Exploration of the Secondary Structure Specific Differential Solvation

Oleksandr Sushko , Rostyslav Dubrovka , and Robert S. Donnan. The Journal of Physical Chemistry B 2013 117 (51), 16486-16492. Abstract | Full Text HTM...
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J. Phys. Chem. B 2006, 110, 20629-20634

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Exploration of the Secondary Structure Specific Differential Solvation Dynamics between the Native and Molten Globule States of the Protein HP-36 Sanjoy Bandyopadhyay,*,† Sudip Chakraborty,† and Biman Bagchi*,‡ Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur-721302, India, and Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore-560012, India ReceiVed: May 31, 2006; In Final Form: July 31, 2006

Recent experiments have shown that the time dependence of fluorescence Stokes shift of a chromophore is substantially different when the chromophore is located in a molten globule (MG) state and in the native state of the same protein. To understand the origin of this difference, particularly the role of water in the differential solvation of the protein in the native and the MG states, we have carried out fully atomistic molecular dynamics simulations with explicit water of a partially unfolded MG state of the protein HP-36 and compared the results with the solvation dynamics of the protein in the folded native state. It is observed that the polar solvation dynamics of the three helical segments of the protein is influenced in a nonuniform heterogeneous manner in the MG state. While the equilibrium solvation time correlation function for helix-3 has been found to relax faster in the MG state as compared to that in the native state, the decay of the corresponding function for the other two helices slows down in the MG state. A careful analysis shows that the origin of such heterogeneous relative solvation behavior lies in the differential location of the polar probe residues and their exposure to bulk solvent. We find a significant negative cross-correlation between the contribution (to the solvation energy of a tagged amino acid residue) of water and the other groups of the protein, indicating a competing role in solvation. The sensitivity of solvation dynamics to the secondary structure and the immediate environment can be used to discriminate the partially unfolded and folded states. These results therefore should be useful in explaining recent solvation dynamics experiments on native and MG states of proteins.

1. Introduction It is now widely accepted that water plays an important role in determining the structure, stability, and function of a protein. Such a role is played through a dynamical coupling that exists between the protein and the water molecules present in its hydration layer (biological water).1-3 A microscopic level understanding of this role in biological activity, particularly in processes such as protein-enzyme interactions, molecular recognition, and folding-unfolding phenomena, is an area of intense research activity.4-8 Several experimental studies have explored solvation dynamics in aqueous protein solutions with the protein in the native state. Three-pulse photon echo techniques have been used by Fleming and co-workers4 to measure the solvation dynamics of an external probe (eosin) in an aqueous solution of lysozyme. They have observed several slow components with one of the order of 500 ps. Using time-resolved fluorescence spectroscopy, Bhattacharyya and co-workers5,6 have observed a slow component in the polar solvation dynamics of proteins, which they attribute to the restricted movement of water molecules near the protein surface. They have noticed a slow solvation dynamics near the active site of an enzyme GlnRS from time-dependent fluorescence Stokes shift measurements at picosecond resolution.6 It is also shown that the solvation dynamics slows down * To whom correspondence should be addressed. E-mail: S.B., [email protected]; B.B., [email protected]. † Indian Institute of Technology. ‡ Indian Institute of Science.

further when the enzyme binds with the substrate. Zewail and co-workers7 have used tryptophan as an intrinsic probe to measure the solvation dynamics of small proteins. The longest component detected by them was much slower than that of tryptophan in bulk water. As a different approach, quasi-elastic neutron scattering (QENS) experiments have been performed by Head-Gordon and co-workers8 to study the hydration water dynamics of model proteins as a function of temperature and concentration. They have shown that the relaxation dynamics of the hydration layer water is nonexponential in nature, while the water translational dynamics exhibits a non-Arrhenius behavior over a wide range of temperatures. In the early stages of unfolding, a protein usually transforms to a nearby (in configuration space) state with a significant fraction of secondary structures and hydrophobic contacts intact. There can be an ensemble of such partially unfolded structures, commonly known as the molten globule (MG) state.9 These are essentially different intermediates along the folding-unfolding pathway. Recently, experimental studies have explored the difference in protein solvation dynamics between the native and the MG states. It is believed that the observed difference in solvation behavior can provide valuable insight into the structure and function of proteins. Bhattacharyya and co-workers10 have shown that the solvation dynamics of the MG state of a protein can be faster or slower than the native state depending on whether the probe is exposed near the protein-water interface or buried within the core. Using femtosecond time-resolved fluorescence spectroscopy, they have shown that different

