J. Phys. Chem. 1994,98, 2210-2214
2210
Correlation between Protein Conformation and Prosthetic Group Configuration As Tested by pH Effects: A Hole-Burning Study on Mesoporphyrin-IX-Substituted Horseradish Peroxidase J. Gafert and J. Friedrich' Physikalisches Institut and Bayreuther Institut fur Makromolekiilforschung, Universitat Bayreuth, 0-95440 Bayreuth, Germany
J. M. Vanderkooi Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Pennsylvania 19104-6089
J. Fidy Institute of Biophysics, Semmelweis Medical University, P.O. Box 263, H-1444 Budapest, Hungary Received: November 15. 1993"
The optical absorption spectrum of mesoporphyrin-IX-substitutedhorseradish peroxidase shows a series of electronic origins with a typical spacing on the order of 100 cm-l. These origins correspond with different tautomer states of mesoporphyrin-IX. A change in the p H from 8 to 5 induces severe changes in structure as well as in the intensity distribution of the tautomer origins. In addition, the pattern of photochemical tautomer transformation changes significantly. The straightforward interpretation is that a change in p H leads to a structural accommodation of the apoprotein. This structural rearrangement influences the energy hypersurface of the prosthetic group leading to the change in the origin spectrum observed. In turn, there seems to be a feedback between the actual structure of the prosthetic group and the substructure of the apoprotein, as is clearly seen in the pressure-tuning behavior of spectral holes in the various tautomer bands.
Introduction Proteins keep the machinery of life running, for instance as enzymes in catalytic reactions, as transport and storage proteins, as photoreceptor proteins and in many other ways. Many of the proteins need prosthetic groups for proper functioning. In native horseradish peroxidase, the protein studied in this paper, the prosthetic group is a heme. It is an interesting problem in protein physics to elucidate the relation between the state of the prosthetic group and the associated structure of the apoprotein: Is there any correlation? If yes, is it restricted to specific structural hierarchies' and is the correlation local or does it have global character in the sense that the overall structure of the protein is linked to the state of the prosthetic group? Finally, one can ask whether such a structural correlation can also exist if the protein is at low temperature. Recently, Henry2has shown by performing molecular dynamics calculations for myoglobin that there is indeed a strong correlation between the dynamics of the heme group and the dynamics of the apoprotein on a time scale of at least nanoseconds. In this paper we will present pressure-tuning hole-burning experiments on horseradish peroxidase at pH 5. We will compare the specific spectral features at pH 5 with those at pH 8.3 These pH values can be considered as two extremes with respect to a heme-linked ionizable group that was shown to titrate with a pK of 7.1 and was suggested to be responsible for the pH dependence of the catalytic activity of the enzyme.4~5 Mesoporphyrin-IX (MP) substituting the native heme group in horseradish peroxidase (HRP) has been proven to be a sensitive monitor for testing the prosthetic groupprotein interaction in the heme pocket. Our experiments will show that the pH changes inducedrasticchanges in the spectral features of the prosthetic group: Some bands related to the tautomeric forms vanish while others come up. The experiments also show that the response of the holes to pressure is sensitive to the pH and depends on the spectral bands selected.
* Abstract published in Advance ACS Abstracrs, February
1, 1994.
0022-365419412098-2210$04.50/0
From this we conclude that it is not simply the configuration of the prosthetic group but the protein structure that changes with PH. The high fluorescence quantum yield and the photochemical tautomerization reactions make M P an ideal chromophore for photochemical hole-burning experiment^.^^ Hence, high-resolution optical techniques can be used to investigate structural effects.
