Residue Specific Interaction of an Unfolded Protein with Solvents in

Article pubs.acs.org/JPCA

Residue Specific Interaction of an Unfolded Protein with Solvents in Mixed Water−Ethanol Solutions: A Combined Molecular Dynamics and ONIOM Study Dayanidhi Mohanta,† Santanu Santra,† G. Naaresh Reddy,‡ Santanab Giri,‡ and Madhurima Jana*,† ‡

Theoretical Chemistry Laboratory, Department of Chemistry, National Institute of Technology, Rourkela 769008, India Molecular Simulation Laboratory, Department of Chemistry, National Institute of Technology, Rourkela 769008, India

†

ABSTRACT: The molecular mechanism of ethanol governed unfolding of an enzymatic protein, chymotrypsin inhibitor 2 (CI2), in water−ethanol mixed solutions has been studied by using combined molecular dynamics simulations and ONIOM study. The residue specific solvation of the unfolded protein and the interactions between the individual amino acid residues of the protein with ethanol as well as water have been investigated. The results are compared with that obtained from the folded state of the protein. Further, emphasis has been given to explore the residue’s preferential site of attraction toward the nature of the solvents. The heterogeneous structuring of water and ethanol around the hydrophobic and hydrophilic surfaces of the protein is found to correlate well with their available surface areas to the solvents. Both hydrophobic and hydrophilic interactions are found to have important contributions in rupturing protein’s secondary structural segments. Further, residue−water as well as residue−ethanol binding energies show significant involvement of the hydrogen bonding environment in the unfolding process; particularly, residue−water hydrogen bonds are found to play an indispensable role.

1. INTRODUCTION Proteins perform their biological activities in solvent medium. Therefore, solvent effect always represents a great importance and challenge. Among several binary mixtures, monohydric alcohols/water are often known to disrupt the tertiary structures of proteins.1−8 Such disruption significantly depends on the nature and concentration of alcohol used.5,8−10 Among several monohydric alcohols, ethanol is an essential solvent which is widely used in biology due to unusual behavior at various concentrations. Such behavior can be elucidated in terms of molecular arrangements as well as the structural transformations driven by molecular interactions such as hydrogen bonding, hydrophobic interactions, etc. The structural and dynamical properties of protein in mixed solution are likely to be changed as a function of the solvent composition. Thus, characterization of protein’s solvation shell in a mixed solution is of real importance to detect their influence in the possible transformations of the protein’s structure and dynamics. A large number of experimental and computational studies have been focused on explaining the anomalous properties of ethanol in water.11−15 Soper et al. infers that rearrangements of hydrogen bonds are responsible for several anomalous behaviors of the dilute alcohol−water solutions.16 However, very few studies have been performed to probe the solvation properties of the protein in the water−ethanol binary solutions and their relationships with protein stability. Recent small-angle © 2017 American Chemical Society

neutron scattering and differential scanning calorimetry experiments describe the solvation properties of protein in water− ethanol binary mixtures and show that ethanol can preferentially bind with both the native and unfolded states.17 Ethanol concentration dependent structural changes of proteins have been quantified by several groups.7,18,19 These studies in general infer that the unfolding of protein is governed by protein−ethanol hydrophobic interactions. Using MD simulation techniques Lousa et. al20 showed that an enzyme that lacks a disulfide bond undergoes larger conformational changes in ethanol solution. Their study infers that such changes have occurred due to the preferential hydrophobic interactions between the protein and ethanol molecule. In an another important recent study, Bagchi and co-workers21,22 have explored the sensitivity of protein unfolding dynamics toward increasing ethanol concentration of a protein, HP-36. The study infers that the partially unfolded states of protein can be attributed to the preferential solvation of the hydrophobic residues of the ethanol. Despite significant efforts, detailed microscopic understanding of the interactions between protein residue and water or ethanol in a mixed solution is not clearly known. Further, how such interaction promotes the protein unfolding process is also Received: June 17, 2017 Revised: July 17, 2017 Published: July 20, 2017 6172

DOI: 10.1021/acs.jpca.7b05955 J. Phys. Chem. A 2017, 121, 6172−6186

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The Journal of Physical Chemistry A

ethanol used. A detailed discussion of this can be found elsewhere.23 At this point we reduced the temperature of s2 simulations to 300 K under the same NVT ensemble, and production trajectories of another 200 ns duration were generated for each system. Hence the trajectory length for each set of s2 simulation becomes 340 ns. All the simulations were carried out using NAMD code26 with a time step of 1 fs. We have used all atom CHARMM36 force field and potential parameters for the protein27 and CHARMM General Force Field for ethanols.28 The nonbonded cross-interactions of the solvent water and ethanol follow Lorentz−Berthelot mixing rules as implemented in NAMD and CHARMM.26,29,30 The modified TIP3P water model as implemented in CHARMM force field was employed for modeling the water molecules.31 The minimum image convention32 was employed to calculate the short-range Lennard-Jones interactions using a spherical cut-off distance of 12 Å with a switch distance of 10 Å. The long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) method.33 2.2. ONIOM Calculation. The residue specific binding energy, BErs, of CI2 with the solvent ethanol and water has been calculated using the following equation:

under investigation. Basically, proteins are sensible probes for measuring solution properties. Therefore, conformational stability of a protein is closely related to the solvation phenomena and the properties of the solvent surrounding it. Further, the characteristic interactions between the protein and the solvent present in mixed solution are important since in binary mixtures the solvents exhibit different behavior than they do as individual components. The aim of the present study is to explore the properties of the water and ethanol molecules, separately, around the residues of the protein CI2 in water− ethanol binary mixtures. Particularly, we explore the role of solvation shell in disrupting the protein’s native conformation in water−ethanol binary mixtures. In a recent study, we have discussed in detail the structural disruption of the protein in various ethanol concentrations.23 However, microscopic investigation of the properties of the surrounding solvents and their preferential site of attraction toward protein surface as well as their binding phenomena has not been explored in detail. Therefore, in this work emphasis has been given to study the residue specific solvation of the protein and the binding with the solvent molecules. Attempts have been made to explore the interaction of solvent with the residues of the protein to specify the underlying mechanism of ethanol governed unfolding of CI2. The residues of CI2 as obtained from the protein data bank are numbered from 1 to 64, which correspond to residues 20 to 83 of PDB entry 1YPC.24 The native structure of the protein contains one α-helix and a 4stranded β-sheet which are connected by turns and loops. For convenience, we denote the α-helix as helix (Ser-12 to Lys-24), and the β-sheet as sheet (Gln-28 to Val-34, Asp-45 to Lys-53, Asn-56 to Ala-58, and Arg-62 to Gly-64) in our study. The protein has an active loop region spanning between the amino acid residues Gly-35 to Ile-44. The rest of the article is organized as follows. In section 2, we describe in brief the setup of the systems and the computational methodology employed. The results are presented and discussed in detail in section 3. The important findings and the conclusions reached from this study are summarized in section 4.