10.1021/jp0633547 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/13/2006

20630 J. Phys. Chem. B, Vol. 110, No. 41, 2006 partially folded states of the protein cytochrome C exhibit different solvation dynamics.11 Peon et al.12 have reported that the longest component of solvation dynamics increases significantly in the denatured state of monellin as compared to its native state. Similar behavior is also observed for the acid denatured state of R-chymotrypsin.13 It has also been suggested that the slower solvation response may arise from the contributions of amino acid side chain residues. Therefore, this appears to be an important problem with several interesting aspects still to be understood. We shall now discuss theoretical studies devoted to protein hydration dynamics. Since the early works of Rossky and Karplus14 and Levitt and Sharon,15 many computer simulations have been attempted to study the dynamics of water present at the surface of a protein and its likely correlation with the dynamics of the protein itself.16-25 Using a combination of molecular dynamics (MD) simulations and QENS measurements, Tarek and Tobias16 have studied water mobility for several proteins in solution as well as in their crystals, dry and hydrated powders. They have shown that for the structural relaxation of a protein, a complete exchange of protein-bound water molecules is necessary. Xu and Berne17 have shown that the kinetics of the water-water hydrogen bonds in the hydration shell of a polypeptide is slower than that in bulk water. Cheng and Rossky18 have demonstrated that two different hydration structures can exist near a protein surface. In a recent study, Marchi et al.19 have found that the rotational dynamics of water around lysozyme in an aqueous solution is much slower than that in the bulk. It is also shown recently that the water in the hydration layer of a protein exhibits subdiffusive motion.20 The solvation behavior of proteins has also been studied in detail by Pettitt and co-workers.21 Recently, we have performed detailed MD simulations to investigate the correlation between the dynamics of the amino acid residues of a protein and the water present in its hydration layer.23 It has been observed that the water near the active site of the protein is less structured and more mobile. It was demonstrated that the lifetimes of protein-water hydrogen bonds differ significantly among different secondary structures of a protein.24 We also showed that the solvation dynamics of different secondary structures of a protein in its native state is sensitive to the relative exposure of the probe residues at the protein surface.25 All the computational studies discussed above deal with proteins in their folded native states. However, not much effort has been made to study the dynamics of the non-native unfolded states of proteins and their solvation behavior. Such studies are important not only to identify the folding intermediates and explore the role played by water in protein folding but also to understand the origin of various malignant diseases. Several theoretical and simulation studies have been attempted in the recent past to understand the role played by water during the folding-unfolding process. Wolynes and co-workers26 have shown that the long-range water-mediated interactions play an important role and help facilitating nativelike packing of supersecondary structures during folding. Harano and Kinoshita27 have pointed out that the gain in entropy of neighboring water molecules can be an important driving force along the folding-unfolding pathway. However, a direct analysis of the microscopic level dynamical correlation between the partially unfolded molten globules and the surrounding water molecules seem not to have been discussed at all. Only recently, we carried out extensive MD simulations to show that the unfolding of a protein molecule influences the mobility of water around it in a heterogeneous fashion.28

Bandyopadhyay et al. In this article, we report atomistic MD simulations to study the solvation dynamics of different segments present in a small 36-residue protein, which is the thermostable subdomain present at the extreme C-terminus of the 76-residue chicken villin headpiece domain, and is popularly known as HP-36.29 Villin is a unique protein which can both assemble and disassemble actin structures.30 HP-36 contains one of the two F-actin binding sites in villin necessary for F-actin bundling activity.30 In this work we number the residues from 1 to 36. Thus, residue numbers 1 to 36 correspond to residues 41 to 76 in the NMR structure.29 The secondary structure of the protein contains three short R-helices. These helices are connected and held together by a few turns and loops and a hydrophobic core. For convenience, the three R-helices are denoted as helix-1 (Asp-4 to Lys-8), helix-2 (Arg-15 to Phe-18), and helix-3 (Leu-23 to Glu-32).23 The biological activity is believed to be centered around helix-3 which contains 10 amino acid residues.29 Solvation dynamics (SD) is an important time-resolved technique that is used to study various dynamical processes that take place when a solute molecule is introduced in a polar solvent. It provides quantitative information on the timedependent response of the solvent reorganization around a solute probe. SD can be studied by characterizing the decay of the equilibrium solvation time correlation function (TCF), CS(t), which according to linear response theory is related to the solvation energy2,3 as

CS(t) )

