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J. Phys. Chem. B 2002, 106, 3510-3514
Deuteration Effects on the Conformational Dynamics of Proteins in a Trehalose Glass J. Schlichter and J. Friedrich* Lehrstuhl fu¨ r Physik Weihenstephan, Technische UniVersita¨ t Mu¨ nchen, D-85350 Freising, Germany
L. Herenyi and J. Fidy Institute of Biophysics, Semmelweis UniVersity, Budapest, Hungary ReceiVed: June 19, 2001; In Final Form: January 17, 2002
We present results of a hole-burning spectral diffusion experiment on a deuterated sample of horseradish peroxidase (substituted with free-base mesoporphyrin-IX) in a trehalose/glycerol glass. Like in earlier experiments, the spectral diffusion dynamics is well-described by a diffusion picture; the most noticeable observation is a power-law dependence of the hole-broadening on time. In addition, the comparison of the present data with our earlier experiments allows for a microscopic interpretation of the influence of trehalose on protein dynamics. Here, internal water molecules in the proteins seem to be of great importance.
Introduction In a multitude of experiments, optical techniques such as single-molecule spectroscopy, three-pulse photon echo, or spectral hole-burning proved their usefulness for the investigation of low-temperature dynamics in disordered solids.1-4 Especially, the dynamical properties of proteins attracted attention in the last years because this topic is interesting for both biologists and physicists: protein dynamics is crucial for the understanding of the functioning of these molecules in an organism; on the other hand, low-temperature proteins are complex nonequilibrium systems, which show a rich dynamical behavior and are therefore interesting from a more fundamental point of view as well. In optical measurements at cryogenic temperatures, a chromophore inside a protein serves as a sensitive probe for dynamical processes in the surrounding molecule. These processes cause stochastic shifts in the absorption line of the chromophore. Hence, an ensemble of chromoproteins, which is initially characterized by identical absorption frequencies (prepared for example by spectral hole burning), loses its degeneracy in frequency space with increasing time because of the structural dynamics in the conformation space of the proteins. In a stationary system, this so-called spectral diffusion, that is, the broadening of the frequency distribution with time, depends only on the time elapsed between preparing the ensemble (i.e., hole burning) and measurement; in the following, this time will be referred to as waiting time, tw. In a nonstationary system, there is an additional influence of the socalled aging time, ta, that is, the time span between the moment when the sample has reached its final temperature and the hole burning; in this case, spectral diffusion is characterized by both tw and ta. During the past few years, we have been investigating the dynamical properties of several protein/solvent systems at helium temperature by spectral hole burning (for a review of this technique, see refs 5 and 6). In all of these experiments, we found a very similiar behavior of spectral diffusion, of which the most noticeable feature was a power-law dependence on * To whom correspondence should be addressed. E-mail: J.Friedrich@ lrz.tu-muenchen.de. Fax: 0049-8161-71-4517.
