Exploring the Counteracting Mechanism of Trehalose on Urea Conferred Protein Denaturation: A Molecular Dynamics Simulation Study.

Article pubs.acs.org/JPCB

Exploring the Counteracting Mechanism of Trehalose on Urea Conferred Protein Denaturation: A Molecular Dynamics Simulation Study Subrata Paul and Sandip Paul* Department of Chemistry, Indian Institute of Technology, Guwahati, Assam, India-781039 ABSTRACT: To provide the underlying mechanism of the inhibiting effect of trehalose on the urea denatured protein, we perform classical molecular dynamics simulations of N-methylacetamide (NMA) in aqueous urea and/or trehalose solution. The site−site radial distribution functions and hydrogen bond properties indicate in binary urea solution the replacement of NMA−water hydrogen bonds by NMA−urea hydrogen bonds. On the other hand, in ternary urea and trehalose solution, trehalose does not replace the NMA−urea hydrogen bonds significantly; rather, it forms hydrogen bonds with the NMA molecule. The calculation of a preferential interaction parameter shows that, at the NMA surface, trehalose molecules are preferred and the preference for urea decreases slightly in ternary solution with respect to the binary solution. The exclusion of urea molecules in the ternary urea−NMA− trehalose system causes alleviation in van der Waals interaction energy between urea and NMA molecules. Our findings also reveal the following: (a) trehalose and urea induced second shell collapse of water structure, (b) a reduction in the mean trehalose cluster size in ternary solution, and (c) slowing down of translational motion of solution species in the presence of osmolytes. Implications of these results for the molecular explanations of the counteracting mechanism of trehalose on urea induced protein denaturation are discussed. hydration of protein is getting enhanced.9−12 Interestingly, computer simulation studies show evidence for both of the mechanisms operating together. This means that these two mechanisms are by no means mutually exclusive.15,16 On the other hand, apart from TMAO, sarcosine, etc., some polyols also efficiently protect the structure and function of proteins against extreme environmental conditions such as high or low temperature, desiccation, etc.17 Among these polyols, trehalose (a disaccharide composed of two α−(1− ↔ 1)−α linked D-glucose units, see Figure 1) has received special attention for its efficient bioprotecting ability against such extreme conditions.18−21 A certain number of plants, insects, and microorganisms such as yeasts and nematodes synthesize these sugar molecules to protect the biomolecule from the extreme environmental stresses and maintain the activity of the biomolecules.22,23 In addition, it is also widely used as an additive for long-term preservation of therapeutic proteins, foods, and cosmetics industries.24−26 In order to explain the effective bioprotecting ability of trehalose, mainly three mechanisms have been proposed for, namely, the water replacement mechanism, vitrification hypothesis, and water entrapment mechanism. According to the first one, trehalose molecules form direct hydrogen bonds with protein or lipid

I. INTRODUCTION It is a well-known fact that aqueous solutions of some osmolytes affect the equilibrium protein unfolding/folding reaction, unfolded (U) ⇌ native (N). The protein structure breaker osmolyte such as urea shifts the above equilibrium toward the U state, whereas some structure protectant osmolytes such as trimethylamine-N-oxide (TMAO), sarcosine, etc.,1,2 stabilize the native state of protein. Note that the above interconversion between the protein N-state and U-state does not involve the breaking or formation of any covalent bonds. Therefore, the protein unfolding/folding reaction is different from normal chemical reactions and represents a reequilibration between U- and N-state populations under the influence of different chemical environments. Thus, in order to have proper elucidation of the above equilibrium process, it is important to consider the interactions between the protein unfolded and native state with the solution species.3,4 Though vastly studied,5−14 the mechanism by which urea shows its deleterious effects on protein conformation is little far from conclusive. In regard to urea-conferred denaturation of protein, two mechanisms, namely, the direct and indirect mechanisms, have been proposed. According to the direct mechanism, urea molecules interact directly by forming hydrogen bonds with the peptide backbone or side chains and stabilize the unfolded state of the protein.5−8 In the second possibility, i.e., in the indirect mechanism, urea molecules act indirectly by altering the water structure and thereby the © 2015 American Chemical Society

Received: February 16, 2015 Revised: June 26, 2015 Published: June 26, 2015 9820

DOI: 10.1021/acs.jpcb.5b01576 J. Phys. Chem. B 2015, 119, 9820−9834

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The Journal of Physical Chemistry B