〈δE(t)δE(0)〉 〈δE(0)δE(0)〉

(1)

where δE(t) corresponds to the fluctuation in the polar part of the potential energy of the probe molecule with the rest of the simulation system at time t with respect to the corresponding average equilibrium value. The angular brackets denote averaging over the probe molecules and over different reference initial times. As mentioned in our earlier work,25 we have calculated CS(t) by measuring the polar part of the interaction energy between the atoms of the polar amino acid residues of each of the three helices and the rest of the simulation system. The rest of the article is organized as follows. In the next section we describe the setup of the systems and the simulation methods employed. The results obtained from our investigations are presented and discussed in the following section. In the last section we summarize the important findings and the conclusions reached from our study. 2. System Setup and Simulation Details We have employed a recently developed molecular dynamics code (PINY-MD)31 to perform the classical atomistic molecular dynamics simulations presented in this article. The CHARMM22 all-atom force field and potential parameters for proteins32 were employed to describe the interaction between protein atoms, while the TIP3P model33 which is consistent with the chosen protein force field was employed for modeling water. Two different simulations have been carried out, designated as S1 and S2. In simulation S1 the native state of the protein is studied at 300 K, while the unfolding of the protein is studied in simulation S2. The initial coordinates of the protein for the simulation S1 were taken from the Protein Data Bank (PDB ID: 1VII) from the NMR structure of the villin headpiece subdomain, as reported by McKnight et al.29 The two end residues (Met-1 and Phe-36) of the protein were capped appropriately, and the whole molecule was immersed in a large cubic box of well-equilibrated water by carefully avoiding

Native and Molten Globule States of Protein HP-36 unfavorable contacts. The final system consisted of the 36 residue protein (596 atoms) in a cubic box containing 6842 water molecules. The initial edge length of the cubic simulation cell was 61 Å. At first, the system was equilibrated at constant temperature (T ) 300 K) and pressure (Pext ) 0) (NPT) for about 500 ps. During this run, the volume of the simulation cell was allowed to fluctuate isotropically. At the end of this equilibration run, the volume of the system attained a steady value with a box edge length of 58.92 Å, and the average pressure of the system was 3 atm. At this point we fixed the cell volume, and the simulation conditions were changed from constant pressure and temperature (NPT) to constant volume and temperature (NVT). The NVT equilibration run was further continued at 300 K for another 1 ns duration. This equilibration period was followed by an NVT run of approximately 2.5 ns duration. The details of simulation S1 can be found elsewhere.23 The native state configuration of the protein obtained at the end of simulation S1 was taken to initiate the unfolding simulation, S2. An NPT simulation was first performed at high temperatures of 500-600 K for approximately 1.4 ns duration, during which the protein underwent a transformation from the native state to a partially unfolded structure where the smallest R-helix (helix-2) unfolded into a coil.28 This partially unfolded structure is a member of the large ensemble of structures, known as the molten globule (MG) state. At this point, the temperature of the system was reduced to 300 K, and the NPT run was continued for another 300 ps. The simulation conditions were then changed from constant pressure and temperature (NPT) to constant volume and temperature (NVT). The unfolded state trajectory in NVT ensemble was then generated for about 2.5 ns at 300 K. The analysis was carried out over this last 2.5 ns of the trajectory. The simulations utilized the Nose´-Hoover chain thermostat extended system method.34 A recently developed reversible multiple time step algorithm, RESPA,34 allowed us to employ a MD time step of 4 fs. Electrostatic interactions were calculated by using the particle-mesh Ewald (PME) method.35 The PME and RESPA were combined following the method suggested by Marchi and co-workers.36 The minimum image convention37 was employed to calculate the Lennard-Jones interactions and the real-space part of the Ewald sum, using a spherical truncation of 7 and 10 Å, respectively, for the short- and the long-range parts of the force decomposition.

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Figure 1. Solvation time correlation function, CS(t), for the three helices of the protein in the native (without symbols) and in the partially unfolded MG states (with symbols) as obtained from simulations S1 and S2 at 300 K. The TCFs are calculated by averaging over the polar amino acid residues of the helices.

Figure 2. Solvation time correlation function, CS(t), for the individual polar amino acid residues of the three helices of the protein in the native (a-c) and in the partially unfolded MG states (d-f) as obtained from simulations S1 and S2 at 300 K. The polar amino acid residues are used as intrinsic probes in the calculations.