the waiting time.7,8,9 In addition, a (small) aging effect was observed, which could likewise be characterized by a power law. The broadening of a spectral hole is mathematically described by a spectral diffusion kernel, the shape of which depends on the microscopic model used for the interpretation of the measurements. Contrary to spectral diffusion measurements in glasses, which usually can be described by the so-called standard-tunnel model (or a modified version of it10), our protein data are analyzed in the framework of a diffusion picture.8,11 In this case, diffusive motions of the amino acid residues in the proteins are held responsible for the observed spectral diffusion. This leads to a Gaussian diffusion kernel, of which the time-dependent width, σ(tw,ta), can be obtained from the hole broadening measured in an experiment. (Because a spectral hole immediately after the burning is empirically well-described by a Lorentzian, the convolution with a Gaussian kernel leads to Vogtian hole profiles at later times. By fitting the spectral holes with Voigt functions and doing a numerical deconvolution, the width σ of the diffusion kernel can be extracted from the data.) The observed power-law behavior for the spectral diffusion therefore is of the form σ ∝ twR/2. For the exponent, all of our measurements yielded the same value of R/2 ≈ 0.25. (It should be stated, that the value of this exponent depends on the microscopic picture used for the interpretation of the data, while the power-law behavior itself is an independent experimental fact. Note that in an earlier paper we tried to explain our measurements within the framework of the TLS model, finding then an exponent of about 0.5.12 However, as more proteins were investigated, it turned out that a consistent interpretation of the spectral diffusion dynamics by the TLS theory was not possible.13 The above-mentioned diffusion picture, which leads to a value of about 0.25 for R/2, seemed to be much more appropriate.) Because low-temperature proteins are nonequilibrium systems, two different kinds of dynamic processes contribute to the spectral diffusion, fluctuations (i.e., transitions between different (local) equilibrium states of the molecule) and relaxations (i.e., movements, by which the protein is approaching its global equilibrium). In our model, the equilibrium fluctuations
10.1021/jp012316e CCC: $22.00 © 2002 American Chemical Society Published on Web 03/13/2002
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J. Phys. Chem. B, Vol. 106, No. 13, 2002 3511
are characterized by a correlation time, τ0. The additional contribution of relaxation processes to the spectral diffusion, which becomes obvious in the experiments by the (small) influence of the aging time on the results, can phenomenologically be included in the model by allowing for an algebraic aging time dependence of the correlation time:
τ(ta) ) τ0(ta/T)2β/R
(1)
Here, T is a typical time-constant for the change of the correlation time with ta; the exponent β is found from the experiment to be on the order of 0.1. As stated above, spectral diffusion and conformational changes (e.g., of a protein) are related to each other.14 It can be shown, that σ2, the variance of a spectral hole, is proportional to the (time-dependent) meansquare displacement of the amino acid residues of the protein;15 the respective proportionality factor depends strongly on the distance between chromophore and residues. Therefore, only movements of the residues in the direct neighborhood of the chromophore contribute to the spectral diffusion. These movements are modeled in our picture by diffusive processes. The diffusion, however, is strongly coupled to processes occurring in the rest of the proteinsthe more remote residues must “step aside” to allow motions in the neighborhood of the chromophore. As shown in a classical treatment by Berlin and Burin,16,17 under these circumstances anomalous diffusion occurs; the time-dependence for the square root of the meansquare displacement as well as the broadening of the hole is, in this case, given by a power law with an exponent R/2 * 1/2, which is exactly what we found in our measurements. To summarize all of these results, spectral diffusion (in our model) is characterized by the expression
( ) ()
σ(tw,ta) ) x2σ0
tw τ0
R/2
ta T
-β
(2)
