scattering profiles. Even the initial slope of a Guinier plot of I(R). . 7. 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. i. 173 (1974). Cry. solvent).8. In fact. a molecule is regarded as a shape with a constant 0.3. Ap = p . internal fluctuations will average out. 8. 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.. SMALLANGLE SCATTERING and have provided useful information on the arrangement of the different chemical species in small viruses. In the classic analysis. varies significantly with po in molecules whose internal density fluctuations are as modest as those of a globular protein. The significance of contrast lies in the fact that the visibility of chemically identifiable regions within a scattering specimen. In fact this approximation is a good deal less useful than often thought.3. B. J .9.
' O R H.i. and ? vectors from the center of the uniform solid for the . M. " " L. a = .3. and R . Stuhrmann and R. Carpenter and W.. 905 (1975). and H. 524 (1960). 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 ) .11) where R is the radius of gyration observed when the contrast is Ap.V2P2)/(U. R. Fr. Tardieu. Ci.3. 46. Ci. Moore. D. J. 1"' D. DATA ANALYSIS 373 conditions (see. R . .JoWp'(?)(r)' dr. Substituting this expression into the definition of radius of gyration. first moment of these f l u c t u a t i ~ n s ~ ~ l"o~ u is 'the volume of the molecular shape.r. J . ' I ' C. A. Bcnoit and C . 67 (1977).P/Ap2. 56. H. Sc. Artid.. H. J. Wippler.383 (1976). 105 (1972). 105. K . lo') J.8. Mateu. (Orsuy. 247 (1965). J . Mateu et a1. Koch. V . Chcnz.112: P = (w. 70. Scanu. Tardieu. the radius of gyration of the object at infinite contrast. Luzzati. L. B. and R.S. + u 2 ) . Z . (8. I V fi = ( Jompl(F)Fd r ) 2 . e. B i d . N<irl. Mol. 74. J. 57.3.g. Chim. P. P. fhy. B. ~. M. and V. R. The sum of the two functions p(F) = p + p'(F) then describes the scatteringlength density of the whole molecule. Z . 335 (1967).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 . Mol. Haas. Pro(. R. are thc radii of gyration of the two regions considered separately and F. M.A. Phys. For an object composed of two regions of uniform but different scatteringlength density p1 and p . B. H . Ph. L. + 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. Phyc. whole object to the centers of the two regions whose volumes are v1 and u2111. one can show that R2 = R.. and A.vs. Aggerbeck. I"" I"' . Bid. Cricliton. Biopolymers 16.1°5). Mattice. Engelman. Sardet. Stuhrrnann. (8. Cotton and H. U. Parfait. J .Z + U/Ap . Luzzati. the following expressions hold: where R . is also the radius of gyration of the uniform solid the size and shape of the whole molecule. Stuhrinaiin and R. A. Chem.) 36. Kirste. Benoit. Kirste. 5547 (1977). B.
Z. it is found that: (8. 8.11) retains its utility. When Z(R) is developed from Eq. all of which are sources of uncertainty in ordinary 11) H.. Dependence of the Extended Scattering Profile o n Contrast In studying the full scattering profile of a molecule.(R) is the profile given by a uniform volume of the size and shape of the molecule.1) using this premise.1 0.3. and the value at l/Ap2 = 0 should be l / u . the same decomposition of the molecule’s scatteringlength density distribution into constant and fluctuation parts which leads to Eq. ~ ~ *l ~ The~three com’” 3 ~ ” ponents of I ( R ) are its “characteristic functions. . I. ~ ~ virtually everything else connected with smallangle profiles. 177 (1970). one can fit the values observed for each R with a quadratic expression in A p with secondorder coefficient I . u determination likewise does not require scaled data.14) where Z(R)is the observed data. One obvious point in favor of such a decomposition is that Zc(R). Stuhrmann. Chem. AII the classical methods for the analysis of scattering profiles are rigorously valid for data of this kind. It is clearly the profile to use as a basis for shape determination. like . 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. The contrast match point can be established from data which are not on an absolute scale (see Section 8. where u is the volume of the molecular shape. (8.2.7. Plots of Q/Z(O) versus l/Ap2 should be linear.. relates to the shape of uniform density most closely related to the molecule’s structure.1 1.3. etc. Z.3. 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. and ZJR) the component due to interference between shape and fluctuation s ~ a t t e r . However.3. A p is a calculable quantity once the contrast match point is known..(R) is the scatter due to the internal fluctuations within the molecule. once u and Ap are known. firstorder coefficient I.1). depends on contrast and is affected by scatteringlength density fluctuations within the molecule under investigation. (Wiesbaden)72. .5). SMALLANGLE SCATTERING 8.3. and the constant term I . .374 8. using the chemical composition and density of the solvent..like R. 8. As already observed (Section 8. (8.3. solid angles.” If data are collected at several Ap. Phy. Absolute Scale and Contrast Variation Luzzati et ~ 1 have recently pointed out that Porod’s invariant Q.
1 D2O protein nucleic acid fatty acid carbohydrate " Reproduced. 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. whereas that of 'H is scattering length of 'H is 3.07 +9.11 +4.0 +8.0 +8. .01 +4.44 0. from D. Moore.74 F (1 F = +6. A practical application of these ideas to the analysis of the structure of lipoproteins from smallangle data has been given by Tardieu et al.67 F.3 +12. M. Engelman and P. Scattering Length Densities for Biological Materials" Substance Fully H substituted H2O protein nucleic acid fatty acid carbohydrate Fully D substituted Neutronb X ray 0.44 +6.2 + 14.12.8.3. Table I gives some representative values for p for normal 'H materials and their 'H counterparts. @ 1975 by Annual Reviews. Neutrons versus X Rays As has been emphasized repeatedly in the past.43 8.3. not to mention water.2 + 14. Volume 4. B. with permission. Atztzual Review of Biophysics and Bioengineering.55 +3.1 +9.89 +8.'3 The m).36 $8. TABLE Neutron and XRay I.4 +16.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. are Hrich substances.27 +6.4 + 16.3 +12.54 +7. Inc. DATA ANALYSIS 375 absolutescale determinations. * Neutron scattering length densities are calculated assuming that all exchangeable hydrogen positions are fully deuterated. Since biological macromolecules.
Lipids cannot be contrast matched in an aqueous environment since pI < pHZ0. (An interesting illustration of the difference between the anhydrous and hydrated volumes of a macromolecule was recently given by Stuhrmann and his collaborators. The problem is that all biological macromolecules include significant numbers of hydrogens which exchange at appreciable rates with water protons. SMALLANGLE SCATTERING Inspection of Table I reveals that pHZO less than and pDZ0 is greater than the scattering length of any ‘H biological material. raising p. Thus a wide range of Aps for protonated macromolecules can be explored. 203 (1978). R. the chemical and physical properties of substances inevitably alter with changes in electron density. Ibel. that contrast variation has not been popular in the past in xray solution work.376 8.O and deuterated glucose as the contrast modifying agents. Mol.3. Alteration of p in an xray experiment corresponds to alteration of electron density. J . and R. . therefore.g. Parfait.13.. J. M. Carbohydrate materials (e.O is included in the solvent. these positions deuterate. B i d . The advent of neutrons for study of biological structure has stimulated interest in contrast techniques enormously. opening the possibility of experimental manipulation of the distribution of densities within a macromolecule. Haas. The volume explored by addition of smallmolecule solutes is the hydrated volume. A p = 0. It is not surprising. The 114 H. Manipulations of this kind lead to isomorphous replacement experiments for the study of internal structure. glucose) can be used to raise solvent densities into the region of ppro. H. J . the scatteringlength density of the macromolecule. and do not perturb its structure. 0 method for po adjustment is not ideal. by use of solvents which are H 2 0 / D 2 0 mixtures. R.. 119. but their presence in high molarity is often incompatible with the requirement that a monodisperse solution be examined. Crichton. wider range A can be explored with salts.95 In this connection it should be noted that the D 2 0 / H . Unfortunately. Also. p o . Moreover. B. An “ideal” experiment is one in which alterations in po do not effect p.but can never produce solutions such that po = pRNA. K .) 8. p of a macromolecule can be altered over a wide range in a nontraumatic manner by isotopic labeling. When D. which is manipulated. the range of contrasts available is limited. Stuhrmann. glycerol. Koch.’ l4 who collected contrast variation data on the same object using both D. The Hydrogen Exchange Problem In most contrast variation experiments it is the scatteringlength density of the solvent. including the condition of contrast match.
Mol. Rea. with an aliquot of D20. N. The radius of gyration found at infinite contrast.' The formalism for the perturbations exchange in neutron contrast variation experiments has been worked out to some extent. Ibel and H. but also to an accurate estimate of thc total number of hydrogens that can exchange. Downes. J . Moore. W.'0. owing to steric constraints imposed by molecular folding. Nielsen. Ado. 0.' 1 7 * ' Third. the great majority of the labile hydrogens do ( . M. S. 93. Hvidt and S. Englander."2~"7 Firm estimates for molecular weight and volume can be obtained provided the extent of exchange is properly taken into account.. for example. assuming no exchange and that value compared to po at contrast match.' 5 .8. P. Teitelbaum. If the distribution is nonuniform.3. the perturbation can be significant. Bid. 21. W. within experimental error. Engelman. can be combined with chemical composition data to predict p . Stuhrmann. In most cases.J. If a macromolecule can be obtained both in deuterated and protonated form. Biol. . In a biological macromolecule. 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.75 % or more). 101 (1975). and H . Mnl. Biochem. Prolein C'hcm. the C8 proton in guanine) the rates of exchange are negligible at neutral pH and room temperature. B. V . The difference should be due to exchange. etc. in small organic molecules hydrogens bonded to C are nonexchangeable and those bonded to 0. If they are distributed uniformly through the molecule's volume. however. 01 will be altered only if the center of gravity of the exchange distribution differs from that of the ' ' '' '' 'Is I" A. 41.. tritium exchange methods can be used.g. [Eq. Not all chemically labile hydrogens in a macromolecule will exchange.' Second. and B.1l)] will include contributions due to the distribution of exchangeable protons. 288 (1966). R.104 As a general rule. S. 255 (1975). In the few situations in biological small molecules related to macromolecules where proximity effects labilize Cbonded H (e. no perturbation will be seen. do exchange. then comparison of the density match points for the two forms gives both 0 and the fraction of the protons undergoing exchange.. knowledge of its monomer composition leads not only to an accurate overall atomic composition. D. Annu. ' I 7 K . N . There are a number of ways to obtain this information. 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.3. determined by solution physicalchemical methods. which can then be evaluated. the molecule in question can be deuterated. First. (8. therefore. B. 91. Schoenborn.903 (1972). I L L ( P. it is important to know what fraction of the potentially exchangeable hydrogens d o in fact exchange. 1 In order to make full use of neutron data.