TABLE 1: Average Solvation Time Constants (〈τS〉) of the Three r-Helices of the Protein in Native and Partially Unfolded Molten Globule States 〈τS〉 (ps)

3. Results and Discussion We have calculated the solvation time correlation function (TCF), CS(t), for the three helices of the protein molecule in the partially unfolded MG state, as displayed in Figure 1. The calculations are carried out by averaging over the polar probe residues present in each of them. For comparison, the corresponding decay curves for the native state of the protein are also included in the figure. The presence of long tails indicates slow dynamics of the helices in both the states. However, interesting differences in the solvation behavior of the three helices have been observed in the two states. A closer examination of the decay curves reveals that the solvation TCF decays faster for helix-3 in the MG state as compared to that in the native state. On the contrary, the corresponding TCFs for helices 1 and 2 are slowed in the MG state. This is an interesting observation which shows that the solvation behavior of the three helices are influenced nonuniformly due to partial unfolding of the protein molecule. We have fitted all the decay curves to multiexponentials and obtained amplitude-weighted average solvation time constants (〈τs〉), which are listed in Table 1. It

segment

native

molten globule

helix-1 helix-2 helix-3

26.2 11.0 29.4

40.1 20.0 26.5

may be noted that the 〈τs〉 values are about 1.5-2 times higher for helices 1 and 2 in the MG state as compared to the corresponding values in the native state. In contrast, the 〈τs〉 value has decreased for helix-3 in the MG state. As will be discussed later, there is a microscopic explanation for such differential dynamics. The dynamics exhibited by the individual residues is likely to reflect the differential heterogeneity in solvation dynamics of the three helices as mentioned above. In Figure 2, we display the solvation TCFs for the individual polar probe residues of the three helices in both the states of the protein. A closer examination of the decay curves indicates that the solvation dynamics of the individual residues of the helices are affected differently due to partial unfolding of the protein molecule.

20632 J. Phys. Chem. B, Vol. 110, No. 41, 2006 We observe that the overall heterogeneous dynamics of the polar residues of helix-1 is retained in the MG state with slowing down of the relaxation for all the residues except Asp-6. The degree of slowness is most striking for Lys-8, which is reflected in the difference in the overall relaxation behavior of helix-1 in the two states (see Figure 1). Although the decay curves for the polar residues of helix-2 relax slightly slowly in the unfolded state, they still exhibit homogeneous dynamics as in the native state. Again, the slower relaxation of the residues of helix-2 is consistent with its overall slow solvation dynamics in the MG state, as shown in Figure 1. We observe interesting differences in the solvation behavior of helix-3 residues. The degree of heterogeneity as observed in the native state dynamics of helix-3 residues is greatly reduced in the MG state. Most of the polar residues of helix-3 except Gln-27 and Glu-32 exhibit homogeneous dynamics in the MG state. This indicates that unfolding of helix-2 and the straightening of coil-3 28 resulted in exposure of most of the helix-3 residues to near identical environment. It may also be noted that the relaxation of the solvation TCF is much faster for Lys-30 in the MG state, while that of the other residues are almost identical in the two states. The contribution arising from the faster relaxation of Lys-30 leads to overall faster solvation dynamics of helix-3 in the MG state, as observed in Figure 1. The change of environment of helix-3 residues due to unfolding and its faster solvation dynamics in the partially unfolded state are important observations, which may be correlated with the biological functionality of HP-36, as most of its active site residues are located in helix-3.29 However, further studies are necessary to establish whether such a correlation exists. It is apparent from the discussion so far that although a protein loses only a fraction of its native secondary structures in a MG state, the dynamics of solvation of different segments of the protein can be significantly different from that of the native state. This agrees well with recent solvation dynamics experiments as reported by Bhattacharyya and co-workers.10 They have shown that the average solvation time of a protein in the MG state can differ from that of the native state, depending on the nature of the probe molecules and their locations in the protein. To further understand the microscopic origin of the secondary structure specific differential solvation dynamics of the protein in the MG state as against the native state, we have measured the contributions from different partial solvation time correlation functions for the three helices. These are displayed in Figure 3. It may be noted that the most dominating contribution to the total solvation TCF of the helices in the native state arises from the interaction between the polar amino acid residues and water (P-W interaction). The native state behavior is discussed in detail in our earlier work.25 It is evident from Figure 3 that the P-W interaction plays an important role in the MG state too. This is particularly so for helices 2 and 3. For helix-1, the interaction between its residues and the other parts of the protein molecule (P-P interaction) also contributes significantly to its solvation dynamics. We have also calculated the cross-correlations between the contribution to the solvation energy of tagged probe residues arising from the interaction with water and with other parts of the protein molecule. In Figure 4 we show the decay of this cross-correlation function for the three helices in both the states. It is noticed that at zero time (t ) 0), the crosscorrelations exhibit negative values in most cases. This is an important observation which indicates that the protein and water dynamics are coupled and anticorrelated. In other words, the water and the other parts of the protein play a competing role in solvating the probe residues. The decay of the function is

Bandyopadhyay et al.

