Fluorescence Spectroscopy

CHE5540 Lab exercise 9

See Lakowicz Book Principles of Fluorescence Spectroscopy Mller, M. & Denicola, A. (2002) Biochem. Mol. Bio. Ed 30(3):175-178 Pain, R. H. (2004) Curr. Protocols Protein Sci. 7.7.1-7.7.20

Emission is observed at a 90 angle from the incident beam to separate it from transmitted light. Transmitted I Incident Io The change in intensity may be slight.

Fluorescence (red-shifted). We minimize background by centrifuging or ltering out anything that could scatter Advantages: more selective , combines and emission spectrum and an absorption spectrum, observed on a zero background

Fluorescence is re-emission at a longer wavelength.

State 1
vibrational substates

Absorption (excitation) Internal conversion Fluorescence (emission)

State 0 !
The state formed upon excitation is surrounded by solvent molecules in a conguration that is optimal for the ground state but not the excited state. Rapid internal conversion and vibrational relaxation result in a vibrational ground state of the electronic excited state. That energy is lost. Similar losses may be associated with the emissive transition. Result, the photon emitted does not retain all the energy of the absorbed photon, and is red-shifted. The Stokes Shift.

Kashas rule, the emission spectrum does not depend on the excitation wavelength (but the intensity does).

State 1

Absorption (excitation) Internal conversion Fluorescence (emission) Emission spectrum arises entirely from lowest vibrational substate of excited electronic state, regardless of which vibrational substate was created upon excitation. (Internal conversion is fast.)

State 0

Franck-Condon principle and the mirror image rule

Absorption (excitation) Internal conversion Fluorescence (emission)

State 1

State 0
0-1 5-0 4-0 0-5 0-3 2-0 1-0 0-0 0-4 0-2 0-0

Franck-Condon: all electronic transitions occur without changes in nuclear positions: if a particular transition is favourable in absorption (eg. 0-1), then it is also 8 in emission. favourable

Anthracenes vibrationally-resolved electronic 8 spectra.

3-0

0-2 0-1 0-0

0-0 1-0 2-0

0-3

3-0

4-0

0-5

0-4

5-0

Figure 1.9. Absorption 7) of 1-hydroxypyreneFigure 1.8. Mirror-image rule and Franck-Condon factors. The absorption and emission spectra are for anthracene. The numbers 0, 1, and 2 refer to vibrational energy levels. From [11].

1.3.3. Exceptions to the Mirror-Image Rule

From Lakowicz Book Principles of Fluorescence Spectroscopy

Figure 1.8. Mirror-image rule and Franck-Condon factors. The ab-

Excited-state re can also result in dev Figure 1.9. Absorption ( One example is sho 7) of 1-hydroxypyrene-3 emission spectrum o aniline.13 The structu

The intensity of uorescence depends on the fraction of excited states that decay that way, vs via alternative decay routes.

Fluorescence in context: Energy and light: electronic states of molecules.

IC

Fluorescence takes place on a much longer time scale than absorption, so it is sensitive to a much wider range of interactions and perturbations.1
http://www.olympusmicro.com/primer/java/jablonski/jabintro/index.html

Fluorescence Quantum Yield

!F = kF/(kIC + kIS + kQ[Q] + kF) Fraction of excited state that returns to the ground state via uorescence This is the same as the fraction of photons absorbed that lead to uorescence. kF = A10 kF is the intrinsic uorescence rate constant. radiative lifetime "F=1/kF

Internal conversion: kIC non-radiative decay , mediated via collisions with solvent, internal motions (vibrations). Increases w. temperature. Intersystem crossing: kIS electron spin ip to a triplet state (different manifold of states). Inefcient (forbidden) slow and usually masked by other processes. Usually need low T, rigid glass and low [O2]. Quenching: kQ[Q] dissipation of energy via interactions with other molecules, or functionalities. Since the natural lifetime of most organic uorophores is only 1 - 100 ns, only efcient quenchers are signicant. I-, O2, acrylamide

Because uorescence emission is a slower process, it is more sensitive to the environment and interactions of the chromophore. This makes it more useful. 18

INTRODUCTION TO

human serum albumin (HSA)

1-Anilinonaphthalene-8-Sulfonic Acid

The lower polarity environment in the core of a protein is less quenching of uorescence and less supportive of internal conversion.

Figure 1.21. Emission spectra of TNS in water, bound to apomyoglobin, and bound to lipid vesicles.

ume (or molecular weight) of proteins. This measurement is possible because larger proteins rotate more slowly. Hence, if a protein binds to another protein, the rotational rate decreases, and the anisotropy(s) increases (Figure 1.23). The rotational rate of a molecule is often described by its rotational correlation time , which is related to

Lakowicz Figure 1.21

Figure 1.22. Accessibility of fluorophores to Reprinted with permission by Wiley-VCH, STM

chains. Hence, an increase in the amou

Examples of commonly-used uorophores

22

Examples of biological uses.

