[13] Intramolecular pyrene excimer fluorescence: A probe of proximity and protein conformational change

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and Frasier, 68 for the global analysis and the previous standard deviations are included for each type of data. Final Remarks Stopped-flow fluorescence techniques provide a number of possible strategies for studying the kinetics of protein folding. The signal-to-noise ratios of such experiments can be good and there are a variety of intrinsic and extrinsic reporter groups that can be employed. Probably the most attractive feature of SF fluorescence is that there are several variations on the basic experimental design and these can provide different types of useful structural information regarding the properties of transient folding intermediates.

Acknowledgments This work was supported by NSF Grant MCB 94-07167. We thank Roxana Ionescu, R. Kent Gartin, and Evan Tillman for assistance in collecting data reported in this chapter.

68 M. L. Johnson and S. G. Fraiser, Methods EnzymoL 117, 301 (1985).

[ 13] I n t r a m o l e c u l a r P y r e n e E x c i m e r F l u o r e s c e n c e : A Probe of Proximity and Protein Conformational Change

By S H E R W I N

S. L E H R E R

In~oducUon In addition to emitting fluorescence from the excited monomer state, some fluorophors can form an excited-state dimer or excimer, by a specific interaction between the excited monomer and a ground-state monomer. When attached to proteins at specific sites, fluorophors that can form excimers can be used to provide information about changes in proximity between attachment sites. The most useful fluorophors for this purpose are pyrene and its derivatives. The excimer fluorescence of these compounds was first reported by Kaspar and F6rster in 1954 (reviewed by F6rster 1) and their properties have been extensively studied by Birks. 2 Excimer 1 T. F6rster, Angew. Chem. Int. Ed. 8, 333 (1969). 2 j. B. Birks, "Photophysics of Aromatic Molecules." Wiley-Interscience, London, 1970.

METHODS IN ENZYMOLOGY, VOL. 278

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fluorescence has also been observed for benzene and toluene 2 and phenol and para-substituted phenols, 3 suggesting that one might observe intrinsic excimer formation in proteins from proximal phenylalanine and tyrosine. The efficient excimer fluorescence of pyrene derivatives attached to neighboring protein sites can be used to probe conformational changes associated with a variety of protein interactions. With the use of sitedirected mutagenesis, amino acid residues that selectively react with pyrene derivatives can be substituted at regions of the sequence that are suspected or known to be in close proximity. Although other spectroscopic properties of pyrene derivatives have been used in protein studies, only a limited number of excimer studies have been reported since the first studies of pyrene excimer fluorescence from probes attached to proteins at specific sites4'5 were published. A review has summarized previous studies of excimer fluorescence.6 This chapter emphasizes the practical problems of measuring and interpreting intramolecular pyrene excimer fluorescence in aqueous solution relevant to studies of protein conformational changes.

Intramolecular Pyrene Excimer Formation

In Organic Solvents An excimer is formed if an excited pyrene monomer, during its fluorescence lifetime, interacts in a specific manner with a neighboring groundstate (unexcited) pyrene. Early studies in "good" solvents, in which the pyrenes do not interact in the ground state, showed that as the pyrene concentration was increased, instead of the expected decrease in the monomer fluorescence due to concentration quenching, excimer fluorescence replaced the monomer fluorescence, a special type of concentration quenching. 1'2 Intramolecular excimer fluorescence in organic solvents has also been observed: for phenyl groups attached between propane, 7 for pyrenes

3 S. S. Lehrer and G. D. Fasman, J. Am. Chem. Soc. 87, 4678 (1965). 4 S. Betcher-Lange and S. S. Lehrer, J. Biol. Chem. 253, 3757 (1978). 5 M. Zama, P. N. Bryan, R. E. Harrington, A. L. Olins, and D. E. Olins, Cold Spring Harbor Syrup. Quant. Biol. 42, 31 (1978). 6 S. S. Lehrer, Pyrene excimer change as a probe of protein conformational change. In "Subcellular Biochemistry" (B. B. Biswas and S. Roy, eds.), Vol. 24, pp. 115-139. Plenum, New York, 1995. 7 F. Hirayama, J. Chem. Phys. 42, 3163 (1965).