Pressure Effects and Spectral Hole Burning in Proteins: Basic Aspects Pressure phenomena on spectral holes have been discussed in detail by Laird and Skinner.lo We apply their model on the basis of the following restriction^:*,^.^'-^^ (1) the protein can be considered as a homogeneous, isotropic matrix for the chromophore; (2) the atomic groups interact independently with the chromophore; (3) the pressure variations are small enough not to cause plastic deformations; (4) the pressure-relevant terms in the chromophore-protein interaction fall off as R6. Within this frame of approximations the pressure shift S of a hole is given by
s = 2K(Yb - V,,,)Ap
(1)
with K being the isothermal compressibility of the protein and vb the burn frequency. vvac is the vacuum absorption frequency of the chromophore. Equation 1 is equivalent to the assumption that pressure shift and solvent shift are pr0portiona1.l~ Note that Yb - uYsDis the solvent shift of those molecules selected by the burn frequency Yb.
A plot of the shift per pressure as a function of Vb yields a straight line with slope 2 ~ This . way, K can be measured by purely spectroscopic methods, provided the model is good enough. As to the quality of the model, we note that from the four points above only point 3 has been checked experimentally for 0 1994 American Chemical Society
The Journal of Physical Chemistry, Vol. 98, No. 8, 1994 2211
Prosthetic Group Configuration
HO-C-CH2CH2 II 0
CH2CH2-C-OH II
0
5
0
-5
Figure 1. Structure of mesoporphyrin-IX. a protein.*J3 The others cannot be directly tested. However, comparative experiments with glassesllJ2J5 and comparison with other experimental approaches for measuring K of proteins1&l8 support the validity of these statements. In addition, there are general arguments which offer explanations why the model seems to work well for proteins, too: The average displacement of an atom at the pressure level of our experiment is on the order of 10-4 A. This is by 3 orders of magnitude smaller than the structural uncertainity as obtained from an analysis of the X-ray intensities.19 Hence, on that scale, correlations among the protein building blocks may indeed be neglected. As a consequence, point 2 seems to be a reasonable approximation. If so, point 4 seems to hold reasonably well, too, because only two particle correlations between the chromophore and the protein building blocks need to be considered. In this case, the restriction to the R" terms reduces to the question of why the nearest neighbor shell of building blocks (Le. atoms or so) can be largely neglected in the pressure-induced shift of the spectral hole. It is this shell which feels the repulsive forces and which would lead to deviations from the assumption associated with point 4. The answer is that these building blocks are in the minimum of the potential curve (seen from the chromophore). They would induce contributions to the shift which are quadratic in pressure.20 Since the pressure level is extremely small, these contributions can be neglected. As to point 1, we note that the experiment will yield an average value of K over the range of the interaction forces.
0
-5
-5
5
frequency
/
0
5
GHz
Figure 2. Holes under pressure in the tautomer origins B1,B3,and B4 at pH 5. Note that the holes in B3 become asymmetric under pressure and show an onset of a tendency to split. These asymmetric holes are fitted by a superposition of two Lorentzians. The frequencies where burning was performed are indicated. Temperature: 1.5 K.
-6
.O
.5
1.0
.O
1.5
Ap
/
.5
1.0
1.5
MPa
Figure 3. Linearity of the pressure shift in the B1,B4 (a), and B3 (b) bands. For the B3 data, a fit with a single Gaussian is indicated (crosses) in order to convey an impressionof how the results depend on the evaluation procedure. d
Experimental Section
\
Isoenzyme C2 of H R P has been isolated and purified from horseradish, reconstituted with purified mesoporphyrin-IX (MP) by the Teale method21 and further purified by K . - 0 . Paul,z2who kindly donated this sample for our purposes. It has been shown23 that HRP after M P substitution still maintains its substrate binding ability. MP-HRP has been stored at 200 K in 50 mM ammonium acetate a t pH 5. The pH has been changed to pH 8 by dialysis. The structure of mesoporphyrin-IX is shown in Figure 1. The samples were sealed in small plastic bags to ensure isotropic pressure conditions. Pressure was transmitted via H e gas. The pressure cell was immersed in liquid helium and kept a t a temperature of 1.5 K. The pressure was regulated with an accuracy of 0.001 MPa and was varied up to about 2.0 MPa. Shift and broadening of the holes in this pressure range are completely reversible for MP-HRP.8 Spectral holes have been burnt at 1.5 K with an argon ion laser pumped single-mode CW dye laser (Coherent 699-21) with a line width on the order of lo4 cm-l. The burning power was in the microwatt range. Holes were burnt to a depth of about 40%. They were detected in the fluorescence excitation mode by scanning the laser over the burn frequency within a range of 1 cm-1 at a reduced intensity. Hole profiles, as they change under pressure, are shown in Figure 2 for various burn frequencies.