They should be regarded more as a source of frustration in that were they not present.3. For such an object a contrast variation experiment should yield the radius of gyration of the whole structure. A p for each data set is evaluated from the observed dependence of [l(O)/C]i 2 on solvent composition. e. 11.378 8. and fl will be unaltered. N . 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. ."^ From the solvent composition at which Ap = 0. Polym. Three experimental approaches to this kind of information have been used in the past: (i) solvent density manipulation. The scattering length densities of different chemical species vary according to the type of radiation with which they are studied. It should be emphasized that in practice exchange effects are not overwhelming. The third type of contrast variation experiment deserves further comment. Federov. 8. rather than merely a very good one. A straightline relationl ship will be observed for homogeneous sample^. Sci.645 (1973) 1. will now correspond to a volume of nonuniform p whose nonuniformities relate to the distribution of exchangeable protons. 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 . and the changes are different for each species. H 2 0 / D 2 0 contrast variation would be the perfect answer for many smallangle problems. for which many examples may be cited (for review. SMALLANGLE SCATTERING whole structure.14. Ap can then be calculated for all other solvent conditions from 'Iy lZo 1. Contrast Variation in Practice: Radius of Gyration The simplest contrast variation experiment is the measurement of radius of gyration as a function of contrast. neutrons.1V49 (1976). and an estimate of the separation of the centers of mass of the two regions. This is a particularly useful experiment when applied to structures which are aggregates of two kinds of material differing significantly in p. Biol. It exploits the fact that the physics of the scattering of x rays. 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. Radii of gyration observed will differ with radiation and give information about the issues in question. and light differ significantly. Serdyuk. . see Jacrot". the radii of gyration of the regions occupied by the two species.'" I . 27. Tardieu et (ii) solute density manipulation"2.' l 8. N.. J . 0 composition.A. in the ideal case the characteristic function for the uniform volume. Srrdyuk and 9. t?rookhac>enSymp. pis estimated in absolute terms with due allowance for exchange.g. They are not large enough to upset the qualitative validity of the results of neutron contrast variation experiments.
The slope of the plot I/Ap = 0 gives a and its curvature 7 !. p curvature will always be concave down. Nulure (London)253. S . the opposite is true. 177 (1978). Talchell. P. 72. Marguerie and H. S. .8. K. J. 13. S. and R. Nucluic A d s RPS. in "two phase" aggregates.S.'30'3 1 and fibrinogen. 139 (1977). .2. In general. Crichton. Rex Commun. and IF. J . J . ' J I J . and B. R. Baudy. Koch. Haas. i. Biochm. Brookhuren Symp. Nut/. J . J ... are contrast matched. 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. Mo/. Bram. G .' 1 2 ~ 1 2 8 * 1 2 9rhodopsin in detergent micelles. There are a number of ways of plotting the data to evaluate their trends. Stuhrmann. ''* H . B. . K. Osborne. U .A. R . Ibel. CeN 10. Commun.Petersen. Biol. Wooley. 2163 (1975). 13 in situ) as well as values for R . are obtained from the plot by picking off the values for R at the contrasts where phase 2 and phase 1. R. Worcester. Bram. E. J . Stuhrmann.e.Biol. P.p o . P." ' nucleosomes and other chromatin related materials. 1043 (1975).l [see Eq. 245 (1975). Res. B. Suau. GrunbergManago. Ibel. Knedle. Biophys. J. Biophys. Acud. MichelVillaz. Thus the quantitative estimation of R . De Wolf.13)] from a and . Biochum. Stuhrmann. Nail. Proc. The quality of these estimates can be greatly improved by studying the structure in a variety of deuteriumlabeled forms. M. H.. . D. intercept at I/Ap = 0 is R:. Baudy. respectively. M. Aerrd. and K. Lc Pault. and B. B. C. Proc.112 Neutron contrast variation studies have been done on a variety of twophase structures including ferritin. M . and A . 1113 (1976). Hjelm.. 399 ( I 976). Le Pault. and K. 72. and IF. Van Holde. Ibel. M u / . Baudy. Ibel.  YzJ. and R. 176 (1976). G . and R . 4. G. Bram. 27. J. G .3. 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. 0. P. Hermann. Jacrot.' 22' 2 7 ribosomes. Yeager. Richards. Koch. M. U.I3' "I H. G. M. M . Bradbury. P. Pardon. 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. L. Parfait. H. Biol. Crichton. R. If a is positive. R. Boseley. D. A . B. S. 100. H. E. B.!7 will only be as good as the available information about the volumes occupied by the phases in situ. Sardet.3. 2379 (1976).U. Perhaps the The most convenient is to plot R 2 versus l/Ap (see St~hrmann"~). F. R. P. J. and M. Kitzes. Vastel. Mol. P. and P. J. Sci. 12Y P. 1 3 " M . Nuckic Acids Res. Beaudry. Bradbury. Baldwin. (8. Ap = p . C . K. K. Baldwin. ButlerBrowne. Chabre.. if a is negative. 102. M. 1 3 ' H. DATA ANALYSIS 3 79 knowledge of solvent composition and density. 123. 72. J. and E. 143 (1976). P.e. Such an investigation can lead to accurate estimation of the volume fraction occupied by each phase (i. Bid. high p regions in the particle must lie towards its exterior. 2275 (1978). 391 (1976). Haas. Sci.
Parlhit. and 0. Srruc. 0. The ribosome studies have produced the only data available on the relative distributions of bulk protein and RNA in these structures (see also Serdyuk'"). Nucleic Acids Res. P. A number (for a ? ' ~ ~ of lipoproteins have been i n v e ~ t i g a t e d ~ ~ * ' ~ ~ . 40. W .3. B. ( R ) by model building just as described in Section 8.(R) as its which input. Shapes can bc derived from i . J.73 (1978). Kratky. J . Stuhrrnann. however.380 8. Moras. Laggner and K. the shape scattering profile or at least an approximation to it. 8. Biuphys.r. 2421 ( I 977).Glatter. 13'" H. 213 (1974). J. P. Giege. R. A . 1 3 4 R. 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. As already emphasized. B. Haas.'" The paper of Tardieu et panion theory paper95 contain a particularly detailed exposition of the analysis of two phase systems. . ' 35 K. more deductive approach has been suggested by S t ~ h r m a n n ' ~ ~ " requires I. Thierry. ' ~ ~review see Laggner and Mueller' 3 7 ) . H. 82. Contrast Variation in Practice: Extended Scattering The analysis of contrast variation data sets into characteristic functions is still a relatively new method. Zc(R). 251 (1978). 4. B. a second. Mueller. Biophys.3. Q . The 30 S subunit structure has been recognized as a reasonably homogeneous mixture of RNA and protein. G . Holasek. and recently the rhodopsindetergent system has been and its comstudied by these same means. B i d . some extensive xray studies have also been undertaken on twophase systems. 11. Mueller. Mech.C. and H.15. with protein tending to form the exterior surface of the particle. 0. 1 3 7 p . IV3 (1976). With the increase in interest in contrast variation methods. Kostner. Koch. 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. Zaccai. J . Laggner. and G. Eur. FEBS Lett. Kostner. Recently. 27. R. 371 (1978). Under these conditions it was possible t o show that at low ligase concentrations a 2 tRNA: 1 ligase complex forms.'33 Giege et have done an interesting study on the formation of complexes between tRNA and tRNA ligase by contrastmatching the protein. Stuhrmann Brookhawn Symp. and G. D. while the 50 S subunit shows considerable segregation of the two species.6. Res. Mueller. I"' K. Jacrot. The conccpt behind the approach is to regard the density distribution ' 3 3 M. Glatter. Biochrrn. Laggner. R. 4. Crichton. is the best possible input into a shape analysis.
H. coli ribosome. B. but its limitations are still unclear. Fibrinogen has also been analyzed. Parfait. nucleosomes and other chromatinrelated structures. One of the first was ly~ozyme. Two methods have evolved for studying multisubunit structures which might be described as (i) "synthesis" and (ii) "distance finding. First.297 (1970). Proc. Acra Crystallqr. For an object undergoing exchange. Crichton. H. Haas. R. contrary to the assumption of uniform density which guides the choice of coefficients for harmonics in model building. which are affected by exchange.A 26. and R. and its solution scattering profile directly reflects the magnitudes of the coefficients of this expansion. Sci. viruses. In this class of structure one would include multisubunit enzymes. Fuesa.(R) obtained corresponds to an object of nonuniform density. K.67 (1976). and lipoproteins. Stuhrrnann.3. B.3. 13' . most of the data used have been neutron data. SCTI. DATA ANALYSIS 38 1 of a molecule as the sum of sphericalharmonic density w a ~ e s . M.S.8. Only a few structures have been dealt with in this way.'i2~"8 For structures with more than two subunits. J. ribosomes. Sluhrmann and H. A A32. Natl. Macromolecular Aggregates: Synthesis Methods In the past decade an increasing amount of lowangle work has been done on macromolecular aggregates.16. Koch. Stuhrmann.. At some level of description many of these can be regarded as twophase problems and approached by contrast variation as already indicated.(R) object.A. The results of this kind of analysis are intriguing. 2316 (1977). these methods are not readily applicable and other means must be used. the Z. Ibel.'37aTakingthese into account one can deduce the coefficients from the data within certain limitations and can then calculate models. Acta Crystuiiogr. ' ~ ~ A~ ' ~ ' ~ ' unique decomposition into spherical harmonics exists for any object. considerable restrictions are placedon thevaluesthesecoefficientsmayhave. 1 3 " H. B. For a discussion of the impact of these problems see Stuhrmann et 8. 74. There are two problems with the analyses done so far.I3' as have the subunits of the E .'~* which a shape quite close to for that of the crystal structure was obtained. 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. J. Acud. the impact of error in the input data on the final result has not been worked out. as is true for the Z. R.. U. Second. The 50 S structure'39 obtained resembles the shape the object appears to have in the electron microscope. Sect. multienzyme complexes." Synthesis H. If the object in question has uniform internal scatteringlength density.