ternary mixed trehalose/urea solution trehalose induced protection of biomolecules comes from trehalose induced enhancement in the water structure as well as preferential exclusion of trehalose molecules from the protein surface (along with this expulsion of some urea molecules due to trehalose−urea hydrogen bond interactions). It is to be noted that, though the results of both of these studies are in general consistent with each other, they lack in exploring other mechanisms such as trehalose induced slowing down of dynamical properties of solution species and proper quantitative estimation of the average number of hydrogen bonds between protein and water molecules. The information about the latter, i.e., protein−water hydrogen bonds along with protein−trehalose and protein−urea hydrogen bonds, provides meaningful information that could have directed us to examine if the water replacement hypothesis has any role in trehalose induced protection of biomolecules. Thus, lack of this information makes these studies incomplete and somewhat unidirectional. From the above discussions, it is clear that the underlying mechanism of stabilization of protein native conformation (by trehalose) against the deleterious effect of urea is largely unexplored in terms of the number of studies as well as the information that we gain from the above-mentioned two studies. In view of this, it is necessary to investigate in depth the molecular level understanding of the effect of trehalose molecules on the denaturing effect of urea on protein taking into account considerations of all possible mechanisms. This is the main objective of the present study. By considering the fact that the interactions between protein, water, trehalose, and urea are very complex in nature, in this study, we consider dilute aqueous solutions N-methylacetamide (NMA, see Figure 1) both in presence and absence of the osmolytes urea and trehalose. NMA is the smallest molecule having both a hydrophobic methyl group as well as a peptide linkage. In the context of protein stability, it is a well-known fact that the hydrophobic interactions present in the protein interior as well as hydrogen bonding interactions involving the protein backbone mostly contribute to it. The use of NMA instead of a “real” protein in our study gives us advantages (many folds) as well as disadvantages. The main advantages are the following: (i) the complexity in terms of interactions between solute NMA and different solution species is greatly reduced; (ii) by considering dilute NMA solutions, we essentially mimic a typical solvent exposed state of a real protein in which the solute−solvent interactions (mainly through hydrogen bonds) can be explored in depth. Moreover, the contributions of NMA−NMA hydrogen bonding interactions become very small. We further note that, in determining the extent of protein stabilization and destabilization, the transfer free energy measurement shows that the protein backbone plays a more important role than the side chains. Consequently, it was reported that the osmolyte effect operates predominately on the protein backbone.39−41 Among the disadvantages, the main disadvantage of use of NMA solution (instead of a real protein) is that it may not necessarily represent the “actual” system. However, several studies have successfully mimicked the interactions between the protein backbone and the solution species by using NMA peptide linkage−solvent interactions.42−44 These studies encouraged us to use NMA molecules as solutes for our study. We use classical molecular dynamics (MD) simulations of aqueous NMA solutions in which the molar ratio of trehalose and urea is 1:8. Our main focus is to

Figure 1. Trehalose (top) and NMA (bottom) molecules. For trehalose, all carbon, oxygen, and hydroxyl hydrogen are labeled. The other hydrogen atoms are left off for clarity. For NMA, Me1 and Me2 represent the methyl groups attached to carbonyl carbon and amide nitrogen atoms, respectively.

head groups and replace water molecules from their hydration shells.27 The vitrification hypothesis says that, in aqueous trehalose solution, trehalose and water molecules form a highly viscous glassy matrix to keep the biomolecule fixed and it is the glassy matrix which capsulates the biomolecules. As a result, the biomolecule remains in its native conformation in the capsule.28 The third mechanism, i.e., the water entrapment hypothesis, proposes that water molecules are trapped in the intermediate layer between trehalose and the biomolecule and this helps to concentrate the residual water molecules around the biomolecule.29 Note that, though the antidoting effects of trehalose on extreme environmental conditions are vastly explored without any conclusive generally accepted answer, unlike TMAO (which protects biomolecules from the deleterious effect of urea),2,30−33 trehalose’s effectiveness counteracting against the urea-conferred protein denaturation has not been studied in great detail. Only recently, a few attempts have been made to understand the mechanism of the counteracting effect of trehalose on urea induced denaturation of protein.34,35 These studies revealed trehalose attenuation of the effect of chemical denaturant urea, albeit in two different molar ratios of trehalose and urea. For example, the differential scanning calorimeter (DSC) and circular dichroism (DC) study34 observed trehalose’s effectiveness against the effect of urea at 1:2 molar ratio and claimed that 1 M trehalose cannot protect biomolecules from urea-conferred denaturation of αchymotrypsin (CT) of urea concentration 3−6 M. On the other hand, a molecular dynamics (MD) simulation study35 reported that trehalose’s effectiveness exists even in a 1:8 molar ratio of trehalose and urea and argued (by citing previously reported comparative studies in theoretical and experimental works36−38) that the molar ratio of trehalose and urea may not necessarily be the same for theory and experiment. Nevertheless, both of these studies demonstrated the direct interaction between protein and urea in binary aqueous urea solution and supported the direct interaction mechanism of urea-conferred protein denaturation. This means that the denaturant urea accumulates at the protein surface and binds with the active site of the protein. They further argued that in 9821


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one atmospheric pressure. The Berendsen barostat was used with a pressure relaxation time of 2 ps to maintain the physical pressure of our systems.55 Further, we have applied periodic boundary conditions in all three directions and the temperature was controlled by the Langevin dynamics method with a collision frequency of 1 ps−1. A typical cutoff radius of 10 Å was applied for all nonbonding interactions, and a 2 fs time step was used for all simulations. The particle mesh Ewald (PME) method was used to treat the long-range electrostatic interactions. The bonds involving a hydrogen atom were constrained by applying the SHAKE algorithm.56 To calculate the diffusion coefficients of different solution species, the simulations were further continued for another 15 ns simulation run in the microcanonical ensemble (NVE). Finally, to analyze the trajectories obtained from production runs, we have used the ptraj program of the AMBER12 toolkit and the Visual Molecular Dynamics (VMD) package.57

explore the structural properties involving NMA and different solution species, viz., water, urea, and trehalose and the role of structure protectant osmolyte trehalose on NMA−water and NMA−urea interactions. Further, in order to explore the counteracting effect of trehalose on urea, we calculate the hydrogen bond properties and translational motions of NMA, water, urea, and trehalose and examine what these suggest about the bioprotecting activity of trehalose. The remainder of this paper is organized into three parts. The models and simulation details are briefly described in section II, the results are presented and discussed in section III, and our conclusions are summarized in section IV.