(σ0 is the width of the inhomogeneous band in which the holeburning experiment is performed.) In this article, we report on a hole-burning experiment on horseradish peroxidase (HRP) in a deuterated trehaloseglycerol-water solution. Trehalose is a disaccharide of great biological importance, helping various organisms to survive phases of strong dehydration or of low temperatures. Crucial for this function are probably two properties of trehalose, the high affinity for hydrogen bonds18 and the high glass-transition temperatures, which are typical for solutions of this sugar.19 The influence of trehalose on protein dynamics has been studied before, for example, in measurements investigating the rebinding of CO to myoglobin after flash photolysis20,21 or in neutronscattering experiments on the same protein.22 The behavior of myoglobin in a dry trehalose glass that was found in these scattering experiments equaled that of an harmonic crystal even at room temperature. Obviously, on the short time scales probed in such a measurement, no significant relaxation processes occur in the protein. In our group, the influence of trehalose on the dynamical properties of proteins has been investigated as well. By spectral hole burning, we compared the dynamics of HRP in a protonated trehalose-enriched and a trehalose-free solvent8 on time scales up to two weeks. Contrary to the neutronscattering experiments,22 we observed diffusive structural relaxation of the protein, even at a temperature of 4.2 K. In addition, our experiments allowed for two important conclusions: (1) the dynamics probed by the chromophore inside the proteins was completely decoupled from its surface and from the solvent, and (2) the dynamics in the interior of the protein
was faster in a trehalose-enriched solvent than in a trehalosefree one. This last result can be explained following a hypothesis, which was suggested by Sastry and Agmon23 as an explanation of the above-mentioned CO-rebinding experiments in myoglobin. According to them, the main influence of trehalose on protein dynamics is based on the preservation of the protein internal water molecules during freezing. These waters bind (via hydrogen bonds) to some of the amino acid residues and so prevent the formation of additional hydrogen bonds inside the protein; hence, the internal rigidity of the molecule is lowered, and this is reflected in a faster spectral diffusion dynamics. It should be emphasized that in our experiments only one parameter of the diffusion model was influenced by the trehalose content of the solvent, namely, the time constant of the equilibrium fluctuations, τ0. Of course, faster internal fluctuations of the protein cause smaller values for τ0. The effect of deuteration on protein dynamics has been investigated, too. In comparative hole-burning experiments on a myoglobin-type protein in protonated and deuterated solvents,7 a significant influence of deuteration has been observed, which again caused changes in the time parameter, τ0, only; all of the other parameters of the model remained unchanged. However, in contrast to the trehalose effect, deuteration led to an increase of τ0. The deuteration experiments allowed for an important conclusion, namely, that in our spectral diffusion measurements no explicit proton dynamics in the protein is monitored. If it was, one would expect a change in the exponent R after deuteration. In addition, as will be shown below, it is not the deuteration of protein internal water molecules that leads to a modified dynamical behavior. The effect of deuteration seems rather to be due to a changed dynamics of the hydrogen bonds in the protein, which is not seen directly in the experiments, however, but only influences the movements of heavier masses. These movements in turn couple to the chromophore. Because proton motion at 4 K is based on tunneling dynamics in the respective hydrogen bonds, the system is, of course, highly sensitive to deuteration. The present experiment on HRP in a deuterated, trehaloseenriched solvent has been performed to test the conclusions from these earlier measurements. If the trehalose effect is really due to the occupation of hydrogen-bonded sites in the protein by water-molecules, it should still give rise to an increased spectral diffusion even in the case when the water is deuterated; the deuteration effect on the other hand, which issaccording to the above argumentssindependent of the trehalose environment, should act the opposite way, that is, it should slow the conformational dynamics and, hence, the hole broadening, too. If both effects were of the same order of magnitude, they should therefore (almost) cancel each other. An experimental observation of such a behavior would prove the independence of trehalose- and deuteration-effect and support the above microscopic interpretations. Experimental Section Like in our previous measurements on HRP, slightly modified proteins were used for the deuteration experiment. Because the natural heme group of HRP does not allow high-resolution hole burning, it was replaced by free-base mesoporphyrin-IX. For the preparation of the sample, the lyophilized proteins were dissolved in a saturated solution of trehalose in a deuterated pH 8.0 buffer. To ensure a good glass quality after cooling, (protonated) glycerol was added, resulting in a 40%:50%:10% (v/v) mixture of saturated trehalose solution/glycerol/buffer. Apart from deuteration, this mixture was completely identical
3512 J. Phys. Chem. B, Vol. 106, No. 13, 2002
Schlichter et al.
Figure 1. Long-wavelength absorption bands of horseradish peroxidase in a protonated and deuterated trehalose-enriched solvent (TH), and in a protonated, trehalose-free environment. The chromophore is the freebase mesoporphyrin-IX; temperature is 4.2 K.