288

[ 13]

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A

Ground-State Equilibria

Excited-State Equilibria

Fluorescence

Fluorescence

FIG. 1. Schematic diagrams of the production of intramolecular excited dimer (excimer) fluorescence from excited monomer pyrenes in organic solvents (A) and in aqueous solution (B). (A) In an organic solvent, the pyrenes do not interact in the ground state. On excitation (*), if unhindered, the pyrenes can reorient to form the excimer, in competition with monomer fluorescence. The excimer emits fluorescence at a longer wavelength and dissociates in parallel with emission. (B) In aqueous solution, the pyrenes will tend to stack owing to hydrophobic interactions producing an equilibrium between unstacked and stacked configurations determined by the flexibility of the "loop" between them and the nature of the hindrance. For the unstacked configuration, only monomer fluorescence will be produced. For the stacked configuration, on excitation, either excimer fluorescence or quenching (no fluorescence) will result, depending of the ability of the stacked pyrenes to reorient to the excimeric configuration.

attached to p r o p a n e derivatives, s,9 and for pyrenes attached to the two SH groups of dithiothreitol. 1° Intramolecular excimer formation is schematically depicted for two pyrene moieties attached to a hydrocarbon chain in a organic solvent environ8 H. Dangreau, M. Joniau, M. De Cuyper, and I. Hanssens, Biochemistry 21, 3594 (1982). 9 K. A. Zachariasse, G. Duveneck, and R. Busse, J. Am. Chem. Soc. 106, 1045 (1984). 10y. Ishii and S. S. Lehrer, Intramolecular excimer fluorescence of pyrene maleimide-labeled dithiothreitol. In "Fluorescent Biomolecules" (D. M. Jameson and G. D. Reinhart, eds.), Vol. 51, pp. 423-425. Plenum, New York, 1989.

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B Unstacked

Stacked

Ground-State Equilibria

/ /

~

XC

Excited-State Equilibria

+ Excimer~ Fluorescence "~,

oescence

+ Monomer Fluorescence FIG. 1.

(continued)

ment where the pyrenes do not interact in the ground state (Fig. 1A). On excitation into the absorption band of pyrene (Amax = 340-343 nm) an excited m o n o m e r is produced. During its long lifetime, >90 nsec in the absence of oxygen for pyrene maleimide dithiothreitol, 1° the excited monomer can rotationally diffuse near its neighbor ground-state pyrene, and if unhindered, an excited-state dimer with a precise symmetrical configuration can be formed. The excimer emits fluorescence with a red-shifted unstructured spectrum because it originates from a lower energy excited state than the excited m o n o m e r and dissociates while remaining tethered on returning to the ground state as indicated (Fig. 1A). Two main properties of this "classic" scheme of excimer fluorescence follow from the photophysics: (1) The excitation spectrum of the excimer is identical to that of the monomer. This follows because both excimer and monomer fluorescence originate from the same source--ultraviolet (UV) absorption by a ground-state uncomplexed pyrene; (2) the time dependence of excimer fluorescence, in the nanosecond time domain, shows both a buildup and decrease; i.e., the excimer is formed after a m o n o m e r is excited so that a delay in excimer

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fluorescence compared to monomer fluorescence is observed. 1'2 However, in aqueous solution, the situation with respect to these two properties is altered as discussed in the next section.