C'rysfaNoqr. 'The work of Stockel et ~~ on the hemoglobin of Tuhfex can also be cited.12)]. These two techniques are thus suitable for the analysis of different kinds of structures. 37. separately and the R. J . V. Ger. 14' G . measure its R . of the ternary complex. 21. is to determine the disposition of the subunits within the aggregate. P.3. as well as that of the whole aggregate. in either case. and R. 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. Eur. 309 ( I 968). M.79 (1977). Purschel.479 (1978). Reich. Mecl. (8. Moore.382 8. by Pilz and colleagues is a fine example of ~ y n t h e s i s . Stockel. l ~ ~ ' 4 1 G . G . The objective of these analyses. Witters. One could imagine a third protein in a complex with the other two. Lontie. R. J . but even this approach is of limited a p p 1 i ~ a b i l i t y . Onecan also study the shapes of the subunits and those of the subcomplexes they may form. Appl. The parallelaxis theorem is the proper formula for combining radii of gyration. 11. Biochem. f 2 the corresponding quantity for the inhibitor. whereas distance finding comes into its own for structures containing single copies of several different kinds of subunits. . Berger. A.15) wheref. 193 (1973). A. 21. B. SMALLANGLE SCATTERING is the only approach possible for aggregates containing many copies of only one or two subunits. Pilz. is the fraction of the total scattering length of complex relative to solvent contributed by trypsin. P. V. and for two subunit systems gives results equivalent to those obtainable by contrast variation [Eq. 80. ' 4 3 P. Engelman. 14* J. Ger. I. Fichtner. using extended scattering curves. and R. and D. Actci B i d . Mayer.3. J. J . Model building is then done in which the objective is to match the whole structure's scattering profile by assembling aggregates of its substructures. Purschel. and J. Damaschun. which is an aggregate of 200 subunits. and so work up to a description of the whole complex in terms of subunit radii of gyration and locations based on relative distances. and their distance apart on the complex inferred using the parallelaxis theorem: (8. 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. Acru B i d . Med. Biochrm.865 (1968). Damaschun and H. and A the distance between their centers of mass. Eur. Keller. The analysis performed on haemocyanin. Langer. 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. H .
(R) = CI12W) + w . lsr. 2(R). Sosfenov. Dokl.and for the unlabeled structure I(R).een repeatedly pointed out theoretically and demonstrated in model system^. + D. Let F= 2f1f2 sin 271Rrl 271Rr. B.3. 145 146 1' 4 1 4 ' 0. Chum. the distance between the two labeled points. Mol. C/zmi.17 . 76. 263 (1947). Then Z12(R) A B = Il(R)= A + B 12(R)= A + C I(R) = A. Akad. and f2 are the scattering lengths of the heavy atoms and the sums run over the unlabeled atoms.C I l W + I2(R)1 = F. + + C + D + E + F. + E. Vainshtein. Nauk S S S R 190.f. Kratky and W. Hoppe. DATA ANALYSIS 383 8. Worthmann. N .3. d . . ( R ) and Z2(R). J. wliere. A. Feigin. W. I. Macromolecular Aggregates: Distance Finding Over the years the possibility of measuring distances between points within a large structure by xray solution scattering has b. 78. 1. 10.. and L. Thus measurement of I.574( 1970). W. 581 (1973). F is a damped sinusoidal ripple whose nodes occur at regular intervals R = n/2r12 in R. 321 (1972). Monutslz. 1 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.'^^'^^ The points in question are labeled with heavymetal atoms by chemical means and solution scattering profiles obtained for the doubly labeled species I . K . Thus if profiles are normalized to equal numbers of molecules and total flux.(R) leads to determination of r I 2 . both of the possible singly labeled species I .8. B i d . Hoppe.
Mu/. 149 The signaltonoise ratio for such an experiment is 10 to 100 times better than in its xray analogue. another difficult step. A p j ~ / C'r)~s/d/ogr. Stockel. Langer. and K . In addition. the ribosome and thc DNAdependent RNA polymerase. May. W . . B. So far this requirement has limited work to structures from bacterial sources. neutron radiation. Moore. and P. D. SMALLANGLE SCATTERING H ~ p p e ' ~ ' . J . which is a foursubunit structure. At the time of this writing. H. i. Mol. 2. Cjeka. 176 (1979). Some comments about data interpretation are in order for these experiments. the profile of the mixed samples obtained at high concentration. Engelman and P.e. ' pointed out a unique feature of this type of measurement. Engelman. in order that the required placement of deuterated subunits be achieved. I51 P. Crespi. So far. A . Hoppe. J .I5l The results are generally in good agreement with other data available on these structures. When whole subunits are labeled rather than simple points. L. Moore. the determination ~ ~ placement of 9 of its 21 protein subunits. 12. J . D. J . Strell. U .72. Engclman. has been fully analyzed. Schindler. The structure must come from an organism which will grow in the presence of high concentrations of D. I . R.O. J . 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. ~* namely that it can be carried out with specimens at very high concentration. D. and the same done with the two singly labeled samples at equally high concentration. Moore. I x ( R ) D. ~ ~ l ileading~ to ~ ~ * ~ ~ of the . The biochemical requirements for such measurements are stringent. it must be possible to disassemble it into its subunits and cause it to reassemble. 595 (1979). two systems of considerable biological importance have been worked on. only biosynthesis leads to levels of D incorporation into macromolecules sufficiently high to make labeling useful. M. Proc. over 30 distances have been measured within the 30 S ribosomal subunit of E . 134. S . No//. 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. 119. If the doubly labeled and unlabeled samples are mixed in equivalent amounts. Heumann. Nevertheless. the structure must be reconstitutable.463 (1978). J . AcuJ. B. B. Ibel.. Is" . Sci. and P.384 8.3888 (1972). of course. the subtraction will still yield l x ( R ) . The possibility was envisioned of mapping the positions of subunits within aggregates by triangulation based on distances. W . A. M. 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. H. M. B i d . Zillig. Katz. the DNAdependent RNA polymerase. Bid. E.
M. one can make use of the fact that 7 = Joa)r2p(r) = d f 2 . Langer. contrastmatching out the protonated parts and measuring the necessary Ris directly.3. being influenced by subunit shapes and relative orientations. just as in the twoatom case.' For less complex structures the possibility exists of deuterating single subunits.18.'^^" 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. Mol. B. A. and h.I. 321 (1979). becomes (for neutrons) DATA ANALYSIS 385 where b. and dl. dr + R: + R:. H and 'H. Alternatively. 12.3. and D. will be 5 2 : d. but is not. B. The possibility of analyzing such data for subunit shape and relative orientation has been pointed out. P. . Engelman..8.Appl. are the scattering lengths of .'~' 8. Bid. 112.(R) can be inverted to obtain its corresponding p(r). p(r) in this case will be the length distribution of all vectors joining d in the two subunits.s/u/lo. r = Jomip(r) dr.qr. . First.. lSZa J. where R .. but no general procedure has yet been di~covered. are the radii of gyration of the two subunits as they are configured in sit^. Moore.B. Two methods for obtaining distances commend themselves. and the sums run over all deuteriums in subunit 1 and all deuteriums in subunit 2. C'rj. Moore and E. Schoenborn. This function looks superficially like a damped sinusoidal ripple. I. and R . 199 (1977). The periodicity of its ripples is irregular. respectively. P. the distance between subunit centers of mass. Although this is the most common P. $1. Weinsiein. 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.
with lysozyme coming in a distant third. The lysozyme work of Stuhrmann and Fuess’ 3 8 has been described ’ ’’ H. and (ii) xray and neutron results on the same molecule are consistent. Robbin. A .’ 53156 Given the problems to be overcome in such a calculation. 1 5 4 J. Surnmw Sch. 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. as is the case with proteins. and B. (8. SMALLANGLE SCATTERING application of these techniques. Shchedrin. (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.qr. L. 15‘ W. .51. Tech. Kayushina. Feigin. L. Biopolvtners 16. 1975. in “SmallAngle XRay Scattering” (H.2. p. The purposes of such studies have been several: (i) as benchmark investigations to establish the validity of smallangle analysis. 701 (1973). Myoglobin has been investigated by Beeman” and Stuhrmann. (ii) surface polar residues are often poorly located in a crystallographic structure because of intrinsic disorder. Is’ Yu. Soler.1) is cumbersome to apply for molecules in which the number of atoms 2 1000. K . Gordon & Breach. they have also been used from time to time to study macromolecules whose structures are known at high resolution.). 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. it is perhaps not surprising that calculated radii of gyration commonly differ from measured values by 0. The favorite objects for benchmark studies have been hemoglobin and myoglobin. and (iii) to investigate conformational changes connected with ligand binding or alterations in environmental conditions. Kristallngrufiya 18.0 A in 25 A. This is not a trivial computation for a number of reasons: (i) Eq. Mattice and D. lnt. Watson. C‘omput. New York. M . Brumberger. Carpenter.386 8. A critical step in such investigations is the computation of an expected scattering curve from the crystallographic data. This suggests that myoglobin in solution may be a more flexible structure than it appears to be in the crystalline statc. A. 1967. and (iii) there is the solvent problem. These problems lead to an interaction between goals (i) and (ii) of the preceding paragraph. 81 (1977). A number of investigators have suggested approaches for making these calculations. the highresolution solution scattering curve shows much less structure than anticipated from the crystal structures. C. L. ed. R . 376 (1976). 267. Cryslollo. Proc.