INTRODUCTI

DAPI or 4',6-diamidino-2-phenylindole is a uorescent stain that binds strongly to A-T rich regions in DNA. It is used extensively in uorescence microscopy

Figure 1.32. FCS of an Alexa-labeled be solution and on a single neuronal cell. Re

Figure 1.30. Fluorescence image of RBL-3H3 cells stained with DAPI (blue), Patman (green), and tetramethylrhodamine (red). Courtesy of Dr. W. Zipfel and Dr. W. Webb, Cornell University. Reprinted with permission from [30].

tetramethylrhodamine methyl ester

More cool experiments: FRET, FRAP

Figure 1.30 from Lakowicz
PROTEIN FLUORESCENCE

Wikipedia the local environment determines the spectral properties of

530

Proteins built-in uorophores

Note Y-axes Trp has a larger extinction coefcient

tryptophan. It is also possible to insert tryptophan analogues into proteins. These analogues display unique spectral features, and are observable in the presence of other tryptophan residues. In summary, a growing understanding of indole photophysics, the ability to place the tryptophan residues at desired locations, and the availability of numerous protein structures has resulted in increased understanding of the general factors that govern protein fluorescence. The high

sensitivity of the emission from tryptophan to the details of its local environment have provided numerous opportunities for studies of protein functions, folding, and dynamics.
16.1. SPECTRAL PROPERTIES OF THE AROMATIC AMINO ACIDS

Several useful reviews and monographs have summarized the spectral properties of proteins.19 Proteins contain three amino-acid residues that contribute to their ultraviolet fluo-

Figure 1.31. Fluorescence correlation spectroscopy. Top: observed volume shown as a shaded area. Middle: intensity fluctuations. Bottom: correlation function.

most single-molecule experiments bilized fluorophores, with fluoro high quantum yields and photostab ical instrument for SMD consists o microscope objective, a scanning s and confocal optics to reject unwan being extended to include UVwhich was considered unlikely ju probe 2,2'-dimethyl-p-quaterpheny tion maximum of 275 nm and an em nm. Figure 1.33 (left) shows intens quartz cover slip.35 The spots repre molecules, which can yield signal tons per second. The technology fo rapidly that the lifetimes of singl measured at the same time the int collected (right). The individual DM lifetimes near 1.1 ns. Without the use of SMD, observe a large number of molecul reveal the ensemble average of t When observing a single molecu averaging, allowing for the behavio be studied. Such an experiment is a hairpin ribozyme labeled with Steady-state measurements on a ribozyme would yield the average fer, but would not reveal the pre showing different amounts of energ cule experiments show that an ind cule fluctuates between conformat er amounts of energy transfer.36 changes are seen from simultaneou

Figure 16.1. Absorption (A) and emission (E) spectra of the aromatic amino acids in pH 7 aqueous solution. Courtesy of Dr. I. Gryczynski, unpublished observations.

Figure 16.1 from Lakowicz

Natural uorophores in proteins

Cantor & Schimmel

riboavin enhanced GFP

370, 480 395, 475

12 55

531 (0.3 - 0.37) 509 0.6

36 330

http://omlc.ogi.edu/spectra/PhotochemCAD/html/004.html

http://en.wikipedia.org/wiki/Green_uorescent_protein

http://zeiss-campus.magnet.fsu.edu/print/probes/fpintroduction-print.html http://www.scholarpedia.org/article/Fluorescent_proteins

Figure 16.10. Emission spectra of proteins that lack tryptophan residues. Neutral pH. Revised from [4849].

Tryptophan uorescence differs depending on exposure to solvent It therefore reports on protein unfolding.

phan can vary greatly between proteins. Denaturation of proteins results in similar emission spectra and quantum yields for the unfolded proteins. Hence, the variations in tryptophan emission are due to the structure of the protein. We are not yet able to predict the spectral properties of proteins using the known structures, but some efforts are underway.61 One might expect that proteins that display a blue-shifted emission spectrum will have higher quantum yields (Q) or lifetimes (). Such behavior is expected from

The Stokes shift is larger in a more polar solvent because the energies associated with interactions with polar solvent molecules are larger.
Figure 16.11. Effect of tryptophan environment on the emission spectra. The emission spectra are those of apoazurin Pfl, ribonuclease T1, staphylococcal nuclease, and glucagon, for 1 to 4, respectively. Revised from [59] and [60].

Figure 16.11 from Lakowicz

Quenching of tryptophan in proteins

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 547

Buried (blue-shifted) tryptophan is less accessible to polar quenching agents, so the quenched spectrum is blueshifted.

Figure 16.30. Collisional quenching of buried (W1) and surface accessible (W2) tryptophan residues in proteins.

single-tryptophan azurin Pae. The spectrum of the quenched tryptophan residue can be seen from the difference spectrum, and is characteristic of an exposed residue in a partially hydrophobic environment. In this favorable case, one residue is quenched and the other is not, provid-

16.32). The emission of azurin is essentially unchanged in up to 0.8 M acrylamide. In contrast, the exposed tryptophan residue in adrenocorticotropin hormone (ACTH) is almost completely quenched at 0.4 M acrylamide (Figure 16.32, left panel).

Figure 16.30 from Lakowicz

Dependance on quencher concentration: Stern-Volmer plots

550 PROTEIN FLUORESCENCE

Figure 16.37. Emiss ble and inaccessible length-dependent fra [113].