Figure 3. Partial solvation time correlation function, CPS(t), for the three helices of the protein in the native (a-c) and in the partially unfolded MG states (d-f) as obtained from simulations S1 and S2 at 300 K.

Figure 4. Protein-water cross-correlation function, 〈δEPP(0)δEPW(t) + δEPW(0)δEPP(t)〉 (CCS(t)), for the three helices of the protein in the native and in the partially unfolded MG states as obtained from simulations S1 and S2 at 300 K.

almost identical for helices 2 and 3 in both the states. However, the function behaves noticeably differently for helix-1 in the two states. The large negative value of the function at t ) 0 and its slow decay indicate that the dynamics of helix-1 is strongly coupled with the rest of the system in the unfolded state. The relaxation behavior of the total solvation TCF for the helices in both the native and the MG states is primarily controlled by the interaction between the amino acid residues and water molecules. The breaking of the native tertiary contacts during unfolding leads to weakening of the packing of the hydrophobic core and its exposure to the solvent. Thus, a comparison between the relative exposure of the polar residues of the helices in the two states may provide an explanation for the observed differential solvation behavior. We have calculated the pairwise correlation function, commonly known as the radial distribution function, g(r), of the Cβ atoms of the polar residues of the three helices with water molecules to investigate the degree of exposure of these residues to the solvent. The results are displayed in Figure 5. The figure shows interesting differ-

Native and Molten Globule States of Protein HP-36

Figure 5. Pairwise correlation function, g(r), of water molecules as a function of distance from the Cβ atoms of the polar amino acid residues of the three helices of the protein in the native (a-d) and in the partially unfolded MG states (e-h) as obtained from simulations S1 and S2 at 300 K.

ences in the distribution functions for the helices in the two states. Several polar residues of helix-1 (Asp-4, Glu-5) and helix-2 (Arg-15, Ser-16) exhibit distinct high intensity first peak as against low intensity or no peak for helix-3 residues in the native state. This suggests that the side chains of the polar residues of helices 1 and 2 are primarily exposed to bulk water, while that of the helix-3 residues are either buried or oriented close to the backbone and, therefore, are mostly in contact with the interfacial water molecules. Here, “bulk” water essentially refers to water that is away from the surface of the backbone of the protein, while water within the first hydration layer of the backbone surface is referred to as “interfacial” water. Few polar residues of helix-1 (Asp-6, Lys-8) are also buried within the core in the native state. In our earlier work,25 we showed that the solvation dynamics of the helices in the native state is correlated with the relative exposure of the polar residues to bulk water. Here, we discuss the relative change in exposure due to unfolding and its likely influence on the solvation behavior. The g(r) curves show a lowering of water structure around the residues Glu-5 of helix-1 and Arg-15 of helix-2. This indicates that the side chains of these residues are oriented more toward the protein core or the backbone in the MG state and hence are less exposed to bulk water. As a result, the contribution from the interaction of the polar residues of helices 1 and 2 with the bulk water (P-WB interactions) is reduced and that with the interfacial water (P-WI interactions) is enhanced in the MG state. Due to faster dynamics of bulk water, P-WB interaction fluctuation is faster than P-WI interaction. This results in slower relaxation of the solvation TCFs for helices 1 and 2 in the MG state (see Figure 1). The polar residues of helix-3 exhibit contrasting behavior. A small but noticeable increase in the intensity of the distribution functions near the first peak position occurs due to unfolding. This is particularly true for residues Asn-28 and Lys-30. It indicates that the side chains of these residues are more exposed to the solvent in the MG state. As a result, the overall contribution from rapidly fluctuating P-WB interactions increases for helix-3, which leads to faster relaxation of the corresponding solvation TCF. In Figure 6, we display representative configurations of the proteins

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Figure 6. Representative configurations of the protein highlighting the surface contours of the side chains of the polar amino acid residues of the three helices in the native (a-c) and in the partially unfolded MG states (d-f) as obtained from simulations S1 and S2 at 300 K. The backbone of the protein is drawn in red, while the residues are in light blue. The water molecules are not displayed for visual clarity. Note the differential change in the orientation and hence the exposure of the side chains of the residues of the helices in the two states.