5-
o~'"'"""''"''"''
=-loo
15900
16000
16100
16200
/
cm-'
F
16300
16400
Figure 4. Pressure shift as a function of burn frequency at pH 5 and at pH 8. Temperature: 1.5 K. The corresponding fluorescence excitation spectra are indicated. Broadband excitation spectra were measured with a JobinYvon monochromator, whose resolution was set to about 10 cm-l.
Results and Discussion Influence of pH on the Spectral Pattern of the Tautomers of Mesoporphyrin-IX in Horseradish Peroxidase. Figure 4 shows the origin region of the fluorescence excitation spectrum at 4.2 K at two pH levels, namely 5 and 8, after sudden freezing from room temperature. For convenience, we have labeled the respective bands in an arbitrary fashion with B 1-B4. Apart from a slight difference in maximum position, the B1 band does not change in the studied pH range. The influence of the pH level is reflected in bands B2 and B3, which are negligible a t pH 5 but show up a t pH 8. In our earlier studies? we were able to show
2212 The Journal of Physical Chemistry, Vol. 98, No. 8,1994
0
-50
cd
pI -100
u h I
-150
j, i.
a.
U
\
-150 15900
16000
16100 ij
16200
16300
16400
cm-'
Figure 5. Pressure shift as a function of burn frequency for photochemically treated samples at pH 5 (a) and at pH 8 (b). Temperature: 1.5 K. The corresponding fluorescence excitation spectra are indicated.
by photoconversion that these bands correspond with different tautomer states of mesoporphyrin-IX. Slight differences in fluorescence lifetime values have also been detected.24 Although the structural details of these configurations are not known, one can think of diagonal arrangements, of various geminate pairs, and even of out of plane configurations. All these states of M P may have different ground-state energies, stabilized by different barriers, both affecting their relative populations. At p H 5,which is well below the pK value, the spectrum is dominated by B1 (Figure 4). This indicates that only one tautomeric structure can be effectively populated. Somewhat above the pK (pH 8), other tautomeric bands become populated also. Since B1 does not change with pH, we conclude that the associated inner-ring proton configuration is not changed. On the other hand, B2 and B3 reflect different configurations which seem to be induced by changing the pH level. The conclusion from the simple excitation spectra is that a change of the pH level leads to significant changes in the complicated energy hypersurface of the two inner-ring protons of the prosthetic group. Moreover, this change does not occur in a uniform manner. Instead, it seems that some molecules are changed whereas others arenot, because the B1 band, for instance, remains almost unaffected. Influence of pH on the Tautomer Photochemistry. Figure 5 compares the origin range of the excitation spectrum for the two pH values after heavy bleaching of B1. Bleaching has been performed at 4.2 K by scanning the laser over the band. Power levels were in the range of 10 mW, and irradiation times about 20 min. Under this condition, heating effects were negligible. It is obvious from the results that the photochemical changes in the M P tautomers are also affected by pH. At pH 8, the B2 band becomes preferentially populated, while B3 gains little intensity. At pH 5, on the other hand, B3 gains dominating inten~ity.~ Upon heavy bleaching (= 100 mW), a further band, B4, shows up, which cannot be populated at all a t pH 8. As has been shown earlier,7-9 B4 can also be populated from B3 in the dark, if the temperature is raised above 40 K. A similar phenomenon has been observed in the pH 8 sample, leading to an increased