with the protonated trehalose-enriched sample used in our earlier experiments. Hence, a direct comparison of the results is possible. In addition to the present results, the data measured with a protonated, trehalose-free sample (HRP in a 50%:50% (v/v) buffer/glycerol solution) are presented in this paper as well. Hole-burning experiments were done in an identical fashion as previously: the burning intensity of the laser was approximately 1 µW; for reading the holes, the laser power was reduced by almost 4 orders of magnitude. The spectral holes were burned in a narrow frequency range near the center of the inhomogeneous absorption band. Before freezing the sample, it was kept a few days at room temperature to allow protondeuteron exchange between solvent and proteins. After that, the sample was cooled from room temperature to 4.2 K in a very short time (∼3 min) and kept at this temperature for the rest of the experiment. Five spectral holes (with aging times between 40 min and 192 h) were burned and afterward observed for almost two weeks, that is, over approximately 4 orders of magnitude in tw. Results Figure 1 shows the long-wavelength absorption band of the three different HRP samples, measured at a temperature of 4.2 K. Obviously, neither the trehalose concentration of the solvent nor the deuteration has any significant influence on the width of this band. (A Gauss fit yields a fwhm of about 60 cm-1.) In Figure 2 (top), the results of the hole-burning experiment on the deuterated, trehalose-enriched sample are illustrated. Here, the broadening of five different spectral holes (with aging times between 40 and 11 525 min) is shown in a semilogarithmic representation. The data can be fitted very well by power laws in tw; the fit of the first data set is given in the figure as an example. In addition, a weak influence of the aging time on the results can be recognized. Obviously, the broadening of spectral holes decreases with increasing ta. This aging dynamics is better seen in the lower part of the figure. Here, the broadening of the spectral holes after a constant waiting time of 10 000 min (i.e., the values at a “cut” through Figure 2 (top) at the dashed line) is plotted as a function of the aging time. For comparison, the respective data of our older experiments on HRP are given as well. Each data set is normalized to its initial value, σ1, at ta ) 40 min. All three samples aresin the error
Figure 2. Results of the waiting time experiment on horseradish peroxidase in the deuterated, trehalose-enriched sample (top) for five spectral holes (aging times, see insert). The line represents a powerlaw fit to the first data set. The bottom panel shows the spectral diffusion as function of aging time in the three different samples at tw ) 10 000 min. All data sets are normalized to their initial value, σ1, at ta ) 40 min.
limits of the measurementsscharacterized by the same aging dynamics; the linear behavior in a double-logarithmic representation indicates again a power law (σ ∝ ta-β); the exponent β is found to be 0.07 ( 0.01. It is now possible to scale the data with their aging-time dependence. The results of this procedure, so-called masterplots, which are independent of ta, are given in Figure 3. Again the results of the two older experiments on HRP (scaled in the same manner) are given for comparison. Three observation should be mentioned: (1) After scaling with their aging dynamics, all of the data of one experiment can be described very well by the same power law in tw. (2) For all three experiments, the exponent of this power law is identical; a fit yields R/2 ) 0.25 ( 0.02. Therefore, the different solvents have only an influence on the intercepts in Figure 3. (3) The masterplot for HRP in a deuterated, trehalose-enriched solvent runs below the one of its protonated counterpart but is almost identical with the one of the protonated, trehalose-free sample. Discussion The width of an inhomogeneous absorption band is a measure for the disorder in the local interaction of a chromophore with its environment. Obviously, neither deuteration nor the trehalose content of the solvent have any significant influence on this width in the above HRP experiments (Figure 1). This is an interesting result: A trehalose-enriched solvent has a much higher glass-transition temperature than a trehalose-free one.19
Conformational Dynamics of Proteins
Figure 3. Spectral diffusion in the three samples after scaling with their aging-time dependence (masterplots). A fit with a power law in tw yields for all three samples an identical exponent, R/2 ) 0.25 ( 0.02.