in both the native and the MG states as obtained from the two simulations, highlighting the orientation (surface contours) of the side chains of the polar amino acid residues of the three helices. The greater exposure of the side chains of the polar residues of helices 1 and 2 to bulk solvent as against the side chains of helix-3 residues in the native state is clearly evident from the figure. However, the most interesting aspect of the figure is the relative change in orientation of the residue side chains of the helices in the MG state as against that in the native state. It can be seen that the unfolding of helix-2 has led to a reduction of exposure of the side chains of the polar residues of helices 1 and 2 to bulk water by orienting them more toward either the core or the backbone of the protein. In contrast, the side chains of the polar residues of helix-3 are more exposed and hence accessible to bulk water in the MG state. Thus Figures 5 and 6 provide evidence that the differential relaxation pattern of the solvation TCFs for the three helices among the two states arises primarily due to the relative differences in the degree of exposure of the amino acid residues to bulk water. Recently, we showed that the structural heterogeneity among different secondary structures in the native state of a protein influences the kinetics of hydrogen bonds formed between the amino acid residues of the protein and water.24 The partial breaking of secondary structures in the MG state is likely to influence the behavior of such hydrogen bonds. Currently, we are investigating in detail the influence of unfolding, if any, on the kinetics of protein-water hydrogen bonds. 4. Conclusions In this work we have presented a comparative study of the solvation dynamics of the native state and a partially unfolded structure of a small globular protein, chicken villin headpiece subdomain containing 36 amino acid residues (HP-36) in aqueous solution. The calculations reveal that the unfolding of

20634 J. Phys. Chem. B, Vol. 110, No. 41, 2006 helix-2 of the protein molecule influences the polar solvation dynamics of all the three helices, but in a nonuniform heterogeneous fashion. It is noticed that while the solvation time correlation function for helix-3 relaxes faster in the MG state as compared to the native state, the corresponding functions for helices 1 and 2 relax slowly in the MG state. The differential solvation dynamics of helix-3 residues compared to those of the other two helices due to unfolding is an important observation, as most of the active site residues of HP-36 are located in helix-3. A primary finding from this work is that, the degree of exposure of the polar probe residues to bulk water and the protein-water (P-W) interaction play important roles in determining the relative solvation dynamics of different secondary structures of the protein in both the native and the MG states. Contrary to the normal expectation, it is observed that the exposure of the side chains of some of the polar probe residues of helices 1 and 2 is reduced due to unfolding. This results in a decrease in contribution from the interaction between these residues and bulk water, which leads to slower solvation dynamics of these two helices in the unfolded MG state. The reverse picture is noticed for the polar residues of helix-3. Side chains of some of these residues are more exposed to bulk solvent in the MG state. This results in faster solvation dynamics of helix-3 in the MG state. It may be noted that, by using the polar residues of different helices of the protein as intrinsic probes, we could microscopically identify such apparent counterintuitive differences in the exposure of the residues due to unfolding. Our results also support the recent experimental studies of Bhattacharyya and co-workers,10 who have suggested that the solvation dynamics of the molten globule states of a protein can be faster or slower than the native state depending on whether the probe is exposed near the protein surface or buried within the core. Thus, in this work we have been able to establish that the relative change in the exposure of the polar probe residues of different secondary structures of a protein molecule to the solvent is strongly correlated with their differential solvation dynamics in the native and partially unfolded MG states. It is noticed that the probe residues experience a competing solvation response from the other parts of the protein molecule and water. To the best of our knowledge, this is the first report on such a correlation. The sensitivity of the solvation response of a protein to its immediate environment can be used to discriminate between the native and MG states of the protein. It would also be interesting to explore whether such a correlation exists for different MG states of a protein formed along different unfolding pathways. Currently, we are investigating these aspects in greater detail by studying the unfolding process at different conditions. Acknowledgment. This study was supported in part by grants from the Department of Biotechnology (DBT), Council of Scientific and Industrial Research (CSIR), and the Department of Science and Technology (DST), Government of India. S.C. thanks CSIR for providing a scholarship. References and Notes (1) Pethig, R. Annu. ReV. Phys. Chem. 1992, 43, 177. (2) Nandi, N.; Bagchi, B. J. Phys. Chem. B 1997, 101, 10954. Nandi, N.; Bagchi, B. J. Phys. Chem. 1996, 100, 13914-13919.

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