Gafert et al. population of B3 at the expense of B2, by warming the sample above 30 K in the dark after irradiation. The conclusion is that the energy surface which determines the various low-temperature tautomer states strongly depends on the pH. We think that this change of the energy surface of the prosthetic group is mediated via a pH-induced change in the structure of the apoprotein. (In principle, the opposite could occur as well.) If so, there should be a correlation between the structure of the apoprotein and the configuration of the prosthetic group. With our technique we are able to measure a parameter which is sensitive to the structure of the protein: the isothermal compressibility K . If the surrounding protein structure is different for different tautomers, this parameter should change when the experiment is carried out in different tautomer bands. Frequency-Dependent hessue Phenomena. Presuming the simple pressure shift-solvent shift model outlined above describes the situation in proteins sufficiently well, we can use eq 1 to determine the compressibility of a protein molecule. Note that, with our technique, a correction for the contributions from the host materials is not necessary, because the compressibility is detected via the chromophore-solvent interaction which is of sufficiently short range not to exceed the dimensions of the proteins. First we note that, in the regions where the tautomer bands overlap, the pressure shift vs frequency pattern is more complicated because different shifts from different bands superimpose and, consequently, there are tendencies to a nonlinear behavior with frequency. However, as can be seen from Figures 4 and 5, in the main parts of the bands, the pressure shift is linear with frequency, in line with eq 1. The pressure phenomena show several noteworthy features. First, in the B1 band the slopes of the pressure shift vs frequency are-within experimental accuracy-identical for the two pH values considered: K = 0.1 1GPa-1. This result matches beautifully with theobservation that theassociated bandsdo not significantly change, and most probably, they are equilibrated with very similar protein structures. The population of the B2 band is extensively increased by irradiation a t pH 8. Pressure leads to a splitting of the holes, irrespective of the photochemical enhancement of B2 (compare Figure 4 and Figure 5b). The compressibilities are very similar before and after irradiation: K = 0.29 and 0.25 GPa-I, respectively.3 In the B3 band (Figure Sa), the situation is more complicated. At pH 5, where the band is populated by irradiation at 4.2 K, the holes split under pressure also. However, the respective frequency dependence of the pressure shift is not a clear straight line, probably because several bands overlap. Toward the high energy side of the band, the upper component seems to approach the slope found for the B3 band of the pH 8 sample (Figure Sb) which is of a single component. Thus, it is not clear whether or not the compressibility values, if determined in B3 at pH 5, are significantly different from that of B3 at pH 8. But from the pressure splitting it is clear that the tautomers associated with B3 are different at the two pH levels. Heavy irradiation conditions or heating of the irradiated sample above 40 K9 causes an additional band to appear at p H 5 (B4). It was possible to do frequency-dependent hole-burning experiments even in this low intensity band. In this case, the slope of the linear plot leads to a compressibility of 0.23 GPa-I (Figure Sa). Summarizing the results, we found that the compressibility of HRP depends significantly on the tautomer band selected. It seems that there are two major protein structural groups equilibrated with the tautomers, one being characterized by K = 0.11 GPa-', the other by (0.23-0.30) GPa-1. It is interesting that these structures are present at room temperature (pH 8 sample,
Prosthetic Group Configuration
The Journal of Physical Chemistry, Vol. 98, NO. 8, 1994 2213
broadening
\
s a
u
n ._
16200
16300
v/
16400
cm-'
Figure 6. Pressure broadening in the B1 band at pH 5 . Temperature: 1.5 K.