ONIOM BErs = Ecomplex − EsQM − ErONIOM

(1)

Here, r = amino acid residue, s = w or et when the solvent is water or ethanol, respectively. “Complex” indicates the noncovalent binding between a residue of the folded or unfolded protein in a protein environment with the solvent ethanol or water molecule near its surface. The structure of the complex was isolated from the equilibrated MD trajectories as generated during s1 and s2 simulations at ambient temperature. Particularly, we have chosen the configurations of the protein surrounded by the solvent from the 50% ethanol solution of the folded and unfolded trajectories of the s1 and s2 simulations. It may be noted that in a recent study we have shown that CI2 exhibits its more probable unfolded configuration at 50% ethanol solution.23 Hence, the isolated structures as obtained from the MD trajectories can be assumed as model configurations of the folded and unfolded protein surrounded by the solvent molecules. These models would represent the binding phenomena of each of the amino acid residues of the folded and unfolded protein with its surface solvents. To compute the most favorable binding energies between the residue and solvent molecule we need to identify the pairs that can form complexes. We apply a simple approach to define such complexes. At first we have identified the residue specific solvation layer of the folded and unfolded protein. This was done by calculating all the ethanol and water molecules that are present within the distance of 5 Å from each residue of the folded and unfolded protein, separately. This essentially provides information on all the solvent molecules present in the solvation shell of a particular protein residue. It may be noted that, since the solvent molecules that are present within the 5 Å distance from the surface of a protein are likely to play an important role in the protein’s structure, we have particularly considered these solvent molecules in our calculations. After identification of water and ethanol molecules that are present in the solvation shell of each residue, we have calculated the binding energies between the particular residue in protein environment with a single ethanol or water molecule present in its solvation shell by using eq 1. Such calculations were carried

2. COMPUTATIONAL DETAILS 2.1. Molecular Dynamics Simulation. The starting geometry of the protein CI2 was obtained from the Protein Data Bank (PDB 1YPC).24 Eight separate simulations of the protein were carried out in four different concentrations of ethanol, such as 10%, 25%, 50%, and 75% (v/v) of initial cubic box of edge length 60 Å at ambient temperature (s1 simulations) and at 450 K temperature (s2 simulations). The overall charge of each system was neutralized by adding 2 Cl− ions. To eliminate any initial stress, both the systems were first minimized using the conjugate gradient energy minimization method. Then the systems’ temperatures were gradually increased to the targeted temperatures within a short MD run under isothermal−isobaric ensemble (NPT) conditions at constant pressure of 1 atm. All the systems were then equilibrated at the desired temperature under NPT ensemble conditions. Temperatures of the systems were controlled by using the Langevin dynamics method with a friction constant 1 ps−1, and pressure of the systems was controlled by the Nosé− Hoover Langevin piston method.25 A long production run was carried out under NVT conditions for each system for 140 ns duration. At the end of 140 ns of s2 simulations the protein was found to denature. The rupture of secondary structures of the protein was found to be dependent on the concentration of 6173


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The Journal of Physical Chemistry A out for all the identified ethanol and water molecules present in the solvation shell of each residue of the folded and unfolded protein. It may be noted that, as per the criteria to identify the residue−solvent pair, a single amino acid residue may form multiple “complexes” with different configuration of water and ethanol molecules that are present within the 5 Å distance from its surface or a single solvent molecule may form multiple complexes with different adjacent amino acid residues for being a “common solvent” for those residues. Therefore, as expressed in eq 1, by “complex” we basically mean the configuration of the trapped residue in protein environment along with a single water or ethanol when all other solvents in the solvation layer were deleted, by “solvent” we mean the configuration of that particular water or ethanol after deletion of the protein from the “complex”, and by “residue” we mean the configuration of the residue in the protein environment after deletion of the “solvent” from the “complex” configuration. It may also be noted that we have maintained the protein environment throughout this study by holding the structure of all the residues intact during calculation. The calculations were carried out for all possible residue−solvent pairs. Finally, we have reported the most favorable binding of the ethanol and water with the residues of the protein in its folded and unfolded states, respectively. All the ONIOM34 calculations were carried out using the GAUSSIAN09 program.35 We computed the single point molecular energies of the “complex”, “solvent”, and “residue” to keep the simulated configurations unchanged. The ONIOM investigations were carried out using two-layer ONIOM, a combination of high-level quantum mechanics (HQM) and low-level quantum mechanics (LQM). We have used Møller− Plesset second-order perturbation methods (MP2) in combination with the 6-31G(d,p) basis set to perform HQM calculation for the particular amino acid residue of the protein and the solvent molecule (ethanol and/or water) that forms a complex, whereas the semiempirical AM1 method36 has been used to perform LQM calculation for the rest of the residues of the protein molecule. Since our goal has been to isolate the most favorable binding mode of the solvent to the individual residue of the protein in its folded and unfolded state to explore if such bindings have any influence in the unfolding process, we have employed the most accurate method for the residue and the solvent (water and/or ethanol) that form the complex. Additionally, to compute the environmental effects, such as the molecular environment on the site of interest (i.e., the high layer), we have treated the rest of the protein molecule with an inexpensive model chemistry. We have shown a representative structure of a residue−solvent complex as a model in Figure 1.