Hence, one would expect a higher degree of disorder to be frozen in the trehalose glass andsif there was any coupling between the solvent and the chromophore inside the proteinsa wider inhomogeneous absorption band. Nothing like this is observed in our measurements. Hence, we conclude that the chromophore must be decoupled from the solvent; accordingly, what one measures in a hole-burning/spectral diffusion experiment is only the internal dynamics of the protein in the neighborhood of the chromophore. These findings are in good agreement with our diffusion model.15 As explained in the Introduction, this model predicts a strongly distance-dependent proportionality factor between spectral broadening and conformational disorder; hence, according to this model, it is the coupling between the chromophore and the first shell of amino acid residues around it that is significant. One might speculate, that deuteration of a solvent changes its glass-transition temperature as well, because both, mass density and kinetics, are modified. In this case, a coupling between glass and chromophore should likewise lead to different inhomogeneous widths in protonated and deuterated samples. The absence of such an effect in our measurements might be an additional proof for the decoupling between chromophore and solvent. Interestingly, Reat et al.24 found very similar results in neutron scattering experiments on deuterated bacteriorhodopsin samples containing a few protonated amino acids, which were mostly situated in the inner part of the proteins (near the retinal). By incoherent scattering, it was possible to test the dynamics of this inner part independent from the rest of the molecule. In analogy to our results, this dynamics seemed to be decoupled from the solvent and the protein surface, because melting of membrane waters caused no effect on the inner-part dynamics. From Figure 2 (bottom), it becomes obvious that the agingtime dependence in our HRP experiments is not influenced by deuteration of the sample, too. In the framework of the diffusion model, aging effects are interpreted as a consequence of relaxation processes in proteins. These relaxations, that is, the approach of the molecules to a global equilibrium structure, lead to “more compact” conformations, which prevent to some extent the fluctuations and therefore lead to a decreased spectral diffusion. Because the aging-time dependence is the same in all of our HRP samples, the relaxation processes cannot be
J. Phys. Chem. B, Vol. 106, No. 13, 2002 3513 sensitive to deuteration. This result is easy to understand, however: The relaxation of a densely packed protein in its native state requires the correlated movement of several atoms, that is, the movement of relatively big masses; the small mass effect caused by proton-deuteron exchange can therefore be neglected. Note that our experiments on myoglobin7 yielded very similiar results; there, the deuteration of the sample had no influence on the size of β (eq 1) as well. The most important results of our HRP experiments are given in Figure 3. Here, the spectral diffusion in the three samples is shown after scaling the data with their aging dependence. Obviously, all three samples are governed by the same power law, σ ∝ twR/2 (with R/2 ≈ 0.25), with differences in their intercepts, however. Therefore, by inspection of eq 2, one recognizes that the parameters τ0, that is, the correlation time of the equilibrium fluctuations, or T, the time constant of the relaxation processes, or both can be influenced by the properties of the different solvents. (The inhomogeneous width, σ0, and the exponents of the power laws in waiting and aging time, R and β, which also determine the intercept, are identical for all three samples.) Because the measurements are not very sensitive to changes in the parameter Tsit appears with the very small exponent β ≈ 0.07 in eq 2sthe varying intercepts of the masterplots in Figure 3 should be a consequence of different equilibrium correlation times in the three samples, only. A quantitative analysis shows that τ0 is (in the error limits of the experiments) identical in the protonated, trehalose-free and in the deuterated, trehalose-enriched sample and shorter by a factor of 4 in the protonated, trehalose-enriched one. Interestingly, the deuteration effect as measured in our experiments on myoglobin has almost exactly the same magnitude. There, likewise, only