Figure 4) and can be photochemically produced at low temperature (eeg.B2atpH80rB3andB4atpH5)bypopulatingtheassociated tautomers. A clear example is the B2 tautomer in the pH 8 sample: it seems to be associated with the same protein structure irrespective of whether the tautomer had been there at room temperature or has been produced at low temperature by irradiation. We interpret these peculiar findings on the basis of a model which we call the "correlated phase space model".25 It is assumed that a specific structure of the protein supports a specific tautomer configurationof the prosthetic group or vice versa. In other words there are strong allosteric effects. This means that specific structures of the protein can be selected by irradiation into a specific tautomer band. As our experiments show, some of the physical properties, such as the compressibility, in these conformational substates of the protein are quite different. We stress that protoporphyrin-IX-substituted myoglobin shows a very similar behavior.25 Pressure-Broadening Effects. So far, we have discussed the pressure shift only. Yet the holes show a broadening under pressure also (Figure 2, Figure 6). As to the interaction responsible for broadening, there are several possibilities: the full Lennard-Jones potential with attractive and repulsive terms, the angular degrees of freedom in the dispersion and higher order electrostatic forces, and long-range coupling to random dipoles of the solvent. It seems that the dominating mechanism of line broadening comes from the angular degrees of freedom in the @-type potential.15 The long-range interaction, for example, can be ruled out because there are too many molecules involved so that, according to the central limit theorem, broadening would be vanishingly small.z6 The fact that pressure broadening in a protein does occur is interesting. It means that compression changes the relative arrangement of the protein building blocks in a random fashion. The magnitude of the broadening seems to be comparable to the respective one in glasses. For instance, protoporphyrin-IX in dimethylformamide/glycerol glass shows a broadening of about 1.8 GHz/MPa,Z5 which is not very far from the value in MPHRP (Figure 6). Broadening signals a lack of correlation between the chromophoreenvironment configurations with and without pressure. On the other hand, a lack of correlation signals structural indeterminism. From our results we have to conclude that, on the scale of the respective pressure deformations, proteins and glasses behave in the same fashion. This conclusion needs to be explained. We have stated above that the scale of deformation in our pressure experiments is on the order of 10-4 A, as is estimated from a typical compressibility of 0.1 GPa-1 and typical pressure changes of 1 MPa. On the other hand, we know from an analysis of the Debye-Waller factors in X-ray diffraction that the scale of the (static) structural indeterminism is on the order of some tenths of an A, Le. several orders of magnitude larger.19 Within
this scale the building blocks of the protein are structurally rather uncorrelated. The conclusion is that for any pressuredeformation within this scale of structural indeterminism, proteins behave like glasses. The pressure scale where we expect deviations from glasslike behavior is determined by the relation that the associated deformation surpasses the scale of structural indeterminism, e.g. approximately 0.2 A. It is easily estimated that this occurs for pressure changes typically larger than 1 GPa. At these pressure levels, the correlation among the protein building units becomes essential and pressure broadening should saturate. Significance of Data Evaluation. We feel it necessary to comment on the significance of the results, because in spectral ranges where several bands overlap, the measured behavior of the holes under pressure may strongly deviate from that predicted by the model (eq 1). According to the model, the pressure shift should be linear with frequency whereas pressure broadening should be independent of it.lo If the inhomogeneous bands are not totally separated, overlap regions always occur. Hence, in between two bands, the data are often not significant: the observed shifts behave in a nonlinear fashion, and the broadening is artificially large because two different shifts superimpose and may also become frequency dependent. Hence, as a rule, we neglected these data points for the evaluation of the compressibility. However, also the main part of a band may consist of more than just one tautomer state due to an accidentaldegeneracy. Since these tautomer states usually have different vacuum frequencies, e.g. Y ~ and~ uv,,,2,~ the , hole ~ can split under pressure by an amount
as follows immediately from eq 1. (Note that, for simplicity, we neglected the fact here that also K depends on the tautomer state.) Whether a splitting is observed or not depends on two quantities, namely on the ratio of the pressure splitting L 7 to the associated pressure broadening Ar of the hole and on the relative populations of the tautomer states involved. In some cases, for instance in the B2 band at pH 8, a splitting is clearly detectable and both components can be evaluatedseparately in a straightforward way.3 On the other hand, the splitting in the B3 band at pH 5 reflects the opposite case. The superimposed components manifest themselves only in an asymmetric shape of the hole under pressure. At higher pressure levels, a low intensity shoulder appears on the red side of the hole (Figure 2). Whenever the shape of the hole became significantly asymmetric under pressure, we fitted two Lorentzians and evaluated them separately. For MP-HRP at pH 5 , this was only the case in the B3 band. In the B1 and B4 band, the holes were perfectly symmetric under pressure. It is clear that a single Gaussian fit to an asymmetric line would affect the shift vs frequency slope to some extent. This is demonstrated in Figure 3b, where the results of single Gaussian fits (crosses) are shown for comparison. Note that the pressure dependence of the shift S is perfectly linear in this case, too. Hence, it is also important to consider the shape of the spectra in order to obtain correct values for the shift per pressure. As to the pressure broadening, it does not depend on frequency in the B1 band. This is in line with the fact that the holes broaden symmetrically with pressure, indicating that this band can be associated with a single tautomer state. Furthermore, on average, broadening does, contrary to the shifts, not depend on the tautomer origins. Yet frequency dependencies show up when several components superimpose.