In Aqueous Solution Studies with pyrene-labeled tropomyosin4 showed that in aqueous solution the excimer excitation spectrum is not identical to that of the monomer. The excimer excitation spectrum was somewhat broadened compared to that of the monomer, indicating ground-state hydrophobic interactions between pyrenes. Lifetime studies showed little time delay between monomer and excimer emission for pyrene-tropomyosin.11Thus, in aqueous solution, attaining the excimer configuration appears to require only a slight reorientation of hydrophobically stacked pyrenes. Data obtained with the model compound, pyrene-dithiothreitol, also indicated that excimer fluorescence originates from stacked pyrenes. The model compound studies also showed that monomer fluorescence can be quenched by pyrene-pyrene interaction without formation of excimer I° and data with pyrene-labeled proteins indicate the generality of such quenching. It appears that pyrene-pyrene interaction can result in quenching of monomer without production of excimer fluorescence if the stacked pyrenes are inhibited from reorienting because of conformational restraint. Thus, ground-state pyrene-pyrene interaction can either facilitate or inhibit excimer formation depending on the ability of the pyrenes to reorient. For aqueous systems the "classic" schematic diagram of intramolecular excimer formation illustrated in Fig. 1 must be modified to take into account ground-state pyrene-pyrene interactions. In aqueous solution, depending on the conformation of the protein and the local environment, the pyrenes will equilibrate between unstacked and stacked configurations in the ground state (Fig. 1B). For proteins in which the "loop" separating the pyrenes is quite flexible, the stacked configuration should dominate as it does for pyrene-dithiothreitol.1° However, for a protein in which the loop represents secondary and tertiary structures that inhibit pyrene interaction, the equilibrium will favor the unstacked pyrene configuration. Changes in conformation of the protein near the pyrenes will influence the equilibrium and change the ability to exhibit excimer fluorescence. Although in Fig. 1B one configuration is schematically indicated for the stacked ground state, it represents a distribution of configurations in view of the nonspecific hydrophobic interactions. It is assumed that excimer formation can originate only from stacked pyrenes and monomer emission can originate only from unstacked pyrenes, because of the different H p. Graceffa and S. S. Lehrer, J. Biol. Chem. 255, 11296 (1980).

[13]

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excitation spectra obtained in model systems. However, every stacked configuration may not lead to excimer fluorescence. For some stacked configurations the excited monomer state is quenched before reorientation to the excimeric configuration can take place. Another group of stacked states is therefore indicated in Fig. 1B that are nonfluorescent, i.e., in a "dark complex," when the rings are prevented from attaining the precise excimeric configuration.

Protein-Labeling Procedures N-(1-pyrene) maleimide was one of the first protein pyrene labeling reagents developed, t2 which owing to its maleimide moiety was quite specific for the SH groups of exposed cysteine side chains. It has two useful properties as a general reagent: (1) it provides a low quantum yield in aqueous solutions, which markedly increases as a result of the labeling reaction, 12 and (2) at high pH values ( > p H 8) the resulting succinimide ring undergoes hydrolysis or aminolysis (with a neighboring lysine group). 4'13'14 Conversion of type I (intact succinimide ring) to type II (cleaved succinimide ring) by incubation at high pH values, 13 red-shifts the monomer fluorescence and increases the excimer/monomer ratio for labeled tropomyosin and dithiothreitol. N-(1-pyrene) iodoacetamide is now also available and both reagents are commonly used to label cysteine groups with pyrene. The iodoacetamide derivative is fluorescent in the unreacted state, in contrast to the maleimide derivative, but has spectra and properties similar to those of the maleimide type II label, with monomer fluorescence peaks at 383 and 402 nm and excimer fluorescence at 480-490 nm (Fig. 2). The excitation spectra for the pyrene iodoacetamide-labeled tropomyosin also show similar broadened peaks of excitation of excimer compared to monomer (Fig. 2) previously seen for the maleimide reagent. 4 Owing to the broadening, a greater excimer/monomer ratio can be realized by excitation near 360 nm instead of at the peak value near 340 nm. Cysteine groups are the prime choice for specific labeling of proteins for several reasons: (1) pyrene maleimide and iodoacetamide derivatives that preferentially label cysteine are commercially available (e.g., Molecular Probes, Eugene, OR; Sigma, St. Louis, MO; Aldrich, Milwaukee, WI); (2) only a limited number of cysteines are usually present in proteins; (3) cysteines that are present as the disulfide or that can be oxidized to the 12j. K. Weltman, R. P. Szaro, A. R. J. Frackelton, R. M. Dowben, J. R. Bunting,and R. E. Cathou, J. Biol. Chem. 248, 3173 (1973). 13C. Wu, L. R. Yarbrough, and F. Y. Wu, Biochemistry 15, 2863 (1976). 14y. Ishii and S. S. Lehrer, Biophys. J. 50, 75 (1986).