sio/. 231 (1969). Durchschlag. Phj. Damaschun. Hossfeld. Schmatz. W.~~ have demonstrated a small volume change in yeast Durchschlag et glyceraldehyde3phosphate dehydrogenase as a function of saturation with NAD. Scherm. Sfud. Schmatz. 1. '('' G.V. l b 8 P. G. J . I h h 1. Eicher. 217 (1972). 0. 0. 851 (1969). Both xray and neutron ) . Schneider. Conrad. Purschel. J. "" H . and J .. and F. Bid. 17. Camejo. 350. J. I"' H. J. and R. Mateu. Ninio. H. H. 7 5 3 5 (1977). Vogel. J . Kratky. ~ . B i d . V. Mayer.ler's Z. B i o p h ~ ~ . Biochem. The change observed is once again close to that anticipated from crystal data. Schelten. Mucller. 351. Schmatz. they also demonstrated a substantial alteration in radius of gyration of hemoglobin upon oxygenation and dcoxygenation. other studies of the same kind have been done on lipoprotein^'^^ tRNA. A .'659'6h a n t i b o d i e ~ to name a few other systems. data have been c o l l e ~ t e d ~ ~ ~ ~ ~ ~Damaschun et ~ 1 . most notably by the Julich group. V. Bioclirm. Ruchpaul. R. There have been a whole series of solution scattering experiments done on hemoglobin. Krigbaum and Kugler' 5 7 have also examined lysozyme by xray methods as well as chymotrypsin and chymotrypsinogen.8. Hq~prSiylc. In addition to hemoglobin. Small changes in structure have also been observed in malate dehydrogenase upon substrate binding. Puchwein. Thomas. R . 27 (1974). and Y . I h 4 L. H. Moshe. Schneider. 9 (1971). H. Mayer. P. B i ~ d ~ m i s t r j ) 1436 (1978). Schelten. 9. 30. 1499 ( 1970). Cl7rm. Eur. G i r . Luzzati. 47. 71. A . B. and H. Damaschun. 87. and . In addition to verifying the possibility of collecting neutron data and showing the close correspondence between the solution and crystal structures. Eur. I h Z R . A. "'I R. Durchschlag. W . Mayer. V. ""' J. 119 (1978). Schneider. Mayer. Mol. Conrad. W. and R. T. Zinke. B i d .169 and a useful critique of the information one can derive from studies of changes has becn prepared. J . 69. DATA ANALYSIS 387 above. Simon. ' ~ ~The (see also . PIz~~siol. Kaiser. Chrm. Physiol. von der Haar.' ~ ~ neutron data are significant because they are among the first ever obtained on a protein. Mol. Malnig. K righaum and F. R. and M. Kirchauser. and K . J . and F. 85 (1978). Mayer. Zipper and H. 41. 19. HoppeSeyler's Z. Biocheni. Kirschner. P. Schuster. 845 (1969).I7' The point is that for molecules of known structure. I"' J. Bioc~hmistry 1216 (1970). Biophys. J . Eitr. Chrm.Kratky. Mol. 151 ' Is" . 41. I h " 1. H o p p e ~ Srj. 184 (1972). Sttjckel. K. J. A . S. Biochiw. A. Stud. Kugler.3. and G.225 (1969). Pilz.'68 Efforts have been made to develop an ultrasensitive difference method for detecting small changes. 350. The idea of using smallangle methods to monitor conformation changes is likely to be a significant application of the method in the long term. W. Schwaiger. H .r'sZ.
’’ 8. although the smallangle data must necessarily bc consistent with the correct shape. Osterbcrg. 153 (1973). whatever it is. 77. 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. etc. Lagerkvist. 0. Concluding Remarks 8. 394 (1975). J . SjBberg. 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. W . Mol.1. B. which limit its accuracy in this regard. R . Smallangle data. 59.. 99. distortions during sample preparation. R . The strength of the smallangle approach lies in the accuracy of the size and dimensional information it produces. Garrett. and V. J . SMALLANGLE SCATTERING minor alterations in smallangle scatter which in the absence of other information might appear totally mysterious are often interpretable. J . Bid. Mul. Pilz.4. This R. if a molecule has a highly unusual shape. The resolution of the description produced by a smallangle analysis corresponds roughly to the best electron microscopy can do with favorable macromolecular specimens. The formation of complexes between macromolecules can also be usefully followed by smallangle methods. Biochern. Kratky. L.388 8. of course. ”’ . 63 (1975). G r r . In the case of aggregates it is sometimes possible to go beyond this “whole molecule” description to specify how its subunits are arranged. Osterberg. report structure directly and offer unique opportunities for studying the subtleties of macromolecular action in solution. B i d . its shape is more likely to be identified correctly from electron microscopic data than from smallangle results. 1. It will undoubtedly become common to test hypotheses about the conformational properties of macromolecules by smallangle scattering. In electron microscopy there are always problems of visibility of molecular edges. On the other hand. Summary Table I1 briefly summarizes the most important kinds of information one can extract from smallangle data on macromolecules or macromolecular aggregates. Finally. 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. and G . Rymo. Stoefiler.4. Folkhard. 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.
There is an additional .4. Critique Lowangle scattering.qre. A typical macromolecule contains 103105 atoms and requires 3n . The numerical data given by smallangle experiments constitute solid information.q. subunit shapes and orientations fact makes smallangle techniques useful as a way of testing models and assessing conformational changes. as equivalent ellipsoid or better I. especially the more complex ones. In any case nothing approaching a full description of a macromolecule is ever possible. Models deduced for molecular shapes. 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. may raise that number severalfold more. Macromokcuior a. A wellcollected solution data set might permit one to specify a length distribution as 15 or 20 independent numbers.8. 8. distribution of interatomic distances 5 .2. Internal labeling methods. surface area 6 . SmallAngle DaVa A. CONCLUDING REMARKS 389 TABLE Principal Molecular Parameters Deducible from 11.4.yaiesonly I .6 coordinates and n scattering lengths for its full description. a failing this technique shares with other lowresolution methods. is a lowresolution technique. as already emphasized. A fullcontrast series might increase the number of specifiable parameters by three. molecular weight 2. however. and the effective lumping of vectors into classes according to length does the same for the scatteringlength information. In a crystallographic study the covalent structure of the molecule under investigation eventually emerges. No comparable internal control is available for the models deduced from smallangle data. subunit radii of gyration 3. lowresolution picture of the internal distribution of high and low scatteringlength density regions 0. position of the centers o mass of subunits f 2. All maruomolecules I. which involve large increases in experimental effort. molecular volume 3. Its correspondence with the molecule’s chemistry verifies the correctness of the analysis. radius of gyration 4. molecular shape. must be approached with caution.
First. smallangle methods are a sensitive means for examining the relationship between solution and crystal structures for biological macromolecules. a smallangle opportunity exists at the very outset of an investigation of a new biological macromolecule. Smallangle methods can rapidly yield a molecular weight and good estimates of size and shape. 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. and in such situations the means and the ends are well balanced. Second. traditionally valuable information. have contributed to this work by furthering my education in smallangle scattering: D. P. Engelman. there arc a number of large biological structures which are simply not suitable for crystallography at this time. in the electron microscope. This work was supported by a grant from the National Science Foundation (PCM7810361). smallangle methods have no rival as a means for testing models for structures. M. for example. Third. both consciously and unconsciously. Schoenborn. Many macromolecules have very eccentric shapes when viewed. or both. for reasons of size or lack ofcrystals. . and V.390 8. The ribosome is a prime example of such an object. The validity of these shapes as a description of the molecules in solution can be examined by smallangle means and by virtually no other. Luzzati. B. Similarly. For some of these it is also true that lowresolution information is relevant to the kinds of questions biochemists are asking about them. The fourth opportunity is the study of conformational change and binding interactions of wellunderstood structures. SMALLANGLE SCATTERING shortcoming common to all lowresolution techniques: most biochemical problems ultimately require structural information at atomic resolution. It is in this last area that one anticipates the most growth in the years to come. Acknowledgments 1 wish to acknowledge my indebtedness to those who. It is conceivable that synchrotron radiation sources and highspeed detectors may prove of great value for investigations of this kind.
Hall. that the specimen structure should not change during the period of electron irradiation while the image is being recorded. and particularly for the biophysicist. provided that the specimen materials are prepared in a suitable form. 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. ELECTRON MICROSCOPY By Robert M.” 2nd ed. these experimental methods are in many cases still at an incomplete stage of development. McGrawHill.9. 1966. C. R. that they can be placed into a vacuum of lop6 Torr or better. 39 1 METHODS OF EXPERIMENTAL PHYSICS. Glaeser 9. Therefore. however. D. Highresolution images can only be obtained. “Introduction to Electron Microscopy. 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. Among the requirements that must be met are that the specimens should be very thin. finally. 1964. lnc. 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.1. The purpose of this article is first to describe some of the current developments in methods of electron microscopy. placing much of the emphasis on the new methods that have been developed for molecular structure determinaation. E. As will be seen. Heidenrcich. Such is by no means the case. “Fundamentals of Transmission Electron Microscopy.” Wiley (Interscience). New York. New York. VOL. and. In the field of electron microscopy there still remain many challenges for the experimental physicist. ISRN 0124759629 . 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. All rightr ofrepruduction in any form reserved. ’ 20 Copyright @ 1982 hy Academic Press.