Conclusions Proteins behave indeed peculiarly: From pressure broadening we concluded that they are like glasses. Yet, the frequency dependence of the pressure shift showed that they are, at the same time, very different from glasses. The compressibility is
2214 The Journal of Physical Chemistry, Vol. 98, No. 8, 1994
not well defined, and we had to invoke a strong correlationbetween optical frequency and protein structure. This is quite in contrast to glasses where such a behavior is absent or at least small. In a homogeneous glass it is, as a rule, impossible to select certain configurations through optical selection. So we ask where the similarities come from and how we can explain the differences. We believe that the similarities as well as differences between proteins and glasses can be traced back to the specific features of their structural phase space. In proteins, disorder seems to occur in a hierarchical fashion.' At a sufficiently high hierarchy level, there are distinguished states. In native myoglobin, for instance, the taxonomic states fall into this category.*' In a distinguished state many substructures are possible. As a consequence of hierarchies in disorder, the structural phase space decomposes into islands.2s In our correlation model we assumed that certain substructures of the apoprotein, namely those which correspond to certain islands in structural phase space, support a specific tautomer configuration. In this case the islands can be separately selected. This means that averages over physical parameters (e.g. compressibility),as measured by theexperiment, are averages over the island-regionsonly, and not over the entire phase space. Since the islands correspond with different structures, the parameters determined are subject to variations. What is surprising is the fact that these variations are rather large, although the associated structural variations-from one tautometer band to a n o t h e r d o not exceed the scale of the structural indeterminism as obtained from an analysis of the DebyeWaller factors in the X-ray diffraction pattern. We concluded above that hierarchies in disorder lead to a decomposition of the structural phase space into islands. There may be quite a series of structural hierarchies. Hence, the islands themselves may consist of smaller islands. From the experiment, there is, as of yet, no way to select higher hierarchies. Hence, we can consider an island as a limited yet homogeneous area in phase space. That is the reason why, within an island, a protein behaves like an ordinary glass. From the pressure broadening of holes as well as from the magnitude of inhomogeneous line broadening, we concluded that the scales of structural indeterminism of a glass and of a protein are similar. One is inclined to believe that structural disorder in glasses is much larger than in proteins in order to account for the well-resolved X-ray diffraction patterns of the latter ones. However, this seems not to be the case. From the measured compressibility we found that, on average, a dislocation of an atomic group on the order of 10-4 A accounts for the observed pressure broadening of roughly 1 GHz. From this we can extrapolateand estimate that a variation in structure on the order of 0.1 A accounts for the inhomogeneouswidth, which is typically 100 cm-1. This is exactly the scale of the structural uncertainty as determined from X-ray analysis.19 The conctusion is that the geometrical scales of disorder for glasses and proteins are similar. As to structural disorder on a nanoscale, there is no difference between glasses and proteins.
Gafert et al. It is the finite size of a protein which determines the difference as compared to glasses. Due to their finite size proteins can form crystals with a mesoscopic scale of order. This mesoscopic scale ensura that the correlationbetween a building block in one protein molecule with the same block in another protein pertains throughout the crystal. This explains the sharp X-ray diffraction patterns observed in protein crystals. In a glass, however, there is no such mesoscopic scale because it is essentially infinite. Hence, the two-particle correlation dies out on a scale of some 100 A, and consequently, there are no sharp X-ray diffraction patterns.