Figure 1. Structure of a representative residue−solvent complex (shown in CPK) in protein environment. The HQM layer (the residue of interest and the associated solvent, ethanol) is shown in ball and stick, and the LQM layer (rest of the protein) is shown in wire frame representation.

may be noted that all the results as reported from the MD simulations have been calculated by analyzing the equilibrated trajectories that are generated under ambient conditions. 3.1. Residue Specific Solvation of the Protein. It is wellknown that protein’s first solvation shell or interfacial solvent layer generally plays a significant role in regulating protein’s three-dimensional structure, dynamics, and activity.37−41 The properties of these solvents around the individual amino acid residues of a protein depend on its surface exposures as well as on the degree of their burial within the structure. To detect an extent of solvation for a specific residue of the protein in water or ethanol environment, we have quantified the average number of water and ethanol molecules present within the solvation layer of each residue of the protein in its folded and unfolded forms at different ethanol concentrations, separately. In Figures 2 and 3 we display the average number of water and ethanol molecules present within the distance of 5 Å from each residue of the protein as a part of the secondary structural segments, such as helix, sheet, and loop. The rest of the protein residues are designated as a part of “others”. In general, it is evident from both figures that, irrespective of a protein’s conformational states, with increasing ethanol concentration the number of ethanol molecules in the solvation layer of the residues increases whereas the number of water molecules decreases. This infers that exclusion of water molecules occurs from some residual surfaces of the protein. However, it is evident from the figures that, as the protein unfolds, the number of both water and ethanol molecules around most of the residues increases. This is more prominent for the ethanol around the residues of unfolded protein (see Figure 3). For

3. RESULTS AND DISCUSSION Water as well as ethanol is expected to play a crucial role in the ethanol governed unfolding process of the protein. The structural and dynamical heterogeneities between the protein’s folded and unfolded forms may lead to nonuniform interactions between the residues of the protein and the surrounding solvent molecules. This, in turn, can result in differential properties of water or ethanol around the folded and unfolded states of the protein depending upon the concentration of ethanol used. Therefore, in this section we have explored the properties of water and ethanol around the folded and unfolded states of the protein. Attempts have been made to correlate the properties of the solvent present in the solvation layer of the residues of the protein with their structural transformations. It 6174


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Figure 2. Average number of water molecules, Nwat, present within 5 Å distance from each residue of the folded (left panel) and unfolded (right panel) protein in several water−ethanol mixed solutions. The plots are shown according to the secondary structural segments of the protein for better understanding and visual clarity.

that the number of ethanol molecules in the solvation shell of most of the residues increases by 2−4 times for the unfolded protein as compared to that around the folded protein. Therefore, it is clear that the residues of the protein get solvated in a heterogeneous manner. Such heterogeneity in the residue’s solvation layer might have arisen due to the residue’s preferential site of attraction toward the nature of the solvent as well as their differential accessible surface area to the solvent. 3.2. Solvent around Hydrophilic/Hydrophobic Surfaces. To explore if any correlation exists between the heterogeneity of the residue’s solvation layer and the preferential solvation site of the residue toward the nature of solvents, we have categorized the amino acid residues of the protein according to the available common hydrophobicity scales43 and then calculated the average number of water and ethanol molecules that are present around the hydrophobic and hydrophilic amino acid residues of the protein, separately. The

some of the residues, the number of solvation waters is found to increase in a significant amount; e.g., as observed from Figure 2, residues such as Ala-16 and Ile-20 of helix, Val-47, Ile57, and Gly-64 of sheet, Leu-8 and Pro-61 of others, etc. become hydrated more upon unfolding. This is particularly found to be true when the ethanol concentration increases. However, such increment in hydration water with increasing ethanol concentration may be due to the heterogeneous unfolding phenomena and differential exposures of the particular residue of the protein to the solvent environment. Interestingly, we have noted that the residues of the loop region of the folded and unfolded protein are hydrated with nearly similar number of water molecules. This is due to the fact that the residues in the loop region are not buried inside the protein core and hence are exposed to the solvent more, irrespective of protein’s configurational state. Our such finding correlates well with the previous findings.23 It is further noticed from the figure 6175


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Figure 3. Average number of ethanol molecules, Netoh, present within 5 Å distance from each residue of the folded (left panel) and unfolded (right panel) protein in several water−ethanol mixed solutions. The plots are shown according to the secondary structural segments of the protein for better understanding and visual clarity.

contacts than the folded protein at 10% ethanol. Such marginal changes in solvent contact might have arisen due to the fact that the extent of unfolding of the protein at 10% ethanol is minimal and hence the residues of the protein are not much exposed to the solvent as compared to its folded form. This is in agreement with our previous findings.23 It has been further noticed from Figure 4a that the hydrophilic amino acids of the protein are surrounded by a greater number of water molecules as compared to the hydrophobic sites of the protein, i.e., in general it can be stated that water molecules prefer to occupy the hydrophilic sites of the protein. Interestingly, it can be seen from Figure 4b that ethanol does not have any preferential site of attraction toward protein, i.e., there is an equal propensity of finding ethanol around the hydrophobic and hydrophilic residues of the protein. This is particularly true when the protein retains its folded form. The affection of the ethanol molecules for both the hydrophobic and hydrophilic sites of the

average number of solvent molecules present in the solvation shell of the hydrophobic and hydrophilic amino acid residues of folded and unfolded protein as a function of ethanol concentration is shown in Figure 4. The values are normalized by dividing by the number of hydrophobic or hydrophilic residues present in the protein. It can be seen from Figure 4a that at 10% ethanol the folded state of the protein is surrounded by a higher number of waters compared to its unfolded state. However, as the ethanol concentration increases up to 50%, the opposite trend is observed, and after that the trend again reverts around the hydrophilic sites. This is an interesting observation, and this might be an indication of stabilization of folded form of the protein at 10% ethanol and that of unfolded form above this concentration. Our such observation correlates well with the experimental findings of Bhattacharyya and co-workers.18 It may be further noted from the figure that the unfolded protein has little fewer solvent 6176


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Figure 4. Average number of water (Wn) (a) and ethanol (Etn) (b) molecules present within the first solvation shell of the hydrophobic and hydrophilic amino acid residues of the protein as a function of ethanol concentration as obtained from the equilibrated s1 and s2 trajectories.

Figure 5. Pairwise correlation function of (a, above) water molecules (g(r)P−W) and (b, below) ethanol molecules (g(r)P−Et) as a function of distance from the backbone atoms of the hydrophobic (left panel) and hydrophilic (right panel) surfaces of the folded (solid line, s1 simulation) and unfolded (broken line, s2 simulation) protein in several water−ethanol mixed solutions.

protein may be due to the fact that besides the hydrophilic −OH group there exists a relatively hydrophobic −CH3 group. Due to such amphiphilic character of ethanol it has a propensity to interact with both the hydrophobic and hydrophilic surfaces of the protein. However, note the number of ethanols around the unfolded forms of the protein. It can be seen that with increase in concentration ethanols are found to be present in greater number around the hydrophobic surface of the protein. Such phenomena might have occurred due to the heterogeneous local structuring of the water and ethanol molecules around the hydrophobic and hydrophilic surfaces of the protein as well as due to the favorable interactions between the ethanol and the hydrophobic residues of the unfolded protein. We have discussed this in section 3.4. To investigate the local structuring of the solvent around the hydrophobic and hydrophilic surfaces of the protein we have separately calculated the pair correlation function, known as the radial distribution function, g(r), of the water and ethanol molecules with the hydrophobic and hydrophilic residues of the folded and unfolded protein, separately. The calculations are carried out separately with respect to the backbone and side chain atoms of the protein in its folded and unfolded forms, as shown in Figures 5 and 6 respectively. The results, in general, indicate differential structuring of water and ethanol molecules around the hydrophobic and hydrophilic surfaces of the folded and unfolded protein. Intense first and second peaks around 3.5 and 5 Å for water around the hydrophobic as well as hydrophilic protein backbone are evident from the figures. This is a signature of moderate structuring of water around both the hydrophobic and hydrophilic backbone surfaces of the protein. The peaks become broader and more intense for water around the hydrophobic and hydrophilic protein side-chain surfaces, respectively. This is an indication of higher structuring of water around the hydrophilic surfaces of the protein side chain. This occurs due to the fact that, in general, the side-chain atoms are more exposed to the solvent than the backbone atoms. Further, it may be noted that for all cases the water around the residues of unfolded protein is more structured than the residues of folded protein. This is an expected result since upon unfolding the protein residues become more exposed to the solvents. However, note the heterogeneity in structuring of water for different ethanol solutions. Interestingly, it may be

Figure 6. Pairwise correlation function of (a, above) water molecules (g(r)P−W) and (b, below) ethanol molecules (g(r)P−Et) as a function of distance from the side-chain atoms of the hydrophobic (left panel) and hydrophilic (right panel) surfaces of the folded (solid line, s1 simulation) and unfolded (broken line, s2 simulation) protein in several water−ethanol mixed solutions.

noted from the plots that the water around unfolded protein backbone or side chain in 25% and 50% ethanol is more structured than that around 10% ethanol solution. Such behavior of water is quite surprising as one would expect that, being more hydrated in 10% ethanol solution, the structuring of water will be more around the protein backbone or side-chain surface. Such differential structuring pattern of water might have arisen due to the fact that in 10% ethanol solution the protein unfolds partially23 and hence the residues are not very exposed or are not accessible to the solvents whereas being unfolded fully in higher ethanol concentrations the residues of the protein become more exposed to the solvents and hence there exist two sharp first and second peaks for the surrounding water. Looking into the structuring patterns of ethanol around the hydrophobic and hydrophilic protein backbone and side-chain surfaces, it is clear that the ethanol molecules are struggling enough to be packed around the protein backbone. Ethanol, being a bulky molecule as compared 6177


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tional states. However, note the differences between the exposures of the surfaces when the protein unfolds. Upon unfolding, the hydrophobic surface of the protein gets exposed by 5−15% more per residue whereas the hydrophilic surface of the protein gets exposed by 1−2%. Further, the exposures of both the hydrophobic and hydrophilic surfaces are more pronounced when the protein unfolds in 50% ethanol solutions. Being exposed more upon unfolding, the solvent would have more access to interact/visit the protein surface. However, since the hydrophobic surface gets more exposed, it can be expected that, between water and ethanol, ethanol being amphiphilic in nature would prefer to interact more to the hydrophobic surface by visiting the sites more. Thus, upon unfolding, the ethanol molecules would be expected to be more structured near the hydrophobic surfaces. This is what we have observed from the corresponding g(r) plots (see Figure 6). 3.4. Interaction Energies between Solvent and Hydrophobic/Hydrophilic Residues. In this part of our work we have explored how favorable the interactions between water and ethanol to the hydrophobic and hydrophilic surfaces are. In order to explore this we have estimated the average interaction energies between several pairs, such as (a) hydrophobic amino acid−water (Ehbw), (b) hydrophilic amino acid−water (Ehpw), (c) hydrophobic amino acid−ethanol (Ehbe), and (d) hydrophilic amino acid−ethanol (Ehpe) for all the folded and unfolded forms of the protein. The estimated average interaction energies between several pairs are shown in Figure 7. It can be seen from Figure 7a that for both the folded

to water, has less access toward the protein backbone atoms, and hence there does not exist any intense peak. However, note the structuring of ethanol around the hydrophobic side-chain atoms of the protein. A high intense broad peak of ethanol around the hydrophobic side chain of the protein indicates that ethanols are moderately structured around it. A low intense first peak followed by an intense broad peak for the ethanol around the hydrophilic side chain of the protein infers that the water is less structured at a short distance, say within 3.5 Å from the hydrophilic surface, but relatively more structured within 3.5−5 Å distance from the surface. Being amphiphilic in nature, ethanol has a propensity to interact with both the hydrophobic and hydrophilic surfaces of the protein. Therefore, they are found to be packed in a more structured manner around the hydrophobic surfaces of the protein. It can be further observed from Figure 6 that the side-chain atoms are more accessible to ethanol, particularly when the protein unfolds and hence the peaks broaden and become more intense as compared to that observed for ethanol around the backbone atoms. The intensity of the broader peak is found to increase with increasing ethanol concentrations. Studies done so far have shown that, in the presence of protein, ethanol molecules aggregate to form clusters depending upon the ethanol concentration.12,18,44 However, in the present study we have not explored such behavior of ethanol, if any. A more detailed calculation on the aggregation of ethanol at various concentrations will be undertaken in our future work. It may be noted that in this work we have used a fixed-charge model; however, inclusion of polarizability in which a particle’s charge is influenced by electrostatic interactions with its neighbors is expected to provide the solvent response in a more accurate manner. Explicit treatment of polarizability in such water−ethanol binary mixtures is expected to lead to important observations about contributions to the dielectric constant that are not possible with additive force fields. 3.3. Solvent Accessibility to the Hydrophobic/Hydrophilic Surface. The differential structuring of water and ethanol around the hydrophobic and hydrophilic surfaces of the protein is likely to be dependent on the relative exposures of the residues to the solvent. To investigate that, we have calculated the solvent accessible hydrophobic (SASAhb) and hydrophilic (SASAhp) surface areas of the folded and unfolded protein over the equilibrated trajectories. The details of SASA calculation can be found elsewhere.42 The calculated SASA values are normalized by dividing by the number of the hydrophobic and hydrophilic residues in each case. The average SASAhb and SASAhp values are shown in Table 1. It can be noticed from the table that the hydrophilic residues are always exposed more to the solvent as compared to the hydrophobic surfaces. This is true irrespective of the protein’s conforma-

Figure 7. Average interaction energy between amino acid residue− water (Eres−W) (a) and amino acid residue−ethanol (Eres−E) (b) as a function of ethanol concentration as obtained from the equilibrated folded and unfolded trajectories (res = hb/hp when the amino acid is hydrophobic/hydrophilic).

Table 1. Average Solvent Accessible Surface Area (SASA in Å2) per Residue of Hydrophobic and Hydrophilic Residues of the Protein as Obtained from Different Concentrations of Ethanol Solutions hydrophobic SASA

and unfolded protein the Ehpw values are more negative than the Ehbw values. This implies that the hydrophilic interaction is stronger than the hydrophobic interaction for the water and protein residue. Further, comparing the folded Ehpw energy with the unfolded Ehpw energy, we have noted that the Ehpw value for folded protein is always more negative than that for the unfolded protein, whereas Ehbw values are found to be more negative for the unfolded protein in 25% and 50% solution as compared to those for folded protein. Additionally, for 10% and 75% solution the Ehbw values remain almost similar for both the

hydrophilic SASA

system

folded

unfolded

Δ

folded

unfolded

Δ

10% 25% 50% 75%

116.80 116.90 116.72 116.95

123.17 130.56 137.26 135.04

6.37 13.66 20.54 18.09

151.14 149.55 150.12 148.98

153.04 157.92 160.64 151.92

1.90 8.37 10.52 2.99 6178


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Figure 8. Residue−solvent binding energy, BERW and BERE of the folded (left panel) and unfolded (right panel) protein as obtained from the ONIOM calculations.

folded and unfolded protein. All such observations indicate that although the interaction between water and hydrophilic residues is more favorable than that with hydrophobic residues, the latter become more favorable when the protein unfolds. This is a signature of increasing hydrophobic interactions of the unfolded protein with water. As observed from Figure 7 b, both the hydrophobic and hydrophilic interactions between the ethanol and protein are less favorable for the folded protein than for the unfolded protein, i.e., whenever the protein unfolds the Ehpe as well as Ehbe values become more negative as compared to those for the folded protein. This implies that, being exposed more (see Table 1) upon unfolding, protein’s hydrophobic interaction with water as well as with ethanol increases irrespective of the nature of the solvents. However, the effect is relatively more pronounced for ethanol. It may be further noted that although increase in hydrophobic interaction helps in destabilizing the compact native structure of the protein, interactions through hydrophilic sites are also not

negligible and it has a proficient impact in rupturing protein’s native structure. 3.5. ONIOM Study: Binding Energetic of Residue− Solvent Complex. In this section we study the electronic structure of the binding site of residue−water/residue−ethanol complex. The residue specific binding energy, BErs, between the amino acid residue of the protein and the solvent ethanol− water has been calculated by using ONIOM technique as described in section 2.2. Such calculation would provide comparative binding information between the folded/unfolded protein residue and the solvent. This, in turn, would provide valuable information in detecting the driving force of the ethanol governed unfolding phenomena of the protein. The binding energies of the residue−solvent complexes are calculated by using eq 1. The corresponding values are displayed in Figure 8 as a function of residue number of the protein. Once again the residues and their corresponding binding energies with the solvent are shown as a part of the 6179


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Table 2. Average Binding Energies (in kcal mol−1) of 18 Different Types of Amino Acid Residues Present in the Protein with Solvent as Obtained from the ONIOM Calculations ⟨BErw⟩

⟨BEret⟩

residue name

folded

unfolded

Δ⟨BErw⟩

folded

unfolded

Δ⟨BEret⟩

Met Lys Thr Glu Trp Pro Leu Val Gly Ser Ala Ile Leu Gln Asp Tyr Arg Asn average

−11.88 −15.36 −4.65 −13.69 −4.07 −1.92 −7.58 −6.10 −7.06 −14.04 −6.47 −3.95 −7.58 −5.08 −10.78 −3.75 −10.43 −4.50

−5.61 −7.41 −5.53 −9.56 −9.71 −4.07 −6.47 −4.04 −9.23 −5.82 −5.87 −5.52 −6.47 −7.64 −15.95 −12.22 −7.68 −5.06

6.26 7.95 −0.88 4.13 −5.64 −2.15 1.10 2.06 −1.63 8.22 0.59 −1.57 1.10 −2.57 −5.17 −8.47 2.75 −0.56 8.90

−6.59 −12.87 −5.03 −8.60 −3.61 −2.92 −6.61 −3.66 −3.19 −5.89 −2.78 −4.73 −6.61 −3.13 −14.46 −0.76 −4.00 −0.90

−8.45 −9.68 −5.64 −10.88 −4.69 −4.27 −3.67 −3.66 −5.73 −5.82 −4.99 −5.26 −3.67 −4.66 −11.59 −1.41 −7.28 0.74

−1.86 3.18 −0.61 −2.27 −1.08 −1.35 2.94 0.00 −2.54 0.07 −2.21 −0.53 2.94 −1.53 2.87 −0.65 −3.27 1.64 −8.39

secondary structural segments for clarity. Negative BErs values indicate a favorable binding upon complexation. It may be noted that, as discussed in section 2.2, we have already identified the binding energies of the residue−solvent complexes that are most favorable out of all the possible pairs. Heterogeneous binding phenomena of the residues with the solvents for the secondary segments of the protein are evident from the figure. It may be observed from the figure that, irrespective of the nature of solvents, there are some residues which are very tightly bound to the solvent molecules; therefore for those complexes the binding energies are found to be very high, e.g., complexation between Lys-17 and Lys-18 with both water and ethanol, Glu-26 with water, Asp-55 with water, etc. in protein’s folded state as well as Leu-54 and Asp-55 with water in protein’s unfolded state etc. This is a signature of strong binding between that particular residue and the solvent water or ethanol molecule, and it might have arisen due to the formation of strong hydrogen bonds between the residues and the solvent molecules. We will discuss this in the forthcoming section. We have further calculated average BEret and BErw values over all residue−water and residue−ethanol pairs separately for the folded and unfolded protein. The average residue−ethanol binding energies of the folded and unfolded protein are found to be −5.74 kcal mol−1 and −5.96 kcal mol−1 whereas the average residue−water binding energies of the folded and unfolded protein are found to be −7.88 kcal mol−1 and −6.93 kcal mol−1. The relatively more negative average BEret value for unfolded complexes indicates that such complexation is a favorable driving force toward unfolding whereas more negative BErw for folded complexes indicates favorable driving force toward folding. This further indicates that ethanol binds more strongly with the residues of unfolded protein whereas water bound more favorably with the residues of folded protein. Such finding correlates well with the results as obtained from the MD simulations as discussed in earlier sections. Importantly, it may also be noted that the marginal difference in average binding energies of the residue−ethanol pair for the folded and unfolded protein is an indication of preferential binding of

ethanol molecules at the protein surface for both the folded and unfolded states. Our such observation agrees well with the experimental findings.17 We have also tried to explore if any correlation exists between the nature of amino acid residues of the protein and their bindings with the solvent. For this, we have calculated average “distinct binding energies” for the complexes formed between the solvent and all similar types of amino acid residues present in the protein. Since CI2 has 18 different types of amino acid residues, such averaging would provide 18 average distinct binding energies for 18 distinct residues that form complexes with the solvent water or ethanol. The results are displayed in Table 2. We have then plotted the average distinct binding energies of the residue−water and residue−ethanol pairs as a function of increasing hydrophobicity of the amino acid residues. The linear fit of the plots indicates that there exist correlations between the hydrophobicity of the amino acids with the BEret values for the unfolded protein complexes as shown in Table 3 whereas the BErw values are found to be Table 3. Correlation Coefficient (r2) Values as Obtained from the Linear Fit of the Plot of ⟨BErw⟩ and ⟨BEret⟩ Values as a Function of Increasing Hydrophobicity of the Amino Acid Residues Present in the Protein r2 system

folded

unfolded

water ethanol

−0.54 −0.42

0.56 0.76

anticorrelated with the hydrophobicity of the amino acid residues, implying existence of correlation with the increasing hydrophilic nature of the amino acids. Being amphiphilic in nature, ethanol when bound to the protein residues shows intermediate regression-coefficient values, implying its favorable interactions with both the hydrophobic and hydrophilic amino acid residues. This once again correlates well with the MD results as discussed earlier. Once again favorable complexation 6180


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Figure 9. Average number of amino acid residue−water (HBRW) hydrogen bonds in several water−ethanol mixed solutions as obtained from the equilibrated s1 (left panel) and s2 (right panel) trajectories.

protein−ethanol (RE) hydrogen bonds. The first condition for an atom of the protein to form a hydrogen bond with the solvent (either as an acceptor or as a donor atom of water or ethanol) is that the distance between the tagged atom and the oxygen atom of the solvent molecule with which it is hydrogen bonded be within 3.5 Å. The second condition for an acceptor atom of the protein is that the angle between one of the O−H bond vectors of the solvent and the vector connecting the hydrogen atom and the acceptor atom be within 40°, but for a residue donor atom to form a RW/RE hydrogen bond the angle between one of the O−H bond vectors of the solvent and the vector connecting the solvent oxygen atom and the hydrogen atom attached with the donor atom should be within 80°−140°. We have calculated the average number of RW and RE hydrogen bonds formed over the equilibrated trajectories of all the systems. The time average RW and RE hydrogen bond numbers are shown in Figures 9 and 10 respectively. It can be observed from Figure 9 that there are around 20 residues, e.g.,

between the folded protein residue with water and the unfolded protein residue with ethanol is evident from the table. This can be directly observed from the differences in binding energies between the aqua as well as alcoholic complexes of the unfolded and folded protein as shown in Table 2. Such strong binding between the solvent and protein residues might have occurred due to the formation of hydrogen bonds between the protein residue and the solvent molecules. We have explored it in the next subsection. 3.6. Hydrogen Bond. The ethanol governed unfolding of the protein is likely to be affected by the formation of differential hydrogen bonding pattern between the protein residue and the solvent molecules. To investigate this we have calculated the average number of hydrogen bonds formed between each of the protein residues and the solvent ethanol or water molecules over the equilibrated folded and unfolded trajectories separately. In this study we have employed purely geometric criteria45−47 to define residue−water (RW) and 6181


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Figure 10. Average number of amino acid residue−ethanol (HBRE) hydrogen bonds in several water−ethanol mixed solutions as obtained from the equilibrated s1 (left panel) and s2 (right panel) trajectories.

shorter lifetime the average values become quite small. However, it may be noted that the small average RW and RE hydrogen bond numbers are not an implication of insignificant role of RW and RE hydrogen bonds in the unfolding process; truly microscopic investigation of these hydrogen bond dynamics is now becoming very important to speculate the driving force of the ethanol governed unfolding process. Importantly, these hydrogen bonds may take an important role in the unfolding process by forming very strongly hydrogen bonded complexes, which, particularly, could be true for the RW hydrogen bonds. This perception has been received from the ONIOM calculation in some respects, which we have discussed below. Governed by several interesting differential binding phenomena of water and ethanol with the residues of folded and unfolded protein as obtained from the ONIOM study, here we have tried to explore if there exists any correlation between the high binding energy (BErs) and the formation of hydrogen

Lys-17, Lys-18, Lys-24, Arg-46, Arg-43, etc., that form a significant number of hydrogen bonds all over the equilibrated trajectories. Interestingly, these residues are found to form tight complexes with the solvents as discussed in section 3.5. Further, note the inset plot of the figure, which shows that almost 80% of the total residues of the protein form hydrogen bonds with water throughout the trajectories. However, the average number of RW hydrogen bonds is found to be much smaller. This may be due to the fact that these residues form hydrogen bonds for a much shorter period of time and hence their average values are significantly smaller. As observed from Figure 10, for a particular residue the average number of RE hydrogen bonds formed over the whole time span of calculation is much smaller. The possible reason for it could be the quick breaking of the RE hydrogen bonds. However, it may be noted from the inset plot that although the number of RE hydrogen bonds is much smaller for each residue, almost all the residues generally form hydrogen bonds with the ethanol. However, due to 6182


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Table 4. Details of Hydrogen Bonded Pairs Formed between the Amino Acid Residue of the Protein and Water along with Their Corresponding Binding Energies as Obtained from the ONIOM Calculations folded

unfolded

segment

residue name

donor atom

acceptor atom

BE

residue name

donor atom

acceptor atom

BE

helix

Ser-12 Ala-14 Ala-15 Lys-17 Lys-18 Val-19 Leu-21 Gln-22 Asp-23 Lys-24 Gln-28 Val-31 Val-34 Asp-45 Arg-46 Arg-48 Phe-50 Asp-52 Lys-53 Asn-56 Ala-58 Arg-62 Gly-64

wat−O pr−N pr−N pr−N pr−N wat−O wat−O wat−O wat−O pr−N wat−O pr−N/wat−O wat−O wat−O pr−N pr−N pr−N wat−O wat−O wat−O wat−O pr−N wat−O

pr−O wat−O wat−O wat−O wat−O pr−O pr−O pr−O pr−O wat−O pr−O wat−O pr−O pr−O wat−O wat−O wat−O pr−O pr−O pr−O pr−O wat−O pr−N

−14.04 −10.83 −8.36 −23.10 −20.41 −1.34 −3.92 −5.51 −4.86 −17.46 −5.19 −8.91 −5.64 −12.44 −11.52 −11.57 −7.99 −14.63 −3.28 −4.50 −0.59 −8.73 −17.30

Ser-12 Val-13 Ala-14 Ala-15 Lys-17 Lys-18 Asp-23 Asp-55 Leu-54

wat−O wat−O wat−O wat−O pr−N pr−N wat−O wat−O wat−O

pr−O pr−O pr−O pr−O wat−O wat−O pr−O pr−O pr−O

−5.82 −5.69 −4.86 −15.36 −17.24 −7.22 −14.40 −28.58 −20.36

Gly-35 Thr-36 Ile-37 Val-38 Thr-39 Met-40 Glu-41 Tyr-42 Arg-43 Met-1 Lys-2 Glu-4 Pro-6 Val-9 Gly-10 Lys-11 Glu-26 Ala-27 Leu-54 Asp-55 Gln-59 Val-60

pr−N wat−O wat−O wat−O wat−O pr−N wat−O wat−O pr−N pr−N pr−N wat−O wat−O wat−O wat−O pr−N wat−O wat−O wat−O wat−O wat−O wat−O

wat−O pr−O pr−O pr−O pr−O wat−O pr−O pr−O wat−O wat−O wat−O pr−O pr−O pr−O pr−O wat−O pr−O pr−O pr−O pr−O pr−O pr−O

−1.34 −4.24 −5.96 −4.36 −11.5 −6.262 −10.56 −3.75 −9.91 −17.49 −11.65 −15.21 −2.78 −8.22 −4.15 −16.27 −15.88 −5.07 −15.04 −11.18 −4.53 −4.64

Gln-28 Ile-29 Ile-30 Val-31 Leu-32 Val-34 Asp-45 Arg-46 Val-47 Leu-49 Phe-50 Val-51 Asp-52 Lys-53 Ile-57 Ala-58 Arg-62 Val-63 Gly-64 Gly-35 Thr-36 Thr-39 Met-40 Glu-41 Tyr-42 Arg-43 Ile-44

pr−N wat−O wat−O wat−O wat−O wat−O wat−O pr−N wat−O wat−O wat−O wat−O wat−O wat−O wat−O wat−O wat−O wat−O wat−O wat−O wat−O pr−O wat−O wat−O wat−O pr−N wat−O

wat−O pr−O pr−O pr−O pr−O pr−O pr−O wat−O pr−O pr−O pr−O pr−O pr−O pr−O pr−O pr−O pr−O pr−O pr−O pr−O pr−O wat−O pr−O pr−O pr−O wat−O pr−O

−9.25 −14.19 −7.10 0.35 −4.25 −4.62 −11.22 −18.58 −8.04 −3.29 −3.52 −5.02 −9.58 −2.38 −4.20 −3.65 −2.84 −6.40 −15.14 −5.00 −1.30 −7.62 −4.93 −12.88 −12.22 −8.10 −5.10

Lys-2 Thr-3 Glu-4 Trp-5 Leu-8 Val-9 Lys-11 Pro-25 Glu-26 Gln-59

wat−O wat−O wat−O wat−O pr−N wat−O wat−O wat−O wat−O wat−O

pr−O pr−O pr−O pr−O wat−O pr−O pr−O pr−O pr−O pr−O

−7.85 −7.65 −9.56 −9.71 −4.85 −3.09 −4.29 −4.33 −11.78 −9.45

sheet

loop

others

bonds between the amino acid residue and solvent pairs. For this we have identified all the residue−solvent pairs that form hydrogen bonds and have noted their corresponding binding energies, as displayed in Tables 4 and 5. We have shown representative snapshots of few RW and RE pairs that are bound with hydrogen bonds in Figure 11. It may be noted from

the table that most of the hydrogen bonded complexes show high negative binding energies. This is true irrespective of the nature of the solvent and the configurational state of the protein. We find that the contribution of the binding energies of the hydrogen bonded RW pair for folded and unfolded protein on the total binding energy of the same pair is around 6183


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Table 5. Details of Hydrogen Bonded Pairs Formed between the Amino Acid Residue of the Protein and Ethanol along with Their Corresponding Binding Energies as Obtained from the ONIOM Calculations folded segment helix

sheet

loop others

unfolded

residue name

donor atom

acceptor atom

BE

residue name

donor atom

acceptor atom

BE

Lys-18 Lys-24 Pro-25 Val-31 Asp-45 Asp-52 Val-63 Met-40

pr−N pr−N et−O et−O et−O et−O et−O et−O

et−O et−O pr−O pr−O pr−O pr−O pr−O pr−O

−23.16 −16.11 −4.17 −5.40 −17.82 −16.09 −9.99 −4.96

Ser-12 Asp-23

pr−O et−O

et−O pr−O

−5.82 −13.78

Asp-52 Gly-64

et−O pr−N

pr−O et−O

−11.77 −13.03

Thr-3 Glu-26 Leu-54 Asp-55

pr−N et−O et−O et−O

et−O pr−O pr−O pr−O

−12.58 −22.28 −10.41 −23.62

Ile-37 Glu-41 Lys-2 Glu-7

et−O et−O pr−N et−O

pr−O pr−O et−O pr−N

−9.08 −3.38 −16.01 −19.29

Figure 11. Representative hydrogen bonded (a) residue−water and (b) residue−ethanol complexes. A part of unfolded protein is shown for visual clarity.

80% and 88% respectively. However, the contribution of the binding energies of the hydrogen bonded RE pairs for the folded and unfolded protein on the total binding energy of such pairs is around 45% and 43% respectively. This implies that both RW and RE hydrogen bonds play a significant role in the unfolding process. Therefore, our preliminary investigation on residue−solvent hydrogen bonds infers that formation of RW as well as RE hydrogen bonds plays an important role in the ethanol induced protein unfolding process. Significant deviations in the time average RW and RE hydrogen bond numbers for each residue clearly indicate that the hydrogen bond dynamics plays a crucial role in the ethanol governed unfolding phenomena of a protein. It may be assumed that the long and short time components of the RW and RE as well as EE (ethanol−ethanol) and WW (water−water) hydrogen bond dynamics would play significant roles in such a process. Further, measurement of hydrogen bond lifetime is also expected to play a vital role. However, all these need to be addressed in a more rigorous manner. Presently, we are working on such aspects in our laboratory.

ONIOM techniques. Attempts have been made to correlate the properties of the solvent present in the solvation layer of the residues of the protein with its structural transformations. Our calculation showed that although replacement of water molecules occurs by the ethanol molecules from most residual surfaces of the protein upon unfolding, interestingly, the residues of the loop region of the protein were found hydrated with an almost similar number of water molecules. Such heterogeneity occurs due to the fact that the residues in the loop region are not buried inside the protein core but are exposed to the solvent more, which is true irrespective of the protein’s configurational state. Our study shows that, being amphiphilic in nature, ethanol can solvate both the hydrophobic and hydrophilic surfaces of the protein whereas water has a propensity to solvate hydrophilic surfaces more. The solvent’s preferential site of attraction toward the nature of the protein surface or vice versa is evident from the heterogeneous structuring pattern of water and ethanol molecules around them. Precisely, water was found to be structured moderately around the hydrophobic and hydrophilic backbone surfaces of the protein. However, such structuring pattern of water becomes more proficient around the side-chain atoms of the hydrophilic surfaces of the protein. This is an interesting result, indicating the hydrophilic residue’s preferential attraction toward the water. Interestingly, among several solutions, water around unfolded protein backbone or side chain in

4. CONCLUSIONS In this article we have studied the properties of water and ethanol around a small enzymatic protein, chymotrypsin inhibitor 2, in its folded and unfolded states in several water−ethanol mixed solutions using MD simulation and 6184


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water, hydrogen bond dynamics becomes vital to explain the role of cosolvent like ethanol in protein denaturation. Currently, we are exploring such aspects in our laboratory.

25% and 50% ethanol solution is found to be more structured than that in 10% ethanol solution. Such differential structuring pattern of water might have arisen due to the heterogeneous unfolding phenomena of the protein in the presence of different concentrated ethanol solutions. High intense broad peak of ethanol around the hydrophobic side chain of the protein indicates that ethanols are moderately structured around it. Our calculation shows that upon unfolding the hydrophobic surface of the protein gets exposed by 5−15% more per residue whereas the hydrophilic surface of the protein gets exposed by 1−2% more. Further, the exposures of both the hydrophobic and hydrophilic surfaces are more pronounced when the protein unfolds in 50% ethanol solutions. Being exposed more upon unfolding, the solvent has more access to interact/visit the protein surface, which correlates well with the heterogeneous structuring pattern of the solvents around the surfaces. The comparative investigation of interaction energies between hydrophobic and hydrophilic residues with water as well as ethanol reveals that although increase in hydrophobic interaction helps in destabilizing the compact native structure of the protein, interactions through hydrophilic sites are also not negligible and have a proficient impact in rupturing the protein’s native structure. The relatively more negative average binding energy of residue−ethanol complexes of unfolded protein, as obtained from ONIOM study, indicates that such complexation is a favorable driving force toward unfolding whereas more negative binding energy of residue−water complex of folded protein indicates favorable driving force toward folding. Importantly, our calculation shows that there exists marginal difference in average binding energies of the residue−ethanol pair for the folded and unfolded protein. This is an indication of preferential binding of ethanol molecules at the protein surface for both the folded and unfolded states. Our such observation agrees well with the experimental findings.17 Our study further reveals that there exist correlations between the hydrophobicity of the amino acids and the residue−ethanol binding energy values for the unfolded protein complexes, whereas residue−water binding energy values are found to be anticorrelated with the hydrophobicity of the amino acid residues, implying the existence of correlation with the hydrophilic nature of the amino acids. The hydrogen bond analysis shows that more than 80% residues of the protein form hydrogen bonds with water and/or ethanol over the equilibrated trajectories. Significant deviations in the number of time average residue−water and residue− ethanol hydrogen bonds for each residue clearly indicate that the hydrogen bond dynamics play a crucial role in the ethanol governed unfolding phenomena of a protein. We find that the most tightly bound residue−solvent complexes are generally bound with the hydrogen bonds and hence exhibit very high negative binding energies. Our calculations further showed that the contribution of the binding energies of hydrogen bonded residue−water pair to the total binding energy of the same pair is about 8% more when the protein unfolds, however, the contribution of the binding energies of hydrogen bonded residue−ethanol pair to the total binding energy of the same pair remains almost similar upon unfolding of the protein. This is an interesting observation which further infers that the residue−water hydrogen bonds play an indispensable role in the ethanol governed unfolding process. Therefore, exploration of heterogeneity in long and short time components of the residue−water and residue−ethanol as well as intra- and intersolvent, i.e., ethanol−ethanol, water−water, and ethanol−

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Santanab Giri: 0000-0002-5155-8819 Madhurima Jana: 0000-0002-6567-4490 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study received grant support from the Department of Science and Technology (SB/FT/CS-065/2012 and DSTINSPIRE, IFA14-CH-151), Government of India. The work was partly carried out using the NIT Rourkela central computational facility. D.M. and S.S thank NIT, Rourkela, and G.N.R. thanks DST, India, for providing a scholarship.

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REFERENCES

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