At 100 keV and with a realistic value C. and an electron energy of 100 keV. in all cases the images so obtained are frequently subject to ambiguous or even misguided interpretation. where d is the resolution. is the coefficient of spherical aberration and . where C.2 A. Such highvoltage electron microscopes were initially designed to make it possible to work with thicker specimens than can be used at 100 keV. 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 .5 A.037 A. and a variety of research instruments going to energies as high as 3 MeV have been in operation for several years. In some microscopes.0 mm. the depth of field works out to be approximately 165 A. However. then the depth of field is given by the expression AZ = d2/2A. Improved “resolution” can also be obtained with tilted (nonaxial) illumination and in the darkfield image mode. this resolution limit works out to be about 3. 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.0087 A). the contrast transfer function continues with appreciable magnitude out beyond the point of the first zero. In practice the resolution experimentally obtained with biological specimens is limited in most cases to approximately 2530 A. under proper conditions of instrumental operation. but it is important to have this factor in mind when going for very high resolution. 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. but depends upon the resolution at which the image is being examined.5 A.5 8. It is the spherical and chromatic aberration of the objective lens and not the electron wavelength which limit the resolution. For a resolution of 3. not just a single value.392 9. At this energy the electron has a relativistic wavelength of A = 0. For 100keV instruments this resolution limit is approximately 3. The depth of field is. is the electron wavelength. Electron microscopes are also commercially available which operate at an energy of 1 MeV (A = 0. under conditions of optimal defocus. and it is prudent to assume that the limit of interpretable contrast is given by the expression f(C. or worse. = I 1. A3)”4. If the objective aperture is optimized for the resolution that is of interest. This sort of specimen thickness is not a limiting factor for biological applications if the image resolution is not as good as 3. This very short wavelength is two orders of magnitude smaller than the actual resolution limit of the electron microscope. 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. because the electron optical lens aberrations cannot be corrected in the same way as is possible in light optical systems. of course.
There is no reason. This limitation is not directly due to any instrumental factors. 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. why the spatial averaging technique cannot be used to obtain experimental results down to the instrumental limit of resolution. together with one or more inelastic processes. that is to say. 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. there may be the problem of poor specimen preservation. in principle. The imaging of inelastically scattered electrons therefore provides structural information that is not obtainable from the images produced by elastically scattered electrons. the .1. where the spread in energy loss values can be very great and where multiple scattering events involving one or more elastic processes. A VERSATILE TOOL FOR STRUCTURE DETERMINATION 393 investigation. hydrated specimens. 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. In discussing the information available in the images produced with inelastically scattered electrons. First. cause the inelastic electrons to be scattered out to quite wide angles. The effect is particularly troublesome in the case of thick specimens. Overcoming these technical difficulties represents an area of active research development at the present time. This information is essentially of a spectroscopic nature. one must remember that the spatial resolution is once again limited by radiation damage. With sufficiently thin specimens and careful control of the radiation exposure. 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. However. However. When there are a large number of inelastic scattered electrons. Even higher resolution can be achieved on unstained. numerous technicaI difficulties still stand in the way of this achievement. The inelastically scattered electrons in themselves carry a great deal of useful information about the object structure. a resolution of approximately 1015 A is possible on certain stained and unstained biological specimens. not to mention possible embedding and sectioning. 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. 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.9. A second factor which can limit the resolution attainable with biological materials is the serious problem of radiation damage.
As a result. The preponderent number of inelastic scattering events occur at energy losses of 350 eV. It is true that an energy filter lens can. and H . Opt& 41. At the same time. in principle. 92 (1974) . which may in turn impose a severe limitation on the attainment of high resolution in the image. provide the localization of the “chromophore” to a resolution better than 1020 A. Other areas of major instrumental improvement also deserve mention.394 9. J. An alternative direction to take for improving instruM. for example. in principle. 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 can be said that inelastic electron images arc best obtained with a scanning transmission electron microscope equipped with a suitable energy loss spectrometer. 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. In fact. images produced with these inelastically scattered electrons cannot. The reduced frequency of such highloss events greatly reduces the signaltonoise ratio in the measurements. Langmore. Isaacson. P.3 Higherresolution images can in principle be obtained with electrons that have suffered much larger energy losses. 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. If a resolution of only 2030 A is desired. there also remains considerable need and opportunity for improvement in the design of spectrometers for scanning transmission electron microscopes. 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. Rose. The amount of delocalization of such excitations is in the range of 1020 A. However. The continued development and refinement of highvoltage electron microscopes offers a promising direction for the achievement of higher resolving power. highvoltage electrons have a distinct advantage in overcoming the multiple scattering processes that can seriously confound image interpretation. the cross section for such events is smaller by about a factor of a thousand than is the case for the lowerenergyloss events. energy losses that would be produced in the excitation of innershell electrons. In terms of instrumental approaches. be developed for the normal imaging mode in the transmission electron microscope. But while such highloss events are quite well localized. 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.
In particular. therefore. 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. the effects that can be seen in the image intensity are the same as if the electrons had actually been absorbed in the specimen. 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. On this basis.2. 9. on the basis of considerable experimental experience. it should be mentioned that the resolution limitations of presentday electron microscopes could be quite directly overcome by the use of image processing techniques. the resolution limitation normally associated with the spherical aberration of the objective lens is. Thus. and in the subsequent discussion of contrast transfer function theory and methods of threedimensional reconstruction. 9. where the specimen is thicker or has . Image Formation in the Electron Microscope In discussing image formation in the electron microscope. their experimental realization remains still as a great challenge. one which can be easily corrected for provided that the image formation occurs under conditions of very high coherence. we shall proceed to “forget about” the inelastically scattered electrons.3.7. Finally. we shall restrict our attention to a description of images that are formed by the elastically scattered electrons. in fact. IMAGE FORMATION IN THE ELECTRON MICROSCOPE 395 mental resolution is the development of devices for correcting lens aberrations. only a systematic error.9.2. 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. In this view of the origin of image contrast. The use of images formed by inelastically scattered electrons will be dealt with again in Section 9. Although the theoretical possibilities have been quite clearly defined for some time.1. 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. Fortunately. one can now say.2. 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.
if the specimen is sufficiently thin and the mass density is correspondingly small.” such that phase contrast image effects are negligible. Clearly. where x is a vector in the plane of the image. that is. a linear function of the projected mass density of the structure. it should be stressed that the derivation assumes that the object structure is amorphous.pL(x)I.12. where p(x. z ) d z = kp’(x). According to the approximations of Zeitler and Bahr. In this limiting case.” and the image is correspondingly darker. under suitable conditions. the image intensity would vary exponentially with the local “absorption coefficient. the exponential function can be expanded to give I(x) Z Io[1 . The parameter k is an instrumentdependent constant which depends upon the objective aperture semiangle and the incident electron energy.396 9. Zeitler and G. This whole idea of “ pseudoabsorption” or scattering contrast was translated into terms of practical use to biologists by Zeitler and B a h ~ . the derivation implicitly assumes that the image is taken in the “infocus condition. Furthermore. there is more scattering and therefore more “absorption. the image intensity at a point x in the image is given by w= 10 expc . F. Other possible sources of intensity variation in the transmitted electrons. 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. p(x) = k I p(x. Exp. but which does not depend upon the properties of the specimen itself. ~ who derived the result that.. 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. in the sense that the angular dependence of the scattering intensity should be the incoherent sum of the individual atomic scattering intensities. Bahr. In addition. such as E. z) is the mass density at each point in the object and z is the direction of propagation of the electron beam. Cell RL‘s.k p ’ ( ~ ) ] . more precisely.” 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. ELECTRON MICROSCOPY greater density. the image intensity can be seen to be a linear function of the projection of the structure. 44 (1957) .
. In practice. Jap and Glaeser7): Y.. ’ J . However. even under conditions when its derivation is clearly no longer valid. ” 9. However. A A34. the transmitted wave Y. 1968. Nevertheless. (Oxfi. The wave optical approach to image formation automatically takes into consideration the effects of lens aberrations.9.rd)17. as well as all other factors that may affect image contrast by the elastically scattered electrons. for example. tt is Planck’s constant divided by 2n. B. IMAGE FORMATION IN THE ELECTRON MICROSCOPE 397 inelastic scattering processes. 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. V’(x) is the projection of the Coulomb potential within the object in the direction of propagation of the incident beam. K. that is. 94 (1978). 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. Microsc. M. and the “absorption”ofe1ectronsby the objective aperture.2. can be written as the product of the incident wave Yoand a transmission function which is linear in the scattering potentiad (see. R. which tends now to be more fashionably referred to as “Fourier optics” (see.” McGrawHill. W. This treatment. In the equation above. Glaeser. c is the velocity of light. Sect. Under these conditions. Goodman. 77 (1979). for example. making use of AbbC‘s “diffraction theory” of image formation.2. “Introduction 10 Fourier Optics. automatically includes the “mass thickness effect” described above. this picture of image contrast is useful for the average cell biologist.(x) = Yo(x)[l  (i/hc&V‘(x)]. 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. therefore. A m Crystulloyr. J . Jap and R. particularly at very high resolution. and e is the electron charge. B is the relativistic electron velocity divided by c. Goodman’ or Glaeser6). are also neglected. New York. M. image defocus.2. Glaeser. it is still necessary for us to neglect the effects of inelastically scattered electrons. the massdensity approximation is not at all suitable for modern applications of the electron microscope in the area of molecular structure analysis.
coherent . that is. we pause to introduce some of the complications that can invalidate the approximations which we have so far employed. if we make the approximation of perfect coherence. More explicitly. which can occur even as the wave propagates through the specimen. 9. 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). 2. At present there are two effects that are of concern to us. The first is the fact that the electrons that are inelastically scattered may just as well have been totally absorbed. These strong scattering situations are effectively the same as those which would lead us to declare the failure of the first Born approximation.” ys incorporates the effects of lens aberrations and defocus.2. 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.i y ( s ) ] A ( s ) } . and 9lindicates the inverse Fourier transform. 3. Before proceeding to that discussion. Situations when the effects of inelastic scattering cannot be ignored. Situations where the scattering is too strong for the weak phase object approximation to apply. Such a situation can.3 below. however. on the one hand. the function () A(s) is unity in the open part of the aperture and zero in the opaque part of the aperture. These problems include the following: 1. * 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. A more detailed and explicit discussion of the very useful weak phase object case is given in Chapter 9. so far as a description of the elastically scattered. If we further assume that the incident wave is a plane wave.3. 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. h(x) = 9‘{exp[ . where s is a vector in Fourier space and is called a “spatial frequency. be regarded as the failure of the “zerowavelength” approximation.398 9. Complications A number of complications can arise that make the weak phase object approximation an invalid description of image formation. Situations in which the specimen thickness is so great that we can no longer ignore Fresnel diffraction effects.