292

FLVORESCENCESPECrROSCOPY

U C

° u

(#}

Excitation

Emissio

oO"

n / m-k

/

[ 13]

\

.-" }J ,IJ

I

I

i

300

i

i

i

,

400

i

i

,

I

i

i

i

500

Wavelength (nm) Fic. 2. Fluorescence spectra of pyrene iodoacetamide-labeled•,B-tropomyosin from gizzard muscle in aqueous solution. M, Monomer fluorescence band; E, excimer fluorescence band. (--) Emission spectrum, .~e×c= 340 nm. (©) Excitation spectra, 1era = 385 nm; (O), • e m = 480 nm. Note the broadened excimer excitation spectrum. [N. Golitsina, X. Zhou, and S. S. Lehrer, unpublished data, 1996.)

disulfide will offer a high probability of forming excimer when labeled; and (4) cysteine groups can be introduced into the amino acid sequence by sitedirected mutation techniques. In the case of cysteine present as disulfide, the disulfide cross-link must first be reduced. This can be accomplished with dithiothreitol, most readily in the unfolded state in the presence of denaturant such as guanidinium hydrochloride (GdmC1) or urea. The dithiothreitol is then removed by dialysis or gel filtration and the labeling reaction is carried out in the presence of denaturant; last, the denaturant is removed to refold the protein. Selective labeling of pairs of cysteines is possible by varying the reaction conditions, e.g., at increased denaturant concentration to selectively reduce a pair that becomes exposed in a partially unfolded intermediate. If cysteine groups are present in the reduced form, labeling can be carried out in the native state if the cysteine groups are accessible or in the unfolded state if they are not. The labeling times are controlled by quenching the reaction with excess dithiothreitol, or by dropping the p H to low values ( < p H 4) where the reaction is inefficient. If the labeling reaction is carried out in the native state, the course of the reaction can be monitored fluorometrically, noting that excimer forms only when each molecule is doubly labeled, and the reaction stops when saturation begins to take place. For pyrene maleimide, kinetics can be followed as an increase in m o n o m e r and excimer fluorescence. 12 The great wavelength separation between the excitation (340 nm) and the excimer emission (490 nm) will minimize effects of light scattering owing to the insoluble pyrene maleimide or iodoacetamide, as will the use of low concentrations. Mixed

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organic-water solutions can be explored to keep the reagent soluble and thereby facilitate reaction. Reaction with either pyrene maleimide or pyrene iodoacetamide is usually performed at pH 6.5-8.0 using a 2-10x excess of reagent over the sulfhydryl concentration, diluted - 2 0 × from dimethylformamide, acetone, methanol, or dimethyl sulfoxide. Reaction times will vary, but 1-5 hr at room temperature or below is often sufficient. Both reagents are not soluble in aqueous solution, so a precipitate usually appears and therefore gentle shaking facilitates the reaction. After quenching the reaction, the solution is filtered or spun to remove excess undissolved reagent and dialyzed or gel filtered to remove unreacted dissolved reagent. Owing to the hydrophobicity of pyrene some noncovalent binding of the reagent may take place, so exhaustive dialysis in the presence of dithiothreitol, which increases the solubility of the reagent on reaction, may be necessary. The presence of noncovalently bound label in the protein solution may lead to extrinsic excimer fluorescence, complicating interpretation. It can be detected on polyacrylamide gel electrophoresis as blue fluorescence migrating with the front. If maleimides are used, the different fluorescent properties of type I- and type II-labeled proteins can be explored (see above). Because the labeled protein may have altered properties, activity or other studies to determine if the labels have affected its properties should be performed. Although both maleimides and iodoacetamides react preferentially with cysteine, there is the possibility of reaction with other protein side chains such as lysine. Maleimides and iodoacetamides react with the basic form of both cysteine and lysine but cysteine is preferentially labeled if present. The degree of labeling can be determined with the use of the extinction coefficient for pyrene,/3343 n m = 2.2 × 104 M - l c m -1 and the protein concentration determined with a protein colorimetric assay. The protein should be unfolded with denaturant so that effects of putative pyrene stacking on absorption can be eliminated. To verify specific labeling at cysteine, a control labeling reaction with the protein that has had its cysteine group reversibly blocked with 5,5'-dithiobis-2-nitrobenzoate can be performed. 15 Proteins with blocked cysteine groups should have no fluorescence unless other groups reacted. Standard limited protein cleavage methods and peptide analysis can also be used to determine specificity and location of label. Interpretation of Fluorescence Changes Regardless of whether the pyrenes are stacked in the ground state, if excimer fluorescence is observed, the pyrenes must be in close proximity. For pyrene iodoacetamide-labeled proteins, the extended distance from 15 S. S. Lehrer and Y. Ishii, Biochemistry 27, 5899 (1988).