II. MODELS AND SIMULATION METHOD In order to understand the mechanism of protein conformation protection by trehalose against urea denaturation, we have performed classical MD simulation of NMA in pure water as well as binary and ternary solutions of urea and trehalose. The four different systems considered here are presented in Table 1.

III. RESULTS AND DISCUSSION A. Hydrophobic Interaction. Each NMA molecule possesses two hydrophobic methyl groups. The methyl group attached to the carbonyl carbon of NMA is denoted as Me1, and for the second methyl group which is attached to the amide group, we denote it as Me2. These two methyl groups may contribute to hydrophobic association of NMA molecules in solutions. Thus, in order to have a better understanding of the effect of the presence of urea and trehalose on the modification of hydrophobic association NMA molecules, following earlier works,58,59 we have computed the equilibrium constant, Keq, as

Table 1. N, M, and wT% Represent Number of Molecule, Molar Concentration, and Weight Percentage, Respectivelya system

NN

NU

NT

NW

volume (nm3)

MU

MT

wT%

NW NU NT NUT

20 20 20 20

0 250 0 250

0 0 35 35

1000 950 950 800

32.15 48.25 43.22 56.04

0 8.60 0 7.41

0 0 1.34 1.04

0 0 41 45

a

Subscripts N, U, T, W are for NMA, urea, trehalose, and water, respectively.

r2

Keq

The PACKMOL program was used to prepare the initial configurations of our systems.45 For the trehalose molecule, we have considered the GLYCAM06 force field,46 which is widely used for the simulations of monosaccharides and oligosaccharides. Comparative studies between different disaccharide force fields reveal that the results obtained using the GLYCAM06 force field are in excellent agreement with the DFT and experimental results.47,48 In our study, we have adopted the popular SPC/E49 model for water. This is because the SPC/E water model produces better dynamical properties and structure (over the SPC and TIP3P water model) that match well with the experimental results.50 In regard to the force field compatibility of GLYCAM06 and SPC/E, it is worth noting that Verde et al.,51 in a recent study, have used the GLYCAM06 force field for trehalose and kojiboise combined with SPC/E water. For NMA, we have adopted the all atom force field where methyl hydrogens are considered explicitly.52 Urea has been considered according to Smith model.53 All the solution properties were investigated by performing MD simulations in a cubic box using the AMBER1254 suite of programs at 300 K. The reasonable initial structures for the production run of our systems were obtained by energy minimization for the 5000 steps, where the first 2500 steps used the steepest descent method followed by the same number of steps in the conjugate gradient method. After that, the systems were heated up slowly from 0 to 300 K for 100 ps in the canonical ensemble (NVT) and then the systems were equilibrated in the isothermal−isobaric (NPT) ensemble for 2 ns. For calculating different structural and hydrogen bond properties, we have conducted the 40 ns production runs for all the systems in the NPT ensemble and

∫r r 2g (r ) dr [SSS] = = 1r1 2 [CS] ∫0 r g (r ) dr

(1)

where g(r) represents the site−site radial distribution function (rdf) involving methyl groups of NMA (not shown) and r1 and r2 are the positions of the first and second minima in the corresponding rdf profile. [SSS] and [CS] correspond to the concentration of the solvent-separated state and the contact state, respectively. Note that since each NMA has two methyl groups we get three equilibrium constant values (due to interactions between Me1−Me1, Me1−Me2, and Me2−Me2) for each system and these are shown in Table 2. As is evident, the Table 2. Equilibrium Constant (Keq) for the Methyl Groups of NMA in Different Systems system

Keq(Me1−Me1)

Keq(Me1−Me2)

Keq(Me1−Me2)

NW NU NT NUT

2.30 3.74 1.96 3.19

3.36 4.06 3.76 2.99

3.45 3.54 3.24 3.38

higher the value of Keq, the lower will be the propensity of hydrophobic association. Thus, any perturbation that decreases the value of Keq favors the associated state of solute NMA. From Table 2, we find that in the pure water system (system NW), in comparison to Me2−Me2, the value of Keq is much lower for Me1−Me1. The Keq values for Me1−Me2 fall in the intermediate regions. This implies that in pure water NMA molecules are preferred to be associated through the methyl group attached to carbonyl carbon. The effect of urea and trehalose alone (i.e., systems NU and NT) on the Keq value is 9822