Acknowledgment. We acknowledge support form the DFG (SFB 21 3-Bl5, Graduiertenkolleg "Nichtlineare Dynamik"), from the Fonds der Chemischen Industrie, from the BMFT (WTZ program with Hungary), and from FEFA (265). References and Notes (1) Ansari, A.; Berendzcn, J.; Bowne, S.F.; Frauenfelder, H.; Iben, I. E. T.; Sauke, T. B.; Shyamsunder, E.; Young, R. D. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5000. (2) Henry, E. R. Biophys. J. 1993,64, 869. (3) Friedrich, J.; Gafert, J.; Zollfrank, J.; Vanderkooi, J. M.; Fidy, J. Proc. Null. Acad. Sci. U.S.A., in press. (4) Yamada, H.; Makino, R.; Yamazaki, I. Arch. Biochem. Biophys. 1975, 169, 344. ( 5 ) Shelnutt, J. A.; Alden, R. G.; Ondrias, M. R. 1.Biol. Chem. 1986, 261. -. , 1720. -(6) Zollfrank, J.; Friedrich, J.; Vanderkooi, J. M.; Fidy, J. J. Chem. Phys. 1991,95, 3134. (7) Zollfrank, J.; Friedrich, J.; Vanderkooi, J. M.; Fidy, J. Biophys. J. 1991. 59. 305. (8) kllfrank, J.; Friedrich, J.; Fidy, J.; Vanderkooi, J. M. J . Chem. Phys. 1991, 94, 8600. (9) Fidy, J.; Vanderkooi, J. M.; Zollfrank, J.; Friedrich, J. Biophys. J. 1992.61, 381. (10) Laird, B. B.; Skinner, J. L. J. Chem. Phys. 1989,90, 3274. (11) Gradl, G.; Zollfrank, J.; Breinl, W.; Friedrich, J. J. Chem. Phys. 1991, 94, 7619. (12) Zollfrank, J.; Friedrich, J. J . Phys. Chem. 1992, 96. 7889. (13) Zollfrank, J.; Friedrich, J.; Parak, F. Biophys. 1.1992, 61, 716. (14) Sesselmann, Th.; Kador. L.; Richter, W.; Haarer, D. Europhys. Lett. 1988.5. 361. (lS)'Pschierer, H.; Friedrich, J.; Falk, H.; Schmitzberger, W. 1.Phys. Chem. 1993,97, 6902. (16) Gavish, B.; Gratton, E.; Hardy, C. J. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 750. (17) Yamato, T.;Higo, J.;Seno, Y.; Go, N. Proteins: Structure,Function, and Genetics 1993, 16, 327. (18) Kharakoz, D. P.; Sarvazyan, A. P. Biopolymers 1993, 33, 11. (19) Frauenfelder, H.; Parak, F.; Young, R. D. Annu. Reo. Biophys. Biophys. Chem. 1988, 17,451. (20) Schellenberg, P.; Friedrich, J.; Kikas, J. J . Chem. Phys., submitted for publication. (21) T a l e , F. W. J. Biochim. Biophys. Acta 1959, 35, 543. (22) Paul, K.-G.; Stigbrand, T. Acta Chem. S c a d . 1970, 24, 3607. (23) Horie, T.; Vanderkooi, J. M.; Paul, K.-G. Biochemistry 1985, 24, 793 1. (24) Fidy, J.; Koloczck, H.; Paul, K.-G.; Vanderkooi, J. M. Chem. Phys. Lett. 1987, 142, 562. (25) Gafert, J.; Friedrich, J.; Parak, F. J . Chem. Phys. 1993, 99, 2478. (26) Kador, L. J . Chem. Phys. 1991, 95, 5574. (27) Frauenfelder, H.; Sligar, S. G.; Wolynes, P. G. Science 1991, 254, 1598. ~