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the sulfur of the cysteine to the middle of the pyrene group is about 9 A. It is therefore possible for excimer fluorescence to be observed for labeled SH groups that are about 20 A apart. This has been observed in at least one system. 16'17The ability to observe excimer fluorescence not only depends on the distance between labeling sites, but also on the flexibility of the pyrenes at attachment sites and on the absence of intervening groups. For pyrenelabeled proteins in aqueous solution, excimer fluorescence appears to originate from surface pyrenes that stack hydrophobically in the ground state. However, if the stacked pyrenes cannot reorient on excitation, the fluorescence will be quenched. Thus, in general, the observation of excimer fluorescence provides proximity information but the lack of excimer fluorescence does not preclude pyrene proximity, particularly if absorption spectra are broadened, relative to the singly labeled system, thereby showing evidence of stacked pyrenes. It is possible that the attached pyrenes could locate in the hydrophobic interior of a protein or in the transmembrane region of reconstituted protein-membrane systems and thereby may not tend to stack. In these situations, somewhat analogous to excimer-forming fluorophors in the lipid environment of a membrane, TM the probability of excimer formation would be determined by the local viscosity of the medium in addition to proximity. In this case, in which the schematic of Fig. 1A applies, opposite changes in monomer and excimer would occur, resulting in an isoemissive point in the spectrum. Such changes were observed early for thermal perturbation of tropomyosin conformation,11 which led to an initial model that did not take into account nonfluorescent stacked species. However, a constant amount of stacked nonfluorescent species would also lead to the same result. In aqueous solution, where conformational changes could change the ability of stacked pyrenes to attain the excimer configuration, both monomer and excimer intensity could, e.g., increase or decrease together depending on changes in the contribution of the nonfluorescent stacked state. An increase in both excimer and monomer fluorescence can be explained, e.g., by a conformational change that "loosens" the pyrene-pyrene interaction, facilitating both the formation of the excimeric configuration and reequilibration toward unstacked configurations. The following information is useful to help determine the origin of the fluorescence spectrum of a given labeled system. 16A. D. Verin and N. B. Gusev, Biochim. Biophys. Acta 956, 197 (1988). 17Y.-M. Liou and F. Fuchs, Biophys. Z 61, 892 (1992). 18B. Wieb Van der Meer, Biomembrane structure and dynamics viewed by fluorescence. In "Fluorescence Studies on Biological Membranes" (H. J. Hilderson, ed.), Vol. 13, pp. 38-41. Plenum, New York, 1988.

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1. The degree of labeling: If it is greater than the number of accessible reactive groups, e.g., cysteine, there could be extra extrinsically bound pyrene, either reacted at other groups or noncovalently bound, contributing to the excimer fluorescence. There will also be monomer fluorescence contributions from incompletely labeled proximal cysteine pairs. 2. Excitation spectra of monomer and excimer fluorescence: If the excimer band is broadened, the excimer fluorescence probably originates from stacked pyrenes. 3. Lifetime studies: If there is a lack of appreciable delay between monomer and excimer decay, stacked pyrenes are present. If fluorescence changes do not result in changes in fluorescence decay parameters, changes in ground-state interactions are involved. 4. Effects of the label on protein conformation or activity: The conformation could be distorted by the pyrenes interacting with each other, resulting in altered proximity of the cysteines to which the pyrenes are attached. Whether excimer fluorescence from labeled proteins arises from stacked or unstacked ground-state configurations, its fluorescence can be used to study conformational changes associated with the binding of substrates and cofactors, complex formation, and unfolding/refolding reactions to obtain binding and equilibrium constants and kinetic parameters. 6

Acknowledgment Supported by N I H Grants H L 22416 and A R 41637.

[14] L o n g - L i f e t i m e M e t a l - L i g a n d C o m p l e x e s a s P r o b e s i n Biophysics and Clinical Chemistry

By E W A L D

T E R P E T S C H N I G , H E N R Y K SZMACINSKI,

and JOSEPH R. LAKOWICZ In~oduc~on In the design of a fluorescence experiment one can choose from hundreds of fluorophores that cover a wide range of absorption and emission wavelengths from 300 to 700 nm. However, the diversity of fluorescence decay times is much more limited, with most fluorophores displaying decay times between 1 and 10 nsec. Although this is a useful time scale for many

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