443 (1956).9. 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. Rev. A rigorous derivation of its validity. but pass through it.. but instead the transmitted wave must be written as the sum of waves transmitted through different layers of the object. One can then picture the electrons as penetrating without interaction to a certain depth in the specimen.e. This nonlinear description of the transmittance function of the object is known in the electron microscope literature as the “strong phase object” approximation. and many of them continue down to the image plane. The simplest case to discuss is one where the weak phase object approximation is valid for a thin slice of material. Phys. intensity) variations in the wave transmitted through the object. Schiff. was first given by Schiff. Assuming once again that the incident electrons are described as a plane wave. It should L. IMAGE FORMATION IN THE ELECTRON MICROSCOPE 399 wave is concerned. leads to rather considerable complications in the description of image formation. we can consider what happens if we keep the zerowavelength approximation but allow strong interaction and failure of the first Born approximation to occur.(x) = exp[(  i/hc/?)eV‘(x)]. 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. . The scattered and unscattered electrons then proceed to be transmitted through the remainder of the specimen with no further interaction. 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. 103.” This simple model is described here not with any recommendation of its accuracy or validity. At the same time the inelastically scattered electrons are not truly absorbed in 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.2. I . where they form their own separate image intensity. where a small fraction are scattered. we find that the transmitted wave retains a simple but no longer linear relationship to the projected Coulomb potential. in the sense that the transmitted wave has constant intensity and there are no amplitude (i. which means essentially the admission of wave propagation and Fresnel diffraction within the specimen. Relaxation of the zerowavelength approximation. at each level of the specimen. As a first step.’ albeit in quite a different context. Y. using the summation of the infinite Born series under the stationary phase approximation.
400 9. a trial and error approach can be taken. and therefore what the image intensity would be. However.” This idea is expressed mathematically by where ‘Y. this is an easy task for modern digital computers. 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. The wave that is transmitted through one slice is then imagined to propagate by Fresnel diffraction to the next slice. Of course.. where it serves as the next “incident wave. 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. very thin slices is approximated by the phase object approximation. 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.’ and this description is commonly referred to as the multislice dynamical theory of electron transmission through moderately thin specimens. In the CowleyMoodie multislice formulation. Rather.’ The CowleyMoodie multislice formulation is sufficiently rigorous for any practical application. defocus) of the individual transmitted waves. A fairly rigorous description of the transmitted wave requires not only that one take into consideration the Fresnel propagation (i. for an object of known structure.(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.e. A t /o Crv~talloyr 10. 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. provided that the slice is thin enough. 609 (1957) . Such a description of the transmitted wave was first given by Cowley and Moodie. it is quite impossible to work backward to find out what the object structure is. where different threedimensional models of the object structure can be used to calculate the expected image intensity. the difficulty is that if we are given only the final image intensity. 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. ‘J M Cowiey and A F Moodie.
“pure phase object approximation.1. Since there can be amplitude variations in the transmitted wave. We also discuss at some length the restrictions on the imaging conditions under which linear transfer theory may be applied with validity. 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.3. The performance of a linear system is most elegantly described in terms of the “transfer function” of the system. CONTRAST TRANSFER FUNCTION THEORY 401 Fresnel propagation and spreading of the electron wave front.9. where s is a general spatial frequency. introduces a further important effect in our description of the transmitted wave which does not appear in the zerowavelength. the wave aberration can be described by the function .” is related to a sinusoidal variation. 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. in the transmittance function of the object. by a set of linear equations. For nearly all situations of practical interest in electron microscopy. contrary to the results in the simple phase object approximation.” That is. ” 9. for a given periodicity or “spatial frequency.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)]. This effect arises from the fact that the intensity of the transmitted wave is no longer constant in space when Fresnel diffraction is included. of which the spherical aberration effect is the primary example. of the same peroidicity. The power of the transfer function lies in the fact that it tells us how a sinusoidal variation in the image intensity. 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. under appropriate conditions. these can be faithfully imaged under ideal. The phase distortion y(s) is due in the first instance to any sort of wave aberration associated with the lens. 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. within the finite thickness of the specimen. we show that the image intensity is related to the object structure.3.3. “infocus” imaging conditions.
This. that is. involves nothing more than looking at the Fouriertransform representation of the image intensity. In the spirit of the weak phase object approximation. Taking the Fourier transform of the righthand side of the equation for the image intensity. Our next task is to consider the image intensity as a linear superposition of many different sinusoidal functions.(i/hcp)eV’(x)]* 9= ‘(exp[ . .s is the complex conjugate of the scattered wave at f s . 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[( .(2e/hcfl)P’(s) sin y(s)~(s). In this equation. We thus have I(x) z 1 . The wave function in the image plane is given by Yy. is the coI efficient of spherical aberration.is the electron wavelength. and . that the wave propagation vector is parallel to the optical axis. The image intensity is obtained simply by squaring the image wave function. i(s) is the Fourier transform of the image intensity. We have further assumed that the objective aperture is axially symmetric.[(2/tic&V‘(x)] * .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.. ELECTRON MICROSCOPY where s is the spatial frequency vector and s its magnitude. 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 . p(s) is the Fourier transform of the projected scattering potential. Friedel symmetry applies when the scattered wave at . AZ is the instrumental defocus. Finally. C.k. if we assume that the specimen is a weak phase object.(x) [I . First. we obtain i(s) = 6(s) . we have assumed that the illumination is axial.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.i y ( s ) ] A ( s ) } . we have dropped from further consideration the quadratic image terms.402 9. where k is the scattered wave vector. 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. of course.F’(sin[y(s)]A(s)}.
. 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. Thus a finite energy spread in the illuminating electrons must lead to a corresponding reduction in the degree of coherence. in the factors leading to degradation of temporal coherence. 9. some sinusoidal components may even be transferred with reverse contrast. The spatial coherence is determined primarily by the angular convergence (or divergence) of the illumination. We thus find that the various sinusoidal components of the object structure are each transferred with an individual modulation of contrast. CONTRAST TRANSFER FUNCTION THEORY 403 result that the convolution product in real space is converted to a direct multiplication in Fourier space. of the projected potential. From this result we can see that there is a simple. the function sin[y(s)] is commonly referred to as the “phase contrast” transfer function in electron microscopy. of the same period. The energy spread can arise from the physics of the emission process at the filament (source). For these reasons. but also of importance is the condenserlens aperture size and the conditions of focus of the condenser lens that are used. The finite size of the primary electron source is obviously a factor in determining the divergence. from instabilities in the accelerating voltage. It is also appropriate to include. and that. 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).3. the effect of focus instability in the objective lens. 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)].3. several factors can reduce both the degree of temporal coherence and the degree of spatial coherence. furthermore. 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. Although this does not directly involve the incident electrons. the superposition being weighted according to the distribution of energies and angles of incidence. each calculated for the appropriate wavelength and propagation vector.9. In the real case. Temporal coherence is determined primarily by the monochromaticity of the illumination. which is associated with ripple in the objectivelens current. the amplitude undergoes a change in sign whenever the function sin[y(s)] goes through zero.2. onetoone relationship between any one Fourier component of the image intensity and a Fourier component. and from electronelectron interactions at points where a beam crossover may be formed. and depending upon the defocus value.
so we have suppressed the aperture function. highresolution electron microscope has values close to unity out to a resolution of 45 A. (9. this approach cannot be used where A(s) = 0.(2e/hcp)P’(s) = i(s)/[sin y ( s ) ] ~ ( s ) . we need only obtain the Fourier transform P’(s).’O. The exact functional form of the envelope function depends upon a detailed description of the factors influencing the partial temporal and spatial coherence.3. 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. where .3. (9. Image Restoration The term “image restoration is used here to refer to the process by which the projection of the object structure. (9.3.3. 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. the formal solution is given by V’(x) = 9“P’(s)]. H. 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). the envelope function may also affect lowerresolution details. in addition. 519 (1973).404 9. Optik 38.’l Under normal circumstances the envelope function of a modern. Frank. 81 (1977). one must correct for the fact that the envelope function is not always equal to unity. Now it is clear that in order to obtain the projected potential V’(x). and depends also upon the defocus value. That is.1) above. . In addition. But examining Eq. Wade and J . Frank. Stated more specifically. V’(x).2) excludes any zerofrequency (constant) term.2) Needless to say. In practice this approach to the image restoration problem leads to In ” J .3. is retrieved from the observed image intensity I(x). Optrk 49. ELECTRON MICROSCOPY In the case in which the object’s transmittance function is approximated by the weak phase object approximation. R. and thus lacks the correct “average value” of V’(x). 9. the function V ’ ( x ) obtained according to Eq. At very large defocus values.
that is. of course. It follows. . By way of a practical example. that the objectivelens spherical abberation is the main factor limiting image resolution.5 A) that is actually realized in presentday electron microscopes. we can take advantage of the fact that I sin[y(s)] I takes on values close to one for different spatial frequencies. In the low. Using these figures. This resolution limitation is in fairly good agreement with the practical limit for useful image restoration ( . at the same time sin[y(s)] will be continuing to undergo oscillations. one can always find another defocus value such that I sin[y(s)] I is close to unity. each taken with a different defocus. '. we find that the envelope function has decreased to a value of 0. sin[y(s)] can oscillate. depending upon the value of defocus. provided that one is prepared to engage in the image restoration task. while at the same time the envelope depends quite strongly upon the chromatic aberration coefficient and the energy spread. CONTRAST TRANSFER FUNCTION THEORY 405 noise amplification whenever the product sin[y(s)]E(s) becomes rather close to zero.3. while. at a resolution of 2. therefore.25 at a spatial frequency of 0. sin[y(s)] can never take on appreciable values except for very large defocus. and the coefficient of chromatic aberration normally has a value not smaller than 1. At low spatial frequencies. such that the product sin[y(s)]E(s) is sufficiently close to unity at all spatial frequencies in one or another micrograph. particularly for moderate values of defocus. However.5 mm. And finally.8 A.9.and intermediateresolution regions.5 eV. At intermediate spatial frequencies. This condition can arise in three regions of the Fourier spectrum of the object. at the spatial frequency ofconcern.3. 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. 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. that if 1 sin[y(s)] 1 is close to zero in an image taken with one defocus value. in the latter case the problem being that the envelop function has become too small. It is commonly supposed. the envelope function is not very sensitive to the value of the spherical aberration coefficient. The main limitation to image restoration will then occur either at low resolution or at very high resolution. A typical value for the energy spread of a 100keV microscope is approximately 1. and not without justification. it is worthwhile to insert some numbers into the analytic functions' representing the envelope function.35 A. In this way it is possible to patch together data from many different images. The practical limit on the illumination angle for conditions of iniaging at high magnification is approximately l o p 3 for short exposures rad and rad for long exposures. at very high resolution the envelope function must gradually tend to zero.
in contrast to the weak phase object where the transfer function is sin[y(s)]. it is convenient to consider first the case of a pureamplitude object. (9. Once again taking the Fourier transform of both sides of the equation. the result remains exceedingly simple and convenient to use.406 9. 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.p(x).” Nevertheless. The image intensity for a weak. 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. ELECTRON MICROSCOPY 9. A slightly more complicated case than the weak phase object is obtained by admitting a weak amplitude variation in the transmitted wave.3. 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. The same analysis as was used in the previous section leads to the result that the image intensity is given by I(x) = 1 . where T(x) is the transmitted wave function when the incident wave is a plane wave.3. in addition to a weak phase variation. mixed phase and amplitude object is once again easily derived by the procedures above. since the phase contrast term is characterized by one transfer function while the amplitude contrast term is . but still making the assumption of weak scattering.3) which is the linear combination of the phase contrast and amplitude contrast effects.iq(x) .3).4. In this case the object transmittance function becomes T(x) = 1 .2 ~ [ p ( x )cos[y(s>].2F[p(x)] cos[y(s)]. The result of such analysis is that the Fourier transform of the resulting image intensity is given by i(s) = 6(s) . 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. Thus for a pure amplitude object the contrast transfer function is found to becos[y(s)].3. However.2p(x) * 9(cos[y(s)]}. (9. To understand the effect that the amplitude variation has in the contrast transfer that is obtained in the image.2 ~ [ q ( x ) ]sin[y(s)] . we obtain i(s) = 6(s) .
R. perhaps pathological.Let us make the further. which could be obtained from just two micrographs.Erickson and A. A fairly extensive analysis of the defocusdependent contrast transfer characteristics of uranylacetatestained catalase was carried out by Erickson and Klug. To illustrate this point. Soc. B261. the experimental fit was found to be moderately good. These relative weights were then obtained from a series of micrographs taken at different values of defocus. As can be easily seen from Eq. CONTRAST TRANSFER FUNCTION THEORY 407 characterized by another transfer function. is the possible difference in the phase origin between the phaseobject Fourier coefficients and the amplitudeobject Fourier coefficients. More general cases are easily evaluated by the same sort of analysis. 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. l2 H . 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. . Ser. 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. The analysis of Erickson and Klug might have been an overly simplified approximation of the complete problem.3). 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. (9.' In this work the relative weights of the phase contrast and the amplitude contrast terms were allowed to vary continuously at each spatial frequency. 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. the magnitude of the Fourier transform of the image intensity will not vary with changes in defocus. The mechanism for carrying out image restoration remains as clear and straightforward as it is in the case of the pure phase object. P. however. there is some experimental evidence that the mixed object representation is a practical representation. Klug. Thus the relative proportions of phase and amplitude contrast. For all but a few spatial frequencies. even though theoretical considerations might not encourage one to accept its validity.105 (1971). London. Trans. were used to determine whether the contrast transfer could be correctly predicted at other defocus values.3. assumption that the two Fourier coefficients have equal magnitude. Nevertheless. A third parameter.9. but for which the weak phase and amplitude object provides an accurate representation. However. 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). which perhaps should not be ignored. Philos.3.
The effect that the quadratic terms have on the Fourier transform of the image intensity is to add an expression of the form (9. Darkfield images are usually produced with either a displaced aperture or tilted illumination.2.4) where i.3. under coherent brightfield illumination conditions. These are actually identical to the darkfield image intensity that would be produced by stopping out the central. 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.3. . one cannot possibly hold to the assumption that the magnitude of the scattered wave is negligibly small in comparison to the unscattered wave. ELECTRON MICROSCOPY The effects of the other complications. As may be clearly seen from the autocorrelation (selfconvolution) representation in Eq. the darkfield image intensity. are not so easily discussed in terms of contrast transfer function theory. 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.(s) is the Fourier transform of Id(x). Inclusion of the quadratic terms necessarily prevents the use of a transfer function in the description of the image intensity.3. which have been described in Section 9. This result follows directly from the application of the convolution theorem of Fourier transforms. (9. it is not possible to use a central beam stop to produce a darkfield image.408 9. unscattered beam in the objective lens’s back focal plane. One further complication merits brief mention at this point. 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.4). In those cases whcre the image contrast is quite large. Nevertheless. 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. In practical electron microscopy. In these cases we might truly expect to find nonnegligible contributions from the quadratic image terms. which is applicable in the case of perfectly coherent illumination. and the asterisk indicates the autocorrelation product (integral). 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. the coherent darkfield image. however obtained.
There exist many difficulties in the task of threedimensional reconstruction. to recover the threedimensional Fourier transform of the object. and many different approaches have been taken in an attempt to overcome these difficulties. the conditions of . More precisely. under suitable conditions. 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. THREEDIMENSIONAL RECONSTRUCTION 409 9.4.9.1. ” ” “ 9.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. first. the Fourier transform would still be used during the steps of twodimensional image restoration .4. Fourier (Crystallographic) Methods The Fourier transform methods of threedimensional reconstruction are in many respects similar to the xray crystallographic methods of macromolecule structure analysis. each obtained at a different tilt angle of the specimen.4. the image intensity is a linear function of the projection of the object’s structure. One can distinguish two general classes of methods for threedimensional reconstruction. of course. which is still fundamentally a realspace” method. This class of methods has a great kinship to the welldeveloped methods of macromolecular crystallography. as another example. 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. 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. the phrase “threedimensional reconstruction refers to the task of obtaining a threedimensional model of the object from many different twodimensional projections. As an example. as long as the image resolution extends to sufficiently high spatial frequencies. is contained in the fact that the structural phases are obtained directly from images in electron microscopy. Three. after which the threedimensional structure is obtained directly by a threedimensional inverse Fourier transform. That the structural phases can be obtained from the images is a direct result of the fact that. One important difference. One class of methods attempts. The conditions needed to provide this linear relationship are.
. we do not have the usual problem encountered when a diffraction pattern is produced physically by some form of radiation. 01. each taken at a different angle. In addition.) is the threedimensional Fourier transform of the object potential. if we have many different projections 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.. it is evident that a single projection in a direction perpendicular to the axis will be composed of a superposition of many different. The projection theorem thus states that the Fourier transform of a projection is a central section of the threedimensional Fourier transform of the object. 2 where P(S. proteins). The net result is that the Fourier transform of one image gives a twodimensional slice through the threedimensional Fourier transform of the whole object. Thus. that is. Thus a single projection of a cylindrical or helical structure contains as much information . Such structures are assembled from identical subunits (e. Considering for the moment only one repeating segment of the structure. One of the most favorable instances in which symmetry facilitates a threedimensional reconstruction is provided in the case of cylindrical or helical structures. it is a straightforward matter to obtain the threedimensional Coulomb potential by inverse Fourier transformation. ELECTRON MICROSCOPY validity for the weak phase object approximation. only the modulus of the Fourier transform can be obtained from experimental measurements. Since we assume here that the Fourier transform of the image intensity can be calculated numerically.g. independent projections of the individual subunits.410 9. Once the threedimensional Fourier transform P(s) has been computed. it has some special advantages in those cases when the object has some form of known symmetry..s. In the latter case. Y) m. For a weak phase object we need only to apply the projection theorem of the Fourier transform to obtain the result ‘F2DW. then by computing the Fourier transforms of each of them we can obtain many different twodimensional.. S. S. whereas in the case of numerical calculations both the magnitude and the phase can be computed. which are arranged in a repetitive pattern around and along a cylindrical (helical) axis. The “crystallographic” Fourier transform method described above is completely general in nature and approach. central sections through the threedimensional Fourier transform.
Ser. 107. L. Nature (London) 272. Microscopical Society of Canada. T. A. Narure (London) 217. Crowther. R. Sor. Slayter.’ and helical fibers of proteins such as glutamine synthetase’* and sickle cell hemoglobin.20*2’ A limited amount of work has also been done on the use of Fourier methods for threedimensional reconstruction of individual macromolecules. Klug. R. In the case of the icosahedral (“spherical”) viruses. Truns. Edelstein.Crowther. J . Philos. P. M. T. Vol. asymmetric unit.506 (1978). 130 (1968). 42 (1 970). 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. H . Mol. in effect. and this approach is not to be recommended.B. G. 2 1 R. In the case of a helical structure with a large number of subunits per repeat. the amount of biological information that can be obtained is rather limited. dehydrated specimens. A. in “Electron Microscopy 1978” (J. For this reason it has been necessary to do such work only with stained. J. N. J . 50. Nature.279 (1970). J. DeRosier. Amos.4.221 (1971). T. and A. a single projection is sufficient to give a good threedimensional reconstruction. l 6 H. and S . A. Mol. London. J . J . ’’D. Bid. Biol. Sturgess. Lake and H. Mol. p. Toronto. P. twodimensional periodic arrays of the fundamental. J. P. 20 R. 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. 641 (1974). The method of helical reconstruction has now been applied to a wide variety of biological specimens. in which case the object lacks any special symmetry relationships.'^ microtubules. Biol. A cylindrical or helical structure is. . Cell Biol. Huxley. Klug. D. and D. as was first demonstrated by DeRosier and Klug13 in their study of the structure of the T4 bacteriophage tail. l 9 G. Finch. J. including the stackeddisk protein of tobacco mosaic virus. 87. Unwin and A. 60. Klug. DeRosier and A. 226. Dykes.9. ed. 271 (1972). 1978.I4 ribosome^.’6 myosindecorated actin filaments.). As a consequence. its own “tilting stage” in the electron microscope. a single projection again provides many different views of the identical protein subunits that lie at the surface of the virus. 66. one for each time the tilt angle is changed. Moore. DeRosier. E. H. Highresolution threedimensional reconstructions can only be achieved at the present time if the specimen can be obtained in the form of rather large. Frey. Erickson. 153 (1974).I. l4 l5 . S. Crepeau. Helical structures with only a few subunits per repeat. A. THREEDIMENSIONAL RECONSTRUCTION 41 1 about the basic subunit as could be obtained from a large number of equivalent projections of a single subunit. and all cylindrical (nonhelical) structures require two or more independent projections with a known angle of rotation about the symmetry axis. B261. J. I l l .
Unwin. asymmetric objects as far as the threedimensional reconstruction problem is concerned. These limitations are described in greater detail in Chapter 9. ~ ’ by Henderson rt and (a) Structural model built from the 3D contour maps. T. Nutitre (London)257. Seven rhelical rodsareshown accordingto themodel published by Henderson and Unwin.28 (1975) . photosynthetic bacterium Hulobucterium hulobium.” while the identification o f the cytoplasmic and extracellular surfaces o f the protein was carried out indepcndcntly by Hayward et ~ 1 .22) (h) Linedrawing idealization of the model with the cytoplasmic and extracellular surfaces identified. (From Hendcrson and Unwin.5. ELECTRON MICROSCOPY CYTOPLASMIC SIDE EXTRACELLULAR SIDE lb) FIG. (Courtesy of Dr. and objects with twodimcnsional redundancy must be treated in the same way as general. 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. Henderson and P. Bacteriorhodopsin is a membrane protein of the halophilic. 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. The requirement for twodimensional arrays has nothing to d o with providing additional crystallographic symmetry. Bacteriorhodopsin is particularly well suited for electron microscope structure analysis since the protein naturally 22 R. Threedimensional inodcl of the structure of bacteriorhodopsin at about 7A resulution.412 9. N.) The requirement for twodimensional arrays exists strictly to overcome the limitations arising from radiation damage. Steven Hayward. I .
S. One class of directspace reconstruction methods is built upon the idea of producing a “back projection from many different twodimensional projections.A.4320 (1978). D.259 (1978). Henderson. J. and K.3. Inverse Fourier transform of the sampled data yielded a threedimensional of the structure of the protein with a resolution model. 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. THREEDIMENSIONAL RECONSTRUCTION 413 occurs in twodimensional crystalline arrays. 9. and whether realspace methods are better than the Fourier transform method are questions which have sometimes been the subject of very hot debate. Sci.4. DirectSpace Methods The Fourier transform (crystallographic) method is by no means the only one by which threedimensional reconstruction can be accomplished.S.9. Mul. Proc. J . Glaeser. 75. A. 123. R . Whytock. In subsequent work Henderson and Unwin” collected image data over a range of tilt angles of f 6 0 ” from the normal. 24 R. Natl. and which appear to span the full thickness of the membrane. M . 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. Jubb.2.” This last problem will be treated at further length in Section 9. Whether one realspace method is better than anothcr. U.2’24 of approximately 7 A in the plane of the membrane and approximately 14 A perpendicular to the plane of the membrane. 1. shown in Fig. 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. Acad. . A. The basic idea of the back projection is to project the density of ” 2 3 S. B. 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. and S. 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.” These membrane patches occur in pieces as large as one micron in diameter which contain more than lo4 protein molecules per patch.4. which can be biochemically isolated and which are referred to as “purple membrane. (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.4. B i d . (ii) the uniqueness of the solution that might be obtained. Hayward. Fisher. Grdno.
and the use of the Fourier transform to execute the spatial filtering is only incidental to the philosophy 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. numerical implementation of the approach. ELECTRON MICROSCOPY the object backward along a straight line perpendicular to the plane of the projection itself.” It is to be emphasized that the filtered back projection approach is still philosophically a realspace method. and if they are not very “dense” in reciprocal space.25The optimization of the model to the experimental projections can also be set up as a ” 25 P. while unwanted density makes only a general background underlying the actual object.414 9. 36. becomes invalid if the projections are not equally spaced. One algorithm allows for the simultaneous optimization of the density values to all projections in each iterative step. parallel sections through which are everywhere identical to the original projection.’s. A common approach to this method is to vary the density parameters of the model in an iterative procedure. Theor. J . Gilbert. When the back projections from many different images are superimposed. This simple modification of the back projection procedure is referred to as the “filtered back projection. the inverse Fourier transform of l/s). and then carrying out an inverse Fourier transformation. the true features of the object are reinforced at points of intersection. Biol. 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. These approaches represent essentially the “refinement of a realspace model. the use of the simple back projection method is not a very satisfactory approach to the threedimensional reconstruction problem. 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. Because this background level is rather high. and therefore have a certain approach in common with the reciprocalspace refinement methods that are now becoming commonplace in xray crystallography of macromolecules.” as must be done in any real. multiplying this Fourier transform by s. Complications to the filtered back projection are encountered first in handling the abrupt cutoff at the edge of the “filter function. The unwanted convolution is easily removed by computing the Fourier transform of the naive back projection. Thus a single projection is used to generate a threedimensional density function in real space.
. 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. that is. to the observed summation of density values in individual columns. but it must be said that there is little mathematical justification to believe that it is true. I while giving projections of the object structure that are consistent. Nature (London) 272. In fact. The directspace model refinement procedures may appear. A rather special version of the realspace model fitting procedures is the socalled maximumentropy te~hnique. no study has yet been published on the suitability of the maximumentropy method for threedimensional reconstruction in electron microscopy. with the observed data.490 (1974) ” S . to be more attractive than they really have a right to be. Crowther and A . It is always possible that such a favorable result could actually be the case. 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. to some extent. A formal solution of the problem is obtained by application of Cramer’s rule or by other. The model fitting procedures have no rigorous mathematical foundation by which one might evaluate the limitations of the methods. 686 (1978).4. At this time. 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.~’ rationale in this case is to The find the set of “’quantified” density values m iin real space which serve to maximize the sum s= c mi ln(mi). There are two difficulties confronted by the approach of directly solving the linear equations. more rapid methods of matrix inversion. as unknowns. Daniell. 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. Gull and G . F. Matrix inversion solutions are also formally available when the linear equations are overdetermined. The first difficulty has to do with the size of the matrices that are involved 2 6 R. within experimental errors. 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. This is particularly true when noise has been added to otherwise perfect data. giving in this case the leastsquares solution. Nuture (London) 251. Klug. J. when the tilt views are equally spaced in angle. crystallographic approach.9. to the observed projections. A.
4. for example.416 9. The reason why we refer to this difficulty as the “hollow cone problem” is most clearly seen in the context of the Fourier. It would not be at all uncommon to expect as many as a thousand simultaneous linear equations. for example. 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 matrix inverse can easily be very poorly conditioned. the Fourier transform of each twodimensional projection corresponds to a twodimensional central section in reciprocal space. The object structure retrieved in this way will certainly have some relationship to the real object structure. It is always possible to compute an inverse Fourier transform of just the data accessible to experimental measurement. Hollow Cone Problem The 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.4. ELECTRON MICROSCOPY for problems of interest in highresolution structure analysis. crystallographic method of threedimensional reconstruction.1. since the extension of electronmicroscopic structural studies to atomic resolution might not be possible without an adequate solution. but at the same time there will necessarily be some differences between the reconstructed object and the original object. But by the samc token the limited range of tilt angles make a conical section of reciprocal space inaccessible to direct experimental observation. 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. even if one has an unlimited amount of free computer time. In addition. 2. The precise nature of the relationship can be seen by application of the convolution theorem. Thus the experimentally obtained data are related to the full threedimensional . We elaborate on this point in more detail in the next section. in the case of helical or cylindrical objects. The need to find an optimal solution to this problem is very great. 60”. 9. 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. 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.3. The size of the corresponding matrix equation makes the numerical calculations in the formal solution quite formidable. as is illustrated in Fig. As already described in Section 9.
9. of course. the convolution product (integral) of the real object structure and a threedimensional pointspread function. ( s ) ] [ = 9. on the right. unfortunately.” The threedimensional aperturc function A(s) is unity everywhere within the domain of observation and is zero within the region of the hollow cone. to a family of “central sections” (planes) over the same angular range in Fourier space. The success of interpolation and extrapolation procedures in solving the hollow cone problem is. 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. 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. a family of projections obtained over a limited angular range (say +60”)in real space corresponds. by the use of the convolution theorem.[ P(s)] * F. to result in a loss of resolution in the z direction.4. The limited range ofviews leavesacone about the vertical axis in Fourier space which is inaccessible to experimental incasurement.[ ~ ( s ) ] V(r) * a(r).t b . rather limited. and :!ie application to the threedimensional reconstruction problem is no exception to that generalization. = 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. The primary effect of the hollow cone can therefore be seen. Extrapolation is traditionally known to be a very refractory mathematical problem. The diagrams should be interpreted as figures of revolution about the vertical axis.2. Thc hollow cone problem is illustrated by the fact that. on the left. and in terms of spherical Bessel functions for general . Fourier transform by a product between the real Fourier transform and a threedimensional “aperture function. The inverse Fourier transform of this product is. Thus we have tbs(s) = p(’(s)A(s).
’’ 9. and biological materials are no exception. A . The incident electron beam must. E.435 (1972). to the original data before the realspace threedimensional