the equilibrium correlation time τ0 was influenced by deuteration, and the ratio of deuterated and protonated correlation times equaled the same value of about four. Because the myoglobin experiments were performed in a trehalose-free solvent, we can conclude, that the deuteration effect is independent of the trehalose content or the detailed features of the solvent. In other words, it is not the deuteration of the internal water molecules that brings about the changes in the dynamics as measured in the spectral diffusion experiments. As stated above, the correlation time in the deuterated HRP sample is the same as that in the protonated, trehalose-free one. This can be understood by considering the additional effect of trehalose. As pointed out in the Introduction, we believe (following Sastry and Agmon) that trehalose preserves water molecules inside the proteins during the freezing procedure, this way causing an increased internal flexibility of the molecule. This effect should not depend on deuteration and therefore should be present in both the protonated and deuterated trehalose-enriched sample. The effects of trehalose and of deuteration act in opposite directions, however: while deuteration yields an increase in τ0, the trehalose effect decreases the correlation time. By accident, the magnitude (a factor of 4) of both effects seems to be almost the same in the case of HRP, leading to an almost identical behavior of the trehalose-free sample and the deuterated, trehalose-enriched one: for the first one, trehalose and deuteration effect do not exist; for the latter one, they just cancel each other. The results of the present measurements on a deuterated HRP sample in a trehalose-enriched environment are therefore in complete agreement with our earlier experiments on both HRP and myoglobin; obviously, trehalose and deuteration effect are completely independent of each other, which of course is what
3514 J. Phys. Chem. B, Vol. 106, No. 13, 2002 one would expect following the “internal water thesis” of Sastry and Agmon. Additionally, these new results on HRPslike all of our experiments on heme proteins beforesfit perfectly in the framework of our diffusion model again. Acknowledgment. Support from the Deutschen Forschungsgemeinschaft (SFB 533, B5), from the Fonts der Chemischen Industrie, from the WTZ program for the German-Hungarian collaboration (UNG 005 97), and from the Hungarian Grant OTKA T-032117 is gratefully acknowledged. References and Notes (1) Lu, H. P.; Xie, S. Nature 1997, 385, 143. (2) Thorn-Leeson, D.; Wiersma, D. A. Nat. Struct. Biol. 1995, 2, 848. (3) Thorn-Leeson, D.; Wiersma, D. A. Phys. ReV. Lett. 1995, 74, 2138. (4) Thorn-Leeson, D.; Wiersma, D. A.; Fritsch, K.; Friedrich, J. J. Phys. Chem. B 1997, 101, 6331. (5) Friedrich, J.; Haarer, D. Angew. Chem., Int. Ed. Engl. 1984, 23, 113. (6) Vo¨lker, S. Excited-State Spectrosc. Solids, Soc. Ital. Fis. (Bologna, Italy) 1987, 96, 363. (7) Schlichter, J.; Friedrich, J.; Parbel, M.; Scheer, H. J. Chem. Phys. 2001, 114, 9638. (8) Schlichter, J.; Friedrich, J.; Herenyi, L.; Fidy, J. Biophys. J. 2001, 80, 2011.
Schlichter et al. (9) Schlichter, J.; Fritsch, K.-D.; Friedrich, J.; Vanderkooi, J. M. J. Chem. Phys. 1999, 110, 3229. (10) Silbey, R. J.; Koedijk, J. M. A.; Vo¨lker, S. J. Chem. Phys. 1996, 105, 901. (11) Skinner, J. L.; Friedrich, J.; Schlichter, J. J. Phys. Chem. A 1999, 103, 2310. (12) Fritsch, K.; Eicker, A.; Friedrich, J.; Kharlamov, B. M.; Vanderkooi, J. M. Europhys. Lett. 1998, 41, 339. (13) Schlichter, J.; Friedrich, J.; Herenyi, L.; Fidy, J. J. Chem. Phys. 2000, 112, 3045. (14) Stephens, M. D.; Saven, J. G.; Skinner, J. L. J. Chem. Phys. 1997, 106, 2129. (15) Schlichter, J.; Friedrich, J.; Parbel, M.; Scheer, H. Photonics Sci. News 2001, 6, 100. (16) Berlin, Y. A. Chem. Phys. 1996, 212, 29. (17) Berlin, Y. A.; Burin, A. L. Chem. Phys. Lett. 1997, 267, 234. (18) Crowe, J.; Carpenter, J.; Crowe, L. Annu. ReV. Physiol. 1998, 60, 73. (19) Miller, D.; de Pablo, J.; Corti, H. J. Phys. Chem. B 1999, 103, 10243. (20) Hagen, S. Science 1995, 269, 959. (21) Hagen, S.; Hofrichter, J.; Eaton, W. J. Phys. Chem. 1996, 100, 12008. (22) Cordone, L.; Ferrand, M.; Vitrano, E.; Zaccai, G. Biophys. J. 1999, 76, 1043. (23) Sastry, G.; Agmon, N. Biochemistry 1997, 36, 7097. (24) Reat, V.; Patzelt, H.; Ferrand, M.; Pfister, C.; Oesterhelt, D.; Zaccai, G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4970.