Figure 16.36 from Lakowicz

Figure 16.36. Stern-Volmer and modified Stern-Volmer plots for apomyoglobin quenching by iodide or trichloroethanol (TCE). The data in the upper panel was reconstructed from the data in the lower panel. Data from [113].

Figure 16.37. Emission spectra of apomyoglobin and of the accessible and inaccessible components. The lower panel shows the wavelength-dependent fractional accessibility to quenching. Revised from 16.6.3. Resolution of Emission Spectra by [113].

In this expression of the total emiss value Ki() (Sectio This procedur S. aureus that con emission waveleng to the different 16.38). The data a the values of Ki() range of emission calculate the emis

represent and the fraction this expression values ofthe fi() tionInof the emission the spectra of quenched unof the total emission quenchable at wavelength that with a quenched components. For apomyoglobin we assumed In the absence of quencher value K ( ) (Section 8.8). i some fraction of the emission was completely inaccessible was applied to a metalloprotease from toFquenching. A more general procedure allows the emis!F = kF/(kIC + kIS + kF) = k " This procedure 116 For all S. aureus that contains two tryptophan residues. sion spectra to be resolved even when both residues are par( is the unperturbed lifeplots time the excited state, also called the emission wavelengths the Stern-Volmer plot is curved Figure 16.36 ." Stern-Volmer and modified Stern-Volmer for of 114116 The basic tially accessible to quenching. approach isdue apomyoglobin quenching by iodide or trichloroethanol (TCE). The to the different accessibilities of each residue (Figure uorescence life time) data in the upper panel was reconstructed from the data in the lower to perform a least-squares fit to the quenching data to recov16.38). The data are fit and by least-squares methods obtain er the quenching constant fractional intensity at to each panel. Data from [113]. the values of K ( ) and f ( ). Similar data are collected for a i i wavelength (): In the presence of quencher range of emission wavelengths. These data can be used to the emission spectra of each component: !F kF/(k kQ[Q]) IC + k IS + kF + calculate 16.6.3. Resolution of= Emission Spectra by fi ( ) F( ) (16.4) Quenching F0 i 1 Ki ( ) Q F ( ) F0 ( ) fi ( ) (16.5) The ratioof of uorescence in the absence of Q vs. ini the presence of Q is Selective quenching tryptophan residues allows resolution of the emission spectra of the quenched and unwhere F0() is the unquenched emission spectrum. For met+ k ISalloprotease + kQ [Q ]this procedure yielded two well-resolved emisquenched components. we k assumed that F0 For apomyoglobin kF F + k IC = was completely inaccessible sion spectra some fraction of the emission (Figure 16.39). F general kF + k IC + kallows to quenching. A more procedure the emis- kF IS The use of quenching-resolved spectra may not always sion spectra to be resolved even when both residues are parbe successful. One possible reason for failure would be if a 114116 The basic approach is tially accessible toF quenching. k + k + k + k [ Q ] kQ [Q ] was not in a unique environment. In this tryptophan residue IS Q 0 = fitFto the IC = 1 +case each to perform a least-squares quenching data to recovtryptophan residue may display more than one F + kIS at each emission kF + kspectrum, er the quenching constant and k fractional F + k ICintensity IC + k IS each of which would be quenched to a wavelength (): different extent. Quenching-resolved spectra have been

The Stern-Volmer Equation for Quenching Selective quenching of tryptophan residues allows resolu-

Quenching

where F0() is the alloprotease this p sion spectra (Figu The use of qu be successful. One tryptophan residue case each tryptop emission spectrum different extent. obtained for prote due.116117 These r protein being pres

F0 = 1 + kQ [Q ]! fi ( ) F F( )
F0
i i

1 K ( ) Q

(16.4)

obtained for proteins that contain a single-tryptophan residue.116117 These results have been interpreted as due to the protein being present in more than a single conformational

The Modied Stern-Volmer Equation

F0 = 1 + kQ [Q ]! F
F= F0 1 + kQ [Q ]!

Observed uorescence is the sum of uorescence from exposed sides and buried sites. F0 For each type of site: F = F = FX + FB 1 + kQ [Q ]!
F=
F=

FX ,0 + FB,0 1 + kQ [Q ]!
FX ,0 + ( F0 " FX ,0 ) 1 + kQ [Q ]!

F0 is the total uorescence when there is no quencher.

F ! F0 = (

1 ! 1 ! kQ [Q ]" 1 ! 1)FX ,0 = FX ,0 ( ) 1 + kQ [Q ]" 1 + kQ [Q ]"

kQ [Q ]# 1 + kQ [Q ]#

F0 ! F = "F = FX ,0

Slope

1 1 1 + kQ [Q ]" = !F FX ,0 kQ [Q ]"

Intercept yields fx, the fraction of uorescence due to exposed sites.

F0 F0 F 1 1 1 = + 0 = + !F FX ,0 kQ [Q ]" Fx ,0 f X kQ [Q ]" f X

F0 1 1 1 1 = + !F f X kQ " [Q ] f X