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behavior of hydrophobic hydration.60,61 Specifically, for the pure water system, the rdf of Me1 and water oxygen (and hydrogen), Ow (and Hw) (Figure 2a), we find that the Me1−Ow rdf starts to rise at 2.75 Å and reaches the bulk density at 3.65 Å, for which g(r) = 1. Thus, below 3.65 Å, water molecules are actually excluded from the solvation shell of the methyl group. The first minimum of this rdf, which appears at 5.45 Å, is considered as the outer limit of the first hydration shell of the Me1 group. We also find that the Me2−Ow rdf (Figure 2b) shows similar hydration characteristics as that of the Me1−Ow rdf. However, the water distribution around the former is slightly better defined as compared to that of the latter, as revealed by an enhancement in the first peak height and the inward movement of the first minimum in the Me2−Ow distribution function. We note that, for these two methyl groups, the water distribution is less narrow when compared to that of methane.60 Furthermore, it is also observed that the water structure around these hydrophobic regions is perturbed due to the presence of other groups.62,63 For example, it has been reported by Pratt and Chandler that the solvent distribution around a methyl group in ethane is somewhat less structured than that of methane.62 Similarly, the study of Trzesniak et al. also revealed that the water peaks are narrower and sharper for spherical molecules (e.g., methane, neopentane, etc.) than those of nonspherical molecules.63 In addition, the appearance of the first peak at similar locations in Me1 (and Me2)−water oxygen and Me1 (and Me2)−water hydrogen rdf’s implies the surface parallel orientation of water molecules near the hydrophobic methyl groups and the appearance of the tail at a shorter distance for water hydrogen reflects a closer approach of it toward these methyl groups. As shown in Figure 2, we also noticed that the addition of urea has very little effect on these rdf’s. On the other hand, the presence of trehalose molecules in solution causes an enhancement in the first peak height, suggesting an improved hydration of NMA methyl groups. A good measurement of hydrogen bonding interactions between NMA and water, albeit indirectly, can be obtained from the site−site distribution functions involving the hydrophilic carbonyl oxygen and amide hydrogen of NMA with water oxygen and hydrogen atoms. The same are displayed in Figure 3. Note that the quantitative measurements of the average number of these hydrogen bonds are also discussed below. Now considering the rdf’s between water hydrogen and water oxygen with NMA oxygen in the pure water system first (see Figure 3a and b), we find the appearance of the first contact peak in rdf profiles at about 1.75 and 2.75 Å, respectively, suggesting CO···HwOw hydrogen bonding interaction.64 The first minimum at about 3.35 Å in the O−Ow rdf indicates the outer limit of the first shell and it decreases to a little more than half of the bulk water density, which indicates a strong radial water structuring around the carbonyl oxygen of NMA. The rdf involving NMA hydrogen−water oxygen (H−Ow) (Figure 3c) has a characteristic hydrogen bonding peak at 2.05 Å, and the weakness of the first peak height suggests the relatively weaker hydration of the amide hydrogen atom by water molecules. Now, focusing on the effect of the osmolytes urea and trehalose individually (systems NU and NUT) on these distribution functions, it is observed that, in comparison to the pure water system, both of them enhance the first peak height of O−Ow and O−Hw rdf’s and make the first valley shallower. In the mixed urea/trehalose system (system NUT), the first peak height is further getting enhanced and the first valley becomes

also quite apparent. Specifically, in comparison to the pure water system, the presence of urea alone increases the values of Keq and the effect is more prominent for Me1−Me1 and Me1− Me2, suggesting the solvent-separated state is favored. On the other hand, for the binary system containing trehalose, the values of Keq decrease sharply for Me1−Me1, whereas, for Me2− Me2 and Me1−Me2, we observe a modest change. These suggest that the presence of trehalose favors the Me1−Me1 contact state of the solute. For the urea/trehalose ternary system, the increased value of Keq for Me1−Me1 (in comparison to the pure water system) indicates the disruption of Me1−Me1 contacts. However, when compared to the binary urea system, the depletion in the value of Keq directs us to suggest trehalose induced enhancement in the hydrophobic interactions between Me1−Me1 contacts. These findings further act as corroborative evidence of trehalose induced reduction in the NMA−urea interactions in the ternary mixed urea/trehalose system (discussed below). These observations further suggest that the effect of urea and trehalose is pronounced for Me1−Me1 contacts followed by Me1−Me2 contacts. Me2−Me2 contact has slight awareness of the presence of urea and trehalose. B. Interaction of NMA with the Solution Species. In order to characterize the solvation of solute NMA molecules in the presence and absence of the osmolytes urea and trehalose, it is important to investigate the interactions of different atomic sites of NMA with different solution species. Since the different site−site radial distribution functions involving different atomic sites of solute and solvent molecules provide very good qualitative information about the interactions between them, in this section, we concentrate on the selected rdf’s involving different atomic sites of NMA and solution molecules. The same are presented in Figures 2−5. Considering the rdf’s between different hydrophobic atomic sites of NMA and water molecules first (see Figure 2), we notice that both of the methyl groups of NMA show the typical

Figure 2. Solid lines represent the site−site distribution function between NMA methyl groups and the water oxygen atom. Parts a and b represent Me1−Ow and Me2−Ow distribution functions, respectively. Dashed lines are for NMA methyl−water hydrogen rdf’s for system NW. 9823


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Figure 4. Site−site radial distribution functions between NMA and urea in aqueous urea (solid) and in mixed urea/trehalose (dashed) solutions. Black, red, and green lines are for carbon (Cu), oxygen (Ou), and nitrogen (Nu) of urea.

Figure 3. Site−site radial distribution functions involving oxygen and amide hydrogen of NMA with water.

The information about the interactions between different atomic sites of NMA and trehalose molecules is probed by means of selected site−site distribution functions, and these are displayed in Figure 5. Since both NMA and trehalose molecules possess groups that participate in hydrogen bonding interactions, in this section, we discuss the rdf’s involving different trehalose oxygen and the carbonyl oxygen and amide hydrogen of NMA for the systems NT and NUT. Note that, since the rdf’s of trehalose hydroxyl oxygen atoms O2, O3, and O4 with the amide group of NMA behave in a similar fashion as that of O6 atom, the rdf’s involving O6, O1, and O5 oxygen atoms of trehalose are considered in Figure 5. The presence of much stronger first peaks of O−O6 and H−O6 rdf’s (see Figure 5a and d) at 2.75 and 1.95 Å suggests that, in the hydrogen bonding interaction between NMA and trehalose molecules, it is the hydroxyl O6 (and O2, O3, and O4) oxygen atom which actively participates. The appearance of peaks with high magnitudes, further, demonstrates that trehalose molecules can interact directly with the NMA molecules through its hydroxyl oxygen atoms. The small peak height of distribution functions involving the ring oxygen atom (O5) (as well as the glycosidic oxygen atom, O1) of trehalose and the amide group of NMA (Figure 5b, c, e, and f) implies much weaker interactions between these atomic sites. Now, for the aqueous urea/trehalose mixture, as per the expectation, we find that the NMA−trehalose hydrogen bonding interaction decreases in the presence of urea. Moreover, from the peak heights of NMA oxygen−trehalose oxygen and NMA hydrogen−trehalose oxygen distribution functions, it is apparent that, in NMA− trehalose hydrogen bonding interactions, the NMA molecule is preferably acting as a donor rather than an acceptor. This observation is further supported by the hydrogen bond property calculations (discussed later).

more shallow. On the other hand, in the presence of urea, there is a modest depletion in the first peak height of the H−Ow pair correlation function, whereas this rdf shows only a slight awareness of the presence of trehalose. Figure 4 demonstrates the interactions of NMA atomic sites with the urea atomic sites. Considering the rdf’s involving the atomic sites of urea and the two methyl groups (Me1 and Me2), we observed that both of the methyl groups exhibit a similar tendency toward the urea molecule. Moreover, the orientation of urea molecules in the vicinity of these hydrophobic methyl groups of NMA is similar to that observed in the case of methane and neopentane.60,61 Further, the very similar locations of the rdf’s of urea oxygen (Ou)−NMA methyl and urea nitrogen (Nu)−NMA methyl imply no preference of these atomic sites of urea near the methyl groups of NMA. These locations and the magnitudes of the first peaks in the corresponding rdf’s suggest the direct interaction of urea with NMA methyl groups. Moreover, in comparison to urea carbon, a slightly closer approach of these two atomic sites of urea toward the methyl groups of NMA is also quite visible. As expected, the distribution of urea molecules around the carbonyl oxygen and amide hydrogen (see Figure 4) indicates the presence of hydrogen bonding interactions between NMA and urea molecules. For carbonyl oxygen, the position of the first peak of the Nu rdf is much closer than Cu and Ou atoms, suggesting the preference of urea nitrogen near the NMA oxygen. Additionally, amide hydrogen shows a preference toward the urea oxygen, indicating the latter accepts the amide hydrogen of NMA in the NMA−urea hydrogen bonding interaction. Interestingly, all of these rdf’s reveal a modest depletion in the urea density near NMA atomic sites upon addition of trehalose. 9824


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Figure 6. (a) Total number of water (solid), urea (dotted), and trehalose (dashed) molecules in the solvation shell of NMA as a function of distance. (b) Total number of solution species in the solvation shell of NMA as a function of distance.

addition of trehalose and urea causes exclusion of water molecules from the NMA solvation shell, and the effect is much more pronounced for the former than the latter. Specifically, for system NT, the presence of two trehalose molecules in the solvation shell causes an exclusion of about 24 water molecules. On the other hand, addition of urea alone to the pure water system (system NU) displaces 34 water molecules (by 17 urea molecules) from the NMA solvation shell. These suggest the displacement of 12 and 2 water molecules, respectively, with the incorporation of one trehalose and one urea molecule in the NMA solvation shell. Due to the presence of a greater number of urea molecules, the total number of excluded water molecules is much higher for the NU system than the NT system (34 against 12). As a result of exclusion of a higher number of water molecules for system NU, the total number of solution species in the solvation shell decreases more for this system (see Figure 6b). These observations are further confirmed from the atomic density analysis discussed below. From the above discussions, it is quite evident that, in the NMA solvation shell, water molecules are replaced by osmolyte urea and trehalose molecules. In view of this, it would be interesting to carry out atomic density analysis of different solution species around NMA. Using VMD,57 the mass density maps of water, urea, and trehalose molecules with a cell side of 0.5 Å, within 3.5 Å, of the NMA molecule are calculated at different time intervals and these are shown in Figure 7. The presence of a large water density around NMA in the pure water system and removal of water molecules on addition of urea and trehalose (as reflected in the depletion of water density) are also quite visible. Further, consistent with the total number of excluded water molecules from the solvation shell of NMA (see above), the reduction in the water density around NMA is relatively higher for the NU system than that for the NT system. Further, on incorporation of trehalose in the NU system (system NUT), in addition to depletion in water density, remarkably, some urea molecules are also replaced,

Figure 5. Site−site radial distribution functions involving oxygen atoms of trehalose with amide oxygen (left panel) and hydrogen (right panel) of NMA for systems NT and NUT.

More insights into the solvation of NMA molecules by water as well as the osmolyte urea and trehalose molecules can be obtained by estimating the number of different solution species, viz., water, urea, and trehalose (central atom only), in the NMA solvation shell. The average number of molecules present around different atomic sites of NMA is calculated from the corresponding site−site distribution function, gαβ(r), by using the relation RCN = 4πρβ

∫0

rc

r 2gαβ (r ) dr

(2)

where RCN represents the running coordination number of atoms of type β in the solvation shell of atom type α in which the solvation shell is extended up to distance rc and ρβ is the number density of atom type β in the system. From the estimations of the above coordination number values, the total number of different solution species that are present in the NMA solvation shell of radius 6 Å is calculated as a function of distance. In Figure 6, we show the change in RCN value for different solution species as a function of distance. Since the size of a water molecule is significantly smaller than the osmolytes urea and trehalose, a much larger radius of exclusion is also observed for these osmolytes. As a result, from the purely geometric point of view, the exclusion of urea and trehalose molecules is observed from a certain volume shell of NMA. Nevertheless, the presence of a considerable amount of osmolyte urea and trehalose molecules in the NMA solvation is quite evident and the abundance of the former is relatively higher than the latter (Figure 6a). As is evident, the number of water molecules in the solvation shell is much higher in the pure water system than in aqueous osmolyte solutions. The 9825


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Figure 7. Contours of water oxygen, urea oxygen, and trehalose hydroxyl O6 density within 3.5 Å around NMA. The systems NW, NT, NU, and NUT are from top to bottom, respectively. From left to right are for 0, 10, 20, 30, and 40 ns time intervals.

albeit, in a small amount, from the solvation shell. These findings are in accordance with the calculations of the average number of different solute NMA−solution species hydrogen bonds (see below). In order to have deeper insights into the interactions of NMA molecules with the solvent molecules, by considering preferential interaction parameters, we have also examined the local environment of NMA and compared it with that of the bulk solution. Following previous works,35,65 the time-averaged normalized ratios of water (gow(r)), trehalose (got(r)), and urea (gcu (r)) are calculated. These parameters provide the information about the relative local distribution of these solution species at a distance r from the NMA molecule, and they can be expressed as gow (r ) =

now (r ) × (Now + Not + Ncu) Now × (now (r ) + not(r ) + ncu(r ))

(3)

got (r ) =

not(r ) × (Now + Not + Ncu) Not × (now (r ) + not(r ) + ncu(r ))

(4)

gcu(r ) =

ncu(r ) × (Now + Not + Ncu) Ncu × (now (r ) + not(r ) + ncu(r ))

(5)

where now(r), ncu(r), and not(r) are the number of water oxygen, urea carbon, and trehalose hydroxyl oxygens, respectively, in the local domain of radius r from the NMA center of mass and Now, Ncu, and Not represent the total numbers of water oxygen atom, urea carbon atom, and trehalose hydroxyl oxygen atoms in the simulation box, respectively. In the close proximity of NMA, if the ratio gow(r) is greater than 1, the NMA is preferentially hydrated by water molecules; conversely, water is preferentially excluded from the NMA surface if the value of gow(r) is lower than 1. In Figure 8, we have shown the normalized ratio of water oxygen, urea carbon, and trehalose hydroxyl oxygens as a function of distance from the NMA center of mass. Focusing on the evolution of gow(r) as the distance changes, we notice that in the vicinity of NMA the value of gow(r) is greater than 1 for all systems considered. Further, the effects of trehalose and urea alone (systems NT and NU) on gow(r) are very much comparable to each other and the values for both of these systems are slightly above 1. 9826


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urea induced denaturation of protein chymotrypsin inhibitor 2 (CI2)35 except for the fact that in our study we find the presence of favorable NMA−trehalose interactions in the NMA solvation shell. However, the findings of ref 35 revealed the exclusion of trehalose molecules from CI2 and argued that in the counteracting effect of trehalose molecules (on urea induced protein denaturation) the preferential hydration (of trehalose) mechanism is operative. C. Hydrogen Bond Properties. In order to have quantitative estimation of interactions between NMA and different solution species through hydrogen bonds, in this section, by estimating the average number of hydrogen bonds formed by a NMA molecule with water, urea, and trehalose, we characterize the solvation pattern of NMA in different solutions. Following earlier works,66,67 by adopting geometric criteria, we consider two molecules are hydrogen bonded if the distance between the donor (D) and acceptor (A) atom is less than or equal to 3.4 Å and, simultaneously, the angle D···H···A is greater than or equal to 120°. Since all solution species including NMA can act as a donor as well as an acceptor, we further decompose each of the types of hydrogen bonds into donor and acceptor contributions. Note that we abbreviate different types of hydrogen bonds as Ix−Jy, where I and J can be N (for NMA), W (for water), U (for urea), and T (for trehalose). x and y can be a (for acceptor) and d (for donor). Further note that the average number of hydrogen bonds is presented with respect to the first species. For example, Na−Wd represents the average number of NMA−water hydrogen bonds per NMA where water acts as a donor and NMA acts as an acceptor and so on. The average number of hydrogen bonds formed between NMA and different solution species is shown in Table 3. Consistent with Figures 3 and 5, we find that, in the formation of NMA−water and NMA−trehalose hydrogen bonds, NMA acts preferably as an acceptor where its carbonyl oxygen accepts hydrogen of water and trehalose hydroxyl groups. For example, in pure water (system NW), each NMA molecule forms 2.46 hydrogen bonds with water in which, on average, it acts as an acceptor and a donor for 1.8 and 0.66 hydrogen bonds, respectively. Thus, in the formation of the NMA−water hydrogen bond, the carbonyl oxygen is solely dominating the hydrogen bond interaction. Addition of urea to the pure water system reduces both of these numbers. As a result, the average number of NMA−water hydrogen bonds decreases. We further observe the formation of a considerable number of NMA−urea hydrogen bonds at the expense of NMA−water hydrogen bonds. In contrast to the pure water system where the carbonyl oxygen of NMA is accepting two hydrogen atoms of water molecules, for system NU, remarkably, the carbonyl oxygen of each NMA is shared almost equally by one water and one urea molecule. Further, we noticed that the total number of

Figure 8. Time-averaged normalized fractions of (a) water, (b) trehalose, and (c) urea as a function of distance from the NMA center of mass.

This suggests a modest preference for water molecules of NMA molecules in binary water/osmolyte systems. Interestingly, in the mixed trehalose/urea ternary system, the value of gow(r) increases sharply, implying that NMA molecules prefer to be hydrated by water molecules. As revealed by the magnitude got(r), the presence of urea molecules in the ternary system increases NMA’s preference for trehalose in its solvation shell. On the other hand, the preferential interaction parameter, gcu(r), decreases slightly for the mixed osmolyte urea/trehalose system (when compared to that of the water/urea binary system) and its value becomes lower than 1 at short-r distance. Thus, from the above observations, we make the following conclusions: In mixed osmolyte systems, some of the urea molecules are expelled from the NMA solvation layer and NMA interacts preferentially with trehalose and water. As a result of this, the direct interactions between NMA and urea molecules are negated by trehalose molecules and NMA molecules are preferentially hydrated. We note that these findings are in general accordance with the results of a recent MD simulation study of the counteracting effect of trehalose on

Table 3. Average Number of Hydrogen Bonds (with Respect to the First Species) between NMA−Water, NMA−Urea, and NMA−Trehalosea system

Na−Wd

Nd−Wa

totalNW

NW NU NT NUT

1.80 1.34 1.47 1.06

0.66 0.47 0.55 0.39

2.46 1.81 2.02 1.45

Na−Td

0.31 0.21

Nd−Ta

0.17 0.11

totalNT

0.48 0.32

Na−Ud

Nd−Ua

totalNU

0.72

0.35

1.07

0.63

0.32

0.95

total 2.46 2.88 2.50 2.77

a

Different hydrogen bond types are defined in the text. TotalNW, totalNT, and totalNU correspond to the total number of NMA−water, NMA− trehalose, and NMA−urea hydrogen bonds per NMA molecule, and total is the sum of the totalNW, totalNT, and totalNU. 9827


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interactions help to accumulate urea molecules in the first solvation shell of the solute. The electrostatic interactions between the solute and solution species play only a minor role.13,72,73 These observations are further supported by another MD simulation study of the effect of trehalose on the protein−urea system, which revealed that Lennard-Jones interactions rather than electrostatic interactions between urea and protein are responsible for urea induced protein denaturation.35 Contrary to these, Das and Mukhopadhyay10 argued that the favorable electrostatic interactions between urea and protein cause protein denaturation. Thus, following the recent analysis of Zhang et al.,35 we have computed direct electrostatic and van der Waals interactions separately between urea and NMA for systems NU and NUT. We consider the interaction energies involving all urea and NMA molecules present in a given system. These energies are plotted as a function of simulation time (see Figure 9). As is evident from

hydrogen bonds formed by each NMA molecule with the solution species is 2.88 (NMA−water plus NMA−urea), whereas this number is 2.46 in pure water. Thus, urea removes the water molecules from the protein solvation shell by making some favorable contact with protein residues, which leads to protein denaturation. In the context of urea conferred protein denaturation, these findings support the direct mechanism.5,7,13,68 In the binary trehalose solution (system NT), the average number of NMA−water hydrogen bonds also decreases from the pure water system. However, this loss is very well compensated by the formation of an equal number of NMA−trehalose hydrogen bonds. For example, the number of Na−Wd per NMA reduces from 1.80 in pure water to 1.47 in binary trehalose solution and there is formation of 0.31 Na−Td hydrogen bonds. It suggests that trehalose is capable of replacing the NMA−water hydrogen bonds by an equal number of NMA−trehalose bonds and this makes the total number of hydrogen bonds formed by a single NMA molecule in the binary trehalose exactly similar to the pure water system. Now, it would be interesting to observe the influence of trehalose on the NMA−water and NMA−urea average number of hydrogen bonds (system NUT). As per the expectation, there is more reduction in the NMA−water hydrogen bond in the mixed urea/trehalose osmolyte system than that of binary solutions. A considerable amount of presence of NMA− trehalose hydrogen bonds is also observed, though this number is much lower than NMA−urea hydrogen bonds (0.32 against 0.95). Further, in the context of NMA−urea hydrogen bonds, we observe that, though there is a modest decrease in the NMA−urea hydrogen bond number, what is interesting is that the presence of trehalose does not reduce it significantly. These suggest that, in the event of trehalose induced protection of urea conferred protein denaturation, trehalose does not prevent formation of protein−urea hydrogen bonds completely. Rather, the replacement of protein−water hydrogen bonds by protein− trehalose hydrogen bonds plays a major role in the trehalose induced protection of protein from the deleterious effect of urea. D. Effect of Trehalose on NMA−Urea Interactions. As mentioned in the Introduction section, the denaturing action of urea molecules can be explained by two different mechanisms, namely, the direct and indirect mechanisms. Within the former, the interactions between protein and urea can further be subdivided into electrostatics and van der Waals interactions. In protein−urea electrostatic interactions, the urea molecules interact directly with the protein backbone via hydrogen bonds or other electrostatic interactions. On the other hand, according to direct van der Waals/dispersion interactions, urea interacts favorably with the protein amino acids through van der Waals interactions. Though there have been debates over which of the forces is dominant,8,69−71 these two direct interactions, viz., electrostatics and van der Waals interactions between protein and urea, are not always mutually exclusive. In this context, it is worth noting that by means of MD simulation studies several attempts have been made in order to understand whether it is the direct electrostatic or van der Waals interactions between urea and protein or a combination of both that is responsible for urea-conferred protein denaturation.10,13,35,72,73 By estimating dispersion and electrostatic interactions between each water/urea molecule (present in the first solvation shell of the solute and bulk) with solute protein/macromolecule separately, it has been proposed that the favorable van der Waals

Figure 9. Intermolecular (a) van der Waals, (b) electrostatic, and (c) total interaction energies between urea and NMA molecules as a function of simulation time for systems NU and NUT. Part d represents van der Waals, electrostatic, and total interaction energies averaged over simulation time.

Figure 9a, the addition of trehalose molecules does not affect urea−NMA electrostatic energy much. Interestingly, there is a modest enhancement (more positive) in the NMA−urea van der Waals energy for the ternary NUT system when compared to the binary NU system (Figure 9b). In the same figure, we also show the total interaction energy as a function of time (Figure 9c) and the average van der Waals, electrostatic, and total interaction energies between NMA and urea where averaging is done over the last 30 ns of simulation time (Figure 9d). It is apparent that, in comparison to the binary NMA−urea system, the addition of trehalose in the NMA−urea system 9828


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logic can be applied for trehalose−trehalose and trehalose−urea hydrogen bondings), the hydroxyl oxygen atoms of trehalose contribute mostly. We note that the appearance of typical hydrogen bond peaks in hydroxyl oxygen and water oxygen rdf’s are already reported in the literature and our results are in fair agreement with those results.74−76 Furthermore, the heights of the first peaks of these rdf’s are getting enhanced in the presence of urea (system NUT). Note that, for these distribution functions, though the height of the first peak and first minimum increases in the presence of urea, the number of first shell water molecules around trehalose decreases from 19.25 (in system NT) to 14.92 (in system NUT) (not shown). This anomaly in the coordination number value is attributed to the effect of reduced water number density for system NUT. As a result, we observe a decrease in trehalose−water average hydrogen bond number as we move from system NT to system NUT (see below). In Figure 11a and b, we have shown the interaction of trehalose O6 oxygen with urea by considering selected site−site

makes NMA−urea interactions unfavorable and much enhanced (more positive) van der Waals interactions play a dominant role in it, with the effect of unfavorable electrostatic energy being minor. As discussed above, since trehalose is known to protect the protein from the deleterious effect of urea, it can be inferred from the above observations that urea− protein direct van der Waals interactions play an important role. These findings are in line with the previous results.13,72,73 E. Interactions between Solution Species. In order to understand the solvation of NMA in different binary and ternary solutions considered here, it is equally important to examine the interactions among different solution species. This is because the interactions among water, urea, and trehalose molecules influence the solvation of NMA molecules, albeit indirectly. In view of this, we also investigate the interactions between these solution species by considering selected site−site pair-correlation functions, and these are shown in Figures 10−12. Since the trehalose molecule possesses hydrophilic

Figure 11. Parts a and b respectively are the site−site radial distribution functions of urea oxygen and hydrogen around trehalose hydroxyl oxygen O6. Parts c and d are the site−site radial distribution functions involving different atomic sites of urea and water.

rdf’s. The magnitude of the first peak height of these distribution functions confirms the presence of a sufficient number of trehalose molecules around urea. The hydrogen bonding interaction between urea and trehalose molecules is also quite evident from rdf’s involving oxygen (Ou) and hydrogen (Hu) of urea and trehalose O6. Further, the comparison of the first peak heights of Ou−O6 and Hu−O6 indicates that, in the hydrogen bonding interaction between trehalose and urea, the latter preferably acts as an acceptor. The distribution of water molecules around the urea atomic sites is displayed in Figure 11c and d. For system NU, the appearance of first contact peaks at 2.85 and 1.85 Å in urea oxygen−water oxygen (Ou−Ow) and urea oxygen−water hydrogen (Ou−Hw) rdf profiles, respectively, reflects the formation of a urea−water hydrogen bond. It is also notable that the first peak in the Ou− Hw rdf is much stronger than the Hu−Ow rdf, which indicates that urea prefers to be a hydrogen bond acceptor rather than a

Figure 10. Site−site radial distribution functions of water oxygen around different oxygen atomic sites of trehalose for systems NT and NUT.

hydroxyl groups that can form hydrogen bonds with both water and urea molecules, in Figure 10, we present the rdf’s involving trehalose hydroxyl oxygens (O2, O3, O4, O6), glycosidic oxygen (O1), and acetalic ring oxygen (O5) atoms and the water oxygen (Ow) atom. It is noticed that each of the hydroxyl oxygen atoms of trehalose shows a characteristic hydrogen bond peak at about 2.75 Å. As revealed by Figure 10a and e, since water molecules cannot be positioned around O1 and O5 oxygen atoms of trehalose effectively because of purely geometrical reasons, a comparatively lower water density is observed around these two trehalose oxygens. Thus, in the hydrogen bonding interactions with water molecules (the same 9829


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these osmolytes with the like molecules also decreases. A close look into the urea−urea and trehalose−trehalose hydrogen bond numbers in binary NU and NT systems reveals that the self-association propensity of urea molecules through hydrogen bonding interactions is somewhat lower than that of trehalose− trehalose self-association. Further, what is remarkable is that, in ternary solution, the presence of trehalose molecules causes a reduction in the urea−water hydrogen bond number. There are about 0.73 urea−water hydrogen bonds, which is lost by per urea molecule as one moves from system NU to NUT. Further, the reduction of 0.11 urea−urea hydrogen bonds is also observed for the mixed urea/trehalose osmolyte system. These lost hydrogen bonds by urea molecules are well compensated by the formation of 0.81 urea−trehalose hydrogen bonds, leaving the NMA−urea hydrogen bonds almost unaffