Methanol Concentration Dependent Protein Denaturing Ability of

Article pubs.acs.org/JPCB

Methanol Concentration Dependent Protein Denaturing Ability of Guanidinium/Methanol Mixed Solution Qiang Shao* Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China S Supporting Information *

ABSTRACT: Mixtures of osmolytes are present in the cell. Therefore, the understanding of the interplay of mixed osmolyte molecules and their combined effects on protein structure is of fundamental importance. In this article, the structure stability of a model protein (BdpA) in the mixture of guanidinium thiocyanate (GdmSCN) and methanol (MeOH) was investigated by molecular dynamics simulation. It was observed that guanidinium (Gdm+) is driven to protein surface by favorable electrostatic interactions and MeOH is driven by both favorable electrostatic and VDW interactions, respectively. The mixture of Gdm+ and MeOH doesnot affect the electrostatic energy distribution of Gdm+ but does reduce the difference in VDW energy of MeOH between the regions of protein surface and bulk solution. As a result, the accumulation level of Gdm+ is not influenced, but the accumulation level of MeOH is lowered in mixed solution. The tertiary structure stability of protein is determined by the accumulated strength of VDW interactions from MeOH to protein side chain, and the secondary structure stability is correlated to the strength of combined electrostatic energies from solvent (water) and cosolvent (Gdm+ and MeOH) to protein backbone, particularly in hydrogen bonding part. The mixture of GdmSCN with low-concentrated MeOH stabilizes native structure of BdpA whereas the further increase of MeOH concentration denatures native structure of protein to expanded unfolded structure. The present study together with our previous study on the mixture of GdmSCN and 2,2,2-trifluoroethanol (TFE) provides novel insights into the effects of mixed osmolytes on protein structure.

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INTRODUCTION The stability of protein structure is sensitive to environmental conditions. Natural osmolytes (e.g., glycine betaine, amino acids, polyols, sugars, and methylamines) are accumulated in the intracellular environment under anhydrobiotic, thermal, and pressure stresses1 and can protect native structures of proteins without perturbing the functional groups.2−4 In contrast, urea ((NH2)2CO), a highly concentrated waste product in mammalian kidneys, can destroy native structures (both tertiary and secondary structures) and interrupt biological functions of proteins. The analogue of urea, guanidinium-containing compound ((NH2)2CNH2+), has similar “denaturing” action toward protein structure. Both species have been widely used in in vitro denaturation and refolding of proteins.5−12 On the other hand, monohydric alcohols including methanol (MeOH) and 2,2,2-trifluoroethanol (TFE) exhibit mixed denaturing (tertiary structure) and stabilizing (secondary structure) behavior toward proteins, generating partially denatured molten globule-like structure.13−22 The molecular interactions between individual osmolytes and protein have been extensively studied in the past several decades, and the understanding of the effects of individual osmolytes on protein structure is well-established.23−39 The © 2014 American Chemical Society

effects of osmolyte mixture on protein structure are, however, less studied. Considering the fact that mixtures of osmolytes are present in the cell,1,2,40,41 the understanding of the interplay of mixed osmolyte molecules and their combined effects on protein structure is of fundamental importance.42 To date, the reported investigations of the behavior of osmolyte mixture have been focused on osmolytes with opposite functions toward protein, to wit, one denaturing osmolyte (mainly urea) and one protecting osmolyte.1,43−51 These studies indicated the counteraction of urea denaturation by protecting osmolytes including trimethylamine N-oxide (TMAO), betaine, sarcosine, and trehalose. The question is, when two denaturing osmolytes are mixed up, could they work collaboratively in denaturing protein or compete with each other to discount the denaturation efficiency? In a recent molecular dynamics (MD) simulation study on two proteins (hen egg-white lysozyme and protein L) in the mixture of two denaturing osmolytes (guanidinium chloride (GdmCl) and urea), Zhou and co-workers found that Received: January 10, 2014 Revised: May 20, 2014 Published: May 20, 2014 6175

dx.doi.org/10.1021/jp500280v | J. Phys. Chem. B 2014, 118, 6175−6185

The Journal of Physical Chemistry B

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perspectives reveal molecular mechanisms of different effects of single and mixed osmolyte solutions on protein structure.

the two denaturants interact with protein competitively and GdmCl is more favorite to accumulate on protein surface than urea.52 The accumulation of two denaturants creates a more crowded environment surrounding protein which results in unexpected structure collapse of denatured protein, different to the extended unfolded structure adopted in either GdmCl or urea solution. We also ran comparative MD simulations on a model protein, the B domain of protein A from Staphylococcus aureus (BdpA), in single-component guanidinium thiocyanate (GdmSCN) and TFE solutions, and their mixture, respectively.53 It was observed that Gdm+ approaches protein and the direct interactions from Gdm+ and water to protein result in large-scale denaturation of protein secondary structure in the absence of TFE. Interestingly, in the presence of both Gdm+ and TFE, the approach of Gdm+ and water to protein is impeded by the preceding accumulation of TFE on protein surface, leading to improved secondary structure stability in solution. Therefore, Gdm+ and TFE work competitively but not collaboratively in their mixed solution.53 In the two above-mentioned cases of denaturing osmolyte mixtures, the osmolyte with stronger accumulation tendency impairs the ability of the other to interact with protein and thus leads to the weakening of the denaturing ability of solution. In the present study, we studied the mixture of GdmSCN and another alcohol, methanol (MeOH) which has lower accumulation tendency than the fluorine-substituted analogue (TFE), to understand the interplay of the two species and their combined effects on protein structure. Two mixed solutions were studied with the constant concentration of GdmSCN (4.0 M) and varied concentrations of MeOH (25% and 40% (v/v)), herein referred to as GdmSCN/MeOH (25%) and GdmSCN/ MeOH (40%) solutions. The concentration of GdmSCN is the same as that in our previous MD simulation studies of GdmSCN/TFE mixture.53 On the other hand, the concentration of MeOH used in previous experimental studies usually spans in a large range from 0% to 80% (v/v).17,19,54,55 Therefore, two concentrations of MeOH within this range were tested in the present study. Moreover, the protein of BdpA in single-component 4 M GdmSCN solution and 25% MeOH solution was also simulated as control tests. Molecular force field is crucial for the accuracy of molecular dynamics simulation. Two popular force fields, namely AMBER FF99 and FF99SB, were employed in our previous MD simulation of GdmSCN/TFE mixture to evaluate the calculation convergence of molecular simulation. The simulation results are consistent with each other, suggesting that both force fields are suitable to model the behaviors of the protein and osmolytes in aqueous solution.53 One of the force fields, FF99, was then used in the present study. The results indicate that the accumulation of guanidinium on protein surface is more competitive than that of MeOH in the mixed solution. The accumulation level of the former species is not affected, but the accumulation level of the latter molecule is declined when the two are mixed in solution. It is also observed that the mixture of GdmSCN with low-concentrated MeOH (e.g., 25% (v/v)) stabilizes native structure of BdpA whereas the further increase of MeOH concentration (e.g., 40% (v/v)) in mixed solution denatures native structure of protein to unfolded structure, which is even more expanded than the denatured structures in single GdmSCN and MeOH solutions. The detailed analyses of interactions between protein and solvent/cosolvent molecules from energetic and structural

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MATERIALS AND METHODS The AMBER 11 suite of program56 was employed to run all MD simulations in the present study of which the force fields involved are FF99 force field for protein57 and SPC/E model for water.58 In addition, the force field parameters of Gdm+ cation were taken from Jorgensen and Tiradorives,59 and those for MeOH molecule were from Caldwell and Kollman.60 In each simulation system, the crystal structure of BdpA (Figure 1) was solvated in a cubic box containing solvent

Figure 1. NMR structure of BdpA protein. The three helices are colored by green (Helix1), yellow (Helix2), and red (Helix3), and the side chains of the hydrophobic core cluster are shown with the VDW representation.

(water) and cosolvent (Gdm+ and SCN− ions, and/or methanol) molecules, which was then neutralized by adding three Na+ cations. The detailed numbers of solvent and cosolvent molecules are listed in Table S1 of the Supporting Information. Three independent trajectories were run for each simulation system. In each trajectory, the constructed system was initially minimized for 2500 steps with the protein being fixed using harmonic restraints (force constant = 500.0 kcal mol−1 Å−2). Second, the system was heated upon 360 K and equilibrated for several nanoseconds with harmonic restrains applied on protein backbone atoms (force constant = 10.0 kcal mol−1 Å−2) to make a good relaxation for the solvent and cosolvent molecules in system. The equilibrium time at high temperature was varied in individual independent trajectories to generate different initial simulation conditions (the coordinates and velocities of solvent and cosolvent molecules). Third, the simulation system was cooled from 360 to 300 K with harmonic restraints still applied on protein backbone atoms. Finally, long-time equilibrium simulation (production run) was performed at constant temperature of 300 K and constant pressure of 1 atm. The SHAKE algorithm61 was used to fix all covalent bonds involving hydrogen atoms, and periodic boundary conditions were used to avoid edge effects. The particle mesh Ewald method was applied to treat long-range electrostatic interactions,62 and the cutoff distance for longrange terms (electrostatic and van der Waals energies) was set as 10.0 Å. The simulation data in each product run was collected every 2.0 ps.

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RESULTS Methanol Concentration Dependent Protein Denaturing Ability of GdmSCN/MeOH Mixture. The structural stability of BdpA protein was measured in two mixed solutions 6176



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four solutions follows the same order: GdmSCN/MeOH (40%) > GdmSCN ≈ MeOH > GdmSCN/MeOH (25%). Consequently, the structural change of protein is severest in GdmSCN/MeOH (40%) solution and is in the middle in single GdmSCN or MeOH solution but is smallest in GdmSCN/ MeOH (25%) solution. The number of native hydrophobic side chain contacts (NHC) and the total number of backbone hydrogen bonds (NHB) formed within BdpA can be used to indicate the change in tertiary and secondary structures of protein, respectively. Two hydrophobic side chains are considered as in contact if any pair of heavy atoms of side chains is close to 5.0 Å. In addition, the criteria used to evaluate the formation of backbone hydrogen bond include a cutoff radius of 3.2 Å between the heavy atoms of hydrogen donor and acceptor and a cutoff of 135° for the N−H−O angle. As shown in Figure 3, the number

containing constant concentration of GdmSCN and varied concentration of MeOH. Moreover, the same protein was also investigated in corresponding single-component solutions as the control tests. Multiple independent simulation trajectories were run for each simulation system to indicate the convergence of simulation, which reached similar (solutiondependent) protein conformations within the simulation times (∼200 ns per trajectory, see simulation paramters in Table S1). Although the real equilibrium states might not be achieved, the present simulation time per system is long enough to display clearly solution dependent protein structural stability. Conclusions could thus be drawn on the basis of the present simulation data. Insead of analyzing individual trajectories, the data in three trajectories of each system were averaged to present more relaible information (Figures 2−12 and Tables

Figure 2. Time series of (a) the root-mean-square deviation (RMSD) corresponding to the NMR structure and (b) the radius of gyration (Rg) of BdpA protein in GdmSCN/MeOH (40%), GdmSCN/MeOH (25%), GdmSCN, and MeOH solutions.

Figure 3. Time series of (a) the number of native hydrophobic side chain−side chain contacts formed and (b) the total number of backbone hydrogen bonds formed in the entire protein of BdpA in GdmSCN/MeOH (40%), GdmSCN/MeOH (25%), GdmSCN, and MeOH solutions.

Table 1. Average Numbers of All Solvent and Cosolvent Molecules in the First Solvation Shell of Protein Surface in All Solutions under Study

MeOH Gdm+ SCN− H2O RMeOH RGdm+ RSCN−

GdmSCN/ MeOH (40%)

GdmSCN/ MeOH (25%)

71.3 ± 5.4 43.0 ± 2.8 31.0 ± 2.6 101.1 ± 6.0 1.31 ± 0.05 1.89 ± 0.02 1.36 ± 0.05

36.2 ± 1.1 41.8 ± 1.3 31.0 ± 0.8 117.0 ± 2.8 1.31 ± 0.02 1.82 ± 0.03 1.35 ± 0.02

GdmSCN

of native hydrophobic contacts decreases slightly in GdmSCN/ MeOH (25%) solution, with the value close to that in pure water (Figure S4a). In contrast, the number drops largely in the remaining solutions, corresponding to the breaking of intraprotein hydrophobic contacts and consequent tertiary structure expansion. Moreover, the intraprotein hydrophobic contact breaking is more apparent in GdmSCN/MeOH (40%) than in single GdmSCN or MeOH solution. On the other hand, the backbone hydrogen bond number of BdpA decreases in GdmSCN and GdmSCN/MeOH (40%) but keeps steady in MeOH and GdmSCN/MeOH (25%) solutions with the value comparable to that in pure water (see Figure S4b). The combined results of Figures 2 and 3 suggest that single MeOH solution destroys the tertiary structure but stabilizes the secondary structure of protein and GdmSCN solution destroys both tertiary and secondary structures, consistent with their specific behaviors toward protein, respectively. In contrast, GdmSCN/MeOH (25%) solution results in slight expansion of the tertiary structure and enhanced stability of secondary structure, implying that the mixture of GdmSCN with lowconcentrated MeOH stabilizes the structure of protein. Nevertheless, the increase in MeOH concentration (e.g., to 40% (v/v)) in the mixed solution largely denatures both tertiary and secondary structures of protein. Undisturbed Accumulation of Gdm+ but Impaired Accumulation of MeOH on Protein Surface in GdmSCN/

MeOH (25%) 61.2 ± 4.5

48.0 ± 1.8 36.4 ± 2.1 184.5 ± 7.0

208.0 ± 8.1 1.62 ± 0.05

1.98 ± 0.05 1.50 ± 0.06

1−3). The individual trajectories for BdpA in two GdmSCN/ MeOH mixed solutions and MeOH solution are depicted in Figures S1−S3 and the trajectories for BdpA in GdmSCN solution are in ref 63 and its Supporting Information. Figure 2 illuminates the trajectories represented by the time dependent root-mean-square deviation (RMSD) and the radius gyration (Rg) of BdpA in four solutions under study. The RMSD value is highly increased within the simulation time in both single GdmSCN and MeOH solutions. Interestingly, the RMSD value keeps small when GdmSCN is mixed with lowconcentrated MeOH (GdmSCN/MeOH (25%)) but becomes largest in the presence of high-concentrated MeOH (GdmSCN/MeOH (40%)). The change in Rg value in the 6177



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Figure 4. Distribution of the number of Gdm+ cations, SCN− anions, and water (MeOH) molecules within the first solvation shell of BdpA in (a) GdmSCN/MeOH (40%), (b) GdmSCN/MeOH (25%), (c) GdmSCN, and (d) MeOH solutions.

MeOH Mixture. The accumulation of MeOH and Gdm+ around protein is believed to play important roles in their respective effects on protein structure. To understand why the mixtures of GdmSCN and varied concentrations of MeOH exert opposite effects on protein structure, we evaluated the number of various solvent and cosolvent molecules in the first solvation shell (FSS) of protein (3.4 Å around protein surface) in the four solutions. The number distribution is represented in Figure 4, and the average number is listed in Table 1 (the number is averaged over three trajectories and the data from individual trajectories are used to calculate error bar). One can see that the number of either Gdm+ or SCN− ions has little change in three solutions containing GdmSCN. The ratio between the numbers of cosolvent (ions or molecules) and water within the FSS of protein which is then normalized according to the total numbers of cosolvent and water in the simulation system (Rcosolvent = (ncosolvent/nwater)surface/(ncosolvent/ nwater)total)) can be used to evaluate the accumulation level of cosolvent around protein: the cosolvent accumulates if the ratio is greater than 1; otherwise, it is disposed from protein surface. The value similarities of the high ratios of both Gdm+ and SCN− ions in solutions indicate that both ions accumulate around protein surface, and their accumulation is not influenced by the addition of MeOH (Table 1). On the other hand, MeOH also accumulates on protein surface, but its accumulation level is lowered in the presence of GdmSCN (see smaller value of RMeOH in GdmSCN/MeOH mixed solutions than that in single-component MeOH solution in Table 1). The time series of Gdm+ and MeOH numbers in the FSS of BdpA protein in GdmSCN/MeOH (40%) solution (Figure S5) indicates an increase in the number of Gdm+ cations from the very early stage of simulation along with a decrease in the

number of MeOH until both numbers reach equilibrium distribution. Therefore, the accumulation of Gdm+ is more competitive than that of MeOH. Accordingly, the preferred accumulation of Gdm+ on protein surface to some extent impairs the accumulation of MeOH. The increase in MeOH concentration in GdmSCN/MeOH mixed solution enlarges the number of MeOH appearing in the FSS of protein, but the accumulation level of MeOH compared to that in bulk region is only little changed. The increase in the fraction of Gdm+, SCN−, and MeOH in the FSS of protein relative to bulk in aqueous solutions can be explained from an energetic perspective.64,65 The electrostatic energy and van der Waals (VDW) interaction energy between each Gdm+/MeOH/water in proximity to protein (defined as the area within 5.0 Å of protein) and in bulk region (6.0 Å away from any protein atoms) with the rest of the system were calculated. A spherical cutoff of 13.0 Å was applied in VDW potential energy calculation, and no cutoff is applied for longrange electrostatic interactions. In all solutions under study (Figure 5 for GdmSCN/MeOH (40%), Figure S6 for GdmSCN/MeOH (25%), and Figure S7 for GdmSCN and MeOH solutions), each solvent (water) and cosolvent (Gdm+ or MeOH) molecule around protein has a distribution of electrostatic energy with a broader peak at lower energy range than that in the bulk. For instance, the difference in the average electrostatic energy between the regions of protein surface and the bulk in GdmSCN/MeOH (40%) solution is −16.80 kcal/ mol for Gdm+, −1.20 kcal/mol for MeOH, and −2.12 kcal/mol for water. On the other hand, in either single-component or mixed solution, the distribution of VDW energy for Gdm+ and water molecule in proximity to protein is centered at the same position as that in the bulk. In contrast, MeOH molecule in 6178



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Figure 6. Distribution of (a−c) electrostatic energy and (d−f) van der Waals energy between protein and each Gdm+/MeOH/water (in proximity to protein) for BdpA in GdmSCN/MeOH (40%) solution.

Figure 5. Distribution of (a−c) electrostatic energy and (d−f) van der Waals energy of each Gdm+/MeOH/water in proximity to protein and in the bulk region, respectively, with the rest of system for BdpA in GdmSCN/MeOH (40%) solution.

electrostatic interactions between protein and all solvent and cosolvent molecules, respectively. Tables 2 and 3 list the detailed electrostatic and VDW energies per Gdm+/MeOH/ water molecule to protein in all solutions under study and the decomposition into protein backbone and side chain parts, respectively. For each kind of solvent and cosolvent molecules, the energy (either electrostatic or VDW part) is more or less similar in all solutions, indicating the trivial effects of the mixture of guanidinium and MeOH on the interactions of individual solvent and cosolvent molecules toward protein. In addition, it is noteworthy that the electrostatic energy of Gdm+ is mainly focused on the backbone whereas the VDW energy of MeOH is largely on the side chain of protein. Although the accumulation level of MeOH on protein surface is impaired in GdmSCN/MeOH mixed solution, the interactions from individual MeOH molecules toward protein and their parking manner around protein are not changed. Previous studies showed that Gdm+ prefers to accumulate around residues possessing planar aromatic side chains or negative charges66−68 whereas MeOH prefers to bind to hydrophobic side chains.12,14,24 The average numbers of Gdm+, SCN−, and MeOH in the first and second (5.0 Å) solvation shells of individual residues in the two GdmSCN/MeOH mixed solutions were calculated and compared to the counterparts in single-component GdmSCN or MeOH solutions, respectively. As shown in Figure 7, the distribution of either Gdm+ or SCN− in first and second solvation shells is residue dependent, and the dependence is very similar in both GdmSCN and GdmSCN/MeOH solutions. For instance, Gdm+ always prefers to bind to the negatively charged side chains (e.g., Glu16, Glu17, Asp28, Asp29, Glu39, and Asp45) and the planar side chains (e.g., Phe5, Tyr6, Asn13, Gln18, and Asn35). Moreover, although the detailed values of MeOH numbers are different, the distribution of MeOH around individual residues is also in the same manner in MeOH and GdmSCN/MeOH solutions, to wit, MeOH prefers to stay around hydrophobic side chains (e.g., Phe5, Tyr6, Leu9, Phe22, Leu26, and so on). Linkage between Protein−MeOH VDW Interactions and Protein Tertiary Structure Stability in MeOHContaining Solutions. Solvent accessible surface area (SASA) of protein and its nonpolar and polar components

proximity to protein has a distribution of VDW energy with a broader peak at lower energy than that in the bulk. Consequently, it is the more favorable electrostatic interactions between Gdm+ and protein that drive Gdm+ cation to protein surface. Methanol molecule, on the other hand, is driven to protein by both more favorable electrostatic and VDW interactions. Interestingly, the average electrostatic energy per Gdm+ (either around protein or in the bulk) is more negative but the VDW energy is more positive in single-component GdmSCN than the counterparts in GdmSCN/MeOH mixed solutions. The energy difference between the regions of protein surface and bulk solution is, however, not changed for all solutions containing GdmSCN. In addition, the difference in VDW energy of methanol between the regions of protein surface and bulk solution in two GdmSCN/MeOH mixed solutions is smaller than that in single-component MeOH solution, implying that the former solutions have weaker driving force for MeOH to approaching protein. This could explain the lowered tendency of MeOH accumulation while it is mixed with GdmSCN. It is worth noting that water around protein surface also has more favorable electrostatic energy than that in the bulk region. Our earlier simulation study demonstrated that the addition of osmolytes rich in proton donors (e.g., Gdm+) in water solution could participate into the hydrogen bonding network among solvent and thus release free water hydrogen bonding donors, which facilitates the hydrogen bonding from water to protein.63 The heavy accumulation of Gdm+ in the region of protein surface further changes the equilibrium among water hydrogen bonding donor and acceptor and thus results in the favorable electrostatic interactions of water around protein surface. The favorable protein−solvent (cosolvent) interactions can be further indicated by the detailed decomposition of interaction energies of single Gdm+/MeOH/water molecule (around protein surface) with protein only. As shown in Figure 6, only the distribution of VDW energy per Gdm+ or water molecule to protein is centered at ∼0 kcal/mol. The peak positions of the VDW energy per MeOH molecule and the electrostatic energy per Gdm+/MeOH/water molecule to protein is more negative, indicating more favorable VDW interactions between protein and MeOH and more favorable 6179



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Table 2. Nonbonded Energy (Electrostatic and van der Waals (VDW) Energies) between Protein and Each Gdm+ (EPG), MeOH (EPM), or Water (EPW) in Proximity to Protein (5 Å) energy (kcal/mol) electrostatic energy

VDW energy

EPG EPM EPW EPG EPM EPW

GdmSCN/MeOH (40%)

GdmSCN/MeOH (25%)

GdmSCN

−77.35 −1.52 −2.39 −0.02 −1.23 −0.04

−76.00 −1.54 −2.47 −0.06 −1.24 −0.05

−76.17 −2.12 −0.05 −0.11

MeOH (25%) −2.01 −2.59 −1.32 −0.10

Table 3. Decompositions of Nonbonded Energy between Each Solvent (Cosolvent) and Backbone and Side Chaina energy (kcal/mol) electrostatic energy

VDW energy

a

EBG ESG EBM ESM EBW ESW EBG ESG EBM ESM EBW ESW

GdmSCN/MeOH (40%)

GdmSCN/MeOH (25%)

GdmSCN

−64.10 −13.25 −0.54 −0.98 −0.88 −1.51 −0.15 0.13 −0.48 −0.75 −0.03 −0.01

−64.70 −11.30 −0.45 −1.09 −0.84 −1.63 −0.16 0.10 −0.54 −0.70 −0.06 0.01

−65.93 −10.24

−0.71 −1.41 −0.11 0.04

−0.06 −0.05

MeOH (25%)

−0.71 −1.30 −0.87 −1.72

−0.50 −0.82 −0.06 −0.04

The energies for backbone and side chain are represented as EB? and ES?, respectively, where ? is G for Gdm+, M for MeOH, and W for water.

Figure 7. Total number of (a, b) Gdm+ cations, (c, d) SCN− anions, and (e, f) MeOH molecules in the first and second solvation shells of individual residues of BdpA in solutions under study.

shows the detailed process of protein structure expansion in the two solutions represented by the time series of nonpolar and polar SASAs. In GdmSCN/MeOH (40%) solution, the nonpolar interactions within protein are detached early (at ∼20 ns) whereas the polar interactions can be fully detached much later in the simulation (∼80 ns). Therefore, the destabilization of tertiary structure in high-concentrated GdmSCN/MeOH solution is the consequence of the impairment of intra-protein hydrophobic interactions. In contrast, it is the polar interactions that are destroyed first in GdmSCN solution, although they fluctuate between detached and reattached states in the remaining simulation. Therefore, one

for BdpA in GdmSCN/MeOH mixed solutions and GdmSCN solution were calculated to demonstrate the detailed effects of solvent and cosolvent molecules on intra-protein side chain− side chain interactions (the profile of MeOH solution has a similar feature as that of GdmSCN/MeOH (40%) solution and is not shown here). As shown in Figure 8b, single peak is presented in the distribution of SASA in GdmSCN/MeOH (25%) solution, corresponding to the compact native structure of protein. The peaks are shifted to larger SASA range in GdmSCN and particularly GdmSCN/MeOH (40%) solutions, indicating partial expansion of protein structure in the former and total expansion in the latter solution (Figure 8a,c). Figure 9 6180



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Figure 8. Distribution of solvent accessible surface area (SASA) of protein and its decomposition (nonpolar and polar parts) for BdpA in (a) GdmSCN/MeOH (40%), (b) GdmSCN/MeOH (25%), and (c) GdmSCN solutions.

constant accumulate on protein surface and have favorable VDW interactions with protein (particularly with protein side chains), which weaken intra-protein nonpolar interactions and lead to the full expansion of protein tertiary structure. In GdmSCN and MeOH mixed solution containing low concentration of MeOH, however, a small amount of MeOH molecules could reach the FSS of protein because of the expelling from Gdm+, and the accumulated VDW interactions are too small to break the hydrophobic core cluster of BdpA protein. Linkage between Protein−Solvent (Cosolvent) Electrostatic Interactions and Protein Secondary Structure Stability. As shown in Figure 6 and Tables 2 and 3, Gdm+, MeOH, and water have favorable electrostatic interactions toward protein. Using GdmSCN/MeOH (40%) solution as an example, we drew the time series of inter-residue electrostatic energy within protein as well as the total electrostatic energies between protein and solvent/cosolvent in proximity to protein to indiacte the correlation between these energies. As shown in Figure 10, along the simultion trajectory, the intraprotein electrostatic energy is increased gradually, accompanied by the decrease of protein−Gdm+ electrostatic energy. The protein− MeOH electrostatic energy is increased in the early stage because of the expelling by Gdm+ accumulation and reaches to the plateau in the remaining simulation. The value of protein− water electrostatic energy is in the middle among all protein− solvent (cosolvent) electrostatic energies, which keeps steady in the entire simulation. Of intra-protein electrostatic interactions, the backbone hydrogen bonding between carbonyl (−CO) and aminde (−NH) groups is the main structural element, and its stability is crucial for secondary structure. Not only Gdm+ cation but also

Figure 9. Time series of nonpolar and polar SASAs for BdpA in (a) GdmSCN/MeOH (40%) and (b) GdmSCN solutions, respectively. Dashed lines demonstrate the positions corresponding to the peaks in Figure 8, and the arrows indicate the starting point for the detachment of nonpolar or polar interactions within protein.

might speculate that the protein structure is expanded via different mechanisms in GdmSCN and GdmSCN/MeOH (40%) solutions. In the former solution, the structure expansion might be induced mainly by the electrostatic interactions from Gdm+ to side chain of protein (because of the weak VDW interactions between Gdm+ and protein, the protein structure could not be fully expanded like in the presence of high-concentrated MeOH). In the latter mixed solution, a great number of MeOH with lower dielectric 6181



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Figure 12. Correlation between the number of backbone hydrogen bonds and the number of protein−solvent (cosolvent) hydrogen bonds in the four solutions under study.

bonds has a general inverse relationship with the number of hydrogen bonds from solvent and cosolvent: the more protein−solvent/cosolvent hydrogen bonds are formed, the less the intra-protein hydrogen bonds are survived.

Figure 10. Time series of (a) intraprotein, (b) protein−Gdm+, (c) protein−MeOH, and (d) protein−water electrostatic energies for BdpA in GdmSCN/MeOH (40%) solution.

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DISCUSSION AND CONCLUSIONS As the representative monohydric alcohols, MeOH and TFE can accumulate around protein surface and destroy tertiary structure but stabilize secondary structure of protein. Guanidinium, as a denturant which is even more effective than urea, can also accumulate around protein and hydrogen bond to protein to denature both tertiary and particularly secondary structures. Given the strong denaturing abilities of alcohols toward tertiary structure and of guanidinium toward secondary structure, one may speculate that the mixture of alcohol and guanidinium could further denature protein to more extended denatured structure. To testify this speculation, we recently ran MD simulations on the stability of a 46-residue BdpA protein in the mixture of GdmSCN and TFE aqueous solution. The surpring observation is that the approach of Gdm+ cations to protein is impeded to a large extent by the preceding accumulation of TFE which leads to the maintenance of protein secondary structure in solution. On the other hand, unlike the complete structure expansion of BdpA in single TFE solution, the less heavy accumulation of TFE on protein surface in GdmSCN/TFE solution only partially expands the tertiary structure of protein. Therefore, the denaturing ability of GdmSCN and TFE toward protein is impaired when the two species are mixed. In the present study, we used the same molecular force field (AMBER FF99) to run MD simulations on BdpA protein in GdmSCN and MeOH mixed solutions. MeOH, which has lower accumulation tendency, was used instead of TFE with the hope to avoid expelling guanidinium from protein by the heavy accumulation of alcohol. It is observed in the present study that the mixture of GdmSCN with low-concentrated MeOH (e.g., 25% (v/v)) stabilizes native structure of BdpA but the further increase of MeOH concentration (e.g., 40% (v/v)) in the mixed solution denatures native structure of protein to unfolded structure, which is even more extended than those in single GdmSCN and MeOH solutions. The MeOH concentration dependent protein secondary structure stabilization has been also observed in previous experiments for single MeOH solution.17,19 For instance, Ozaki and co-workers observed in their circular dichroism (CD), fluorescence, and infrared (IR) spectroscopy experiment that the rmethuG-CSF protein maintains its tertiray and secondary structures in MeOH concentration range of 0 to 20% (v/v). However, when the

water molecule could work as strong hydrogen bond donors and/or acceptors, and their hydrogen bonding toward protein backbone could interrupt backbone hydrogen bonds and denature protein structure (see Figure 7 in ref 63). On the other hand, MeOH is also a weak hydrogen bonding donor, which could form hydrogen bond with the backbone carbonyl group (Figure 9 in ref 69 and Figure 9 in ref 70). An inverse relationship can be seen more clearly between the intraprotein hydrogen bonds and the hydrogen bonds from Gdm+, MeOH, and water to protein. As shown in the time series of those hydrogen bonds in the trajectories of BdpA in GdmSCN/MeOH (40%) solution (Figure 11), the breaking of

Figure 11. Breaking and formation of hydrogen bonds for residues involved in all backbone hydrogen bonds of BdpA in GdmSCN/ MeOH (40%). Black line: the number of backbone hydrogen bonds; red line: the number of hydrogen bonds formed between carbonyl groups and Gdm+ as hydrogen bonding donor; green line: the number of hydrogen bonds formed between carbonyl groups and MeOH as hydrogen bonding donor; blue line: the number of hydrogen bonds formed between carbonyl groups and water as hydrogen bonding donor.

backbone hydrogen bonds is certainly accompanied by the formation of hydrogen bonds between the backbone carbonyl groups and solvent and cosolvent (particularly water and Gdm+). Figure 12 shows the correlation between the averaged number of protein backbone hydrogen bonds and the averaged total number of protein−solvent/cosolvent hydrogen bonds in four solutions (all the hydrogen bond numbers are averaged over three trajectories). The number of intraprotein hydrogen 6182



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radius of gyration (Rg), and the total number of backbone hydrogen bonds formed inside of protein from individual trajectories of GdmSCN/MeOH (40%), GdmSCN/MeOH (25%), and MeOH solutions, time series of the number of native hydrophobic side chain−side chain contacts and the total number of backbone hydrogen bonds formed within BdpA in pure water and the comparison to those in GdmSCN/MeOH (25%) solution, time series of the number of Gdm+ and methanol in the first solvation shell of BdpA protein in GdmSCN/MeOH (40%) solution, and the distribution of electrostatic and van der Waals energies per Gdm+/MeOH/ water in proximity to protein and in the bulk region, respectively, with the rest of system for BdpA in GdmSCN/ MeOH (25%), GdmSCN, and MeOH solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

MeOH concentration is increase upon 30−70% (v/v), the amount of tertiary structure decreases significantly while the αhelix within the protein also decreases a little.17 Moreover, Babu and Douglas also found the same phenomenon for another protein system, myoglobin, in MeOH solution, although the detailed MeOH concentration ranges which exert different effects on protein secondary structure are different: 35−40% MeOH solution maintains native-like scondary structure of myoglobin, whereas 50% MeOH solution slightly decreases helical scondary structure.19 The detailed analyses indicate that both MeOH and guanidinium accumulate around protein surface in the mixed solution, consistent with our speculation. Different than preferential accumulation of TFE over Gdm+ in GdmSCN/ TFE mixed solution, it is Gdm+ that becomes more competitive in accumulation than MeOH does in GdmSCN/MeOH mixed solution. Thus, while the accumulation level of Gdm+ always keeps constant in solutions containing similarly concentrated GdmSCN, the accumulation level of MeOH in GdmSCN/ MeOH mixed solution is lowered in comparison to single MeOH solution. The energy calculation suggests that Gdm+ is driven to protein surface by favorable electrostatic interactions and MeOH is driven by both favorable electrostatic and VDW interactions. The heavy accumulation of Gdm+ around protein makes the nearby environment more polar, reducing the difference in VDW energy of MeOH between the regions of protein surface and bulk solution and thus weakens the driving force for MeOH to approaching protein. Although the accumulation level of MeOH is lowered by Gdm+, the interaction strength of each species with protein is only slightly affected by another. The tertiary structure stability of protein in GdmSCN/MeOH mixed solution should be determined by the accumulated strength of VDW interactions from MeOH (around protein) toward protein side chains. GdmSCN and MeOH mixed solution containing low concentration of MeOH has small amount of MeOH in the first solvation shell of protein because of the expelling from Gdm+, which exerts little influence on the tertiary structure of protein. The increase in MeOH concentration enlarges MeOH number around protein and consequently increases total favorable VDW interactions between nearby MeOH molecules and protein side chains, which weaken intraprotein nonpolar interactions and lead to the full expansion of protein tertiary structure. On the other hand, the strength of intraprotein electrostatic interactions is inversely correlated to the strength of combined electrostatic energies from solvent (water) and cosolvent (Gdm+ and MeOH), particularly in hydrogen bonding part. That is to say, while Gdm+ and MeOH accumulate on protein surface, both species as well as water molecules could directly hydrogen bond to protein and thus influence the stability of intraprotein hydrogen bonds. The mixture of GdmSCN and low-concentrated MeOH has limited hydrogen bonding resource from solution environment (mainly from water molecules) and as a result stabilizes the secondary structure of protein. The increase of MeOH concentration in GdmSCN/MeOH solution increases the hydrogen bonding from solution (mainly from MeOH) and thus destabilizes the secondary structure of protein.

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AUTHOR INFORMATION

Corresponding Author

*Tel +86-21-50806600-1304, e-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by research grants from the National Natural Science Foundation of China (Grants 21373258 and 21003003). The author acknowledges computer time and resources from Shanghai Supercomputer Center (SSC) and TianHe-1 supercomputer in Tianjin.

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S Supporting Information *

Parameters for all simulations under study; figures with time series of the root-mean-square deviation (RMSD) and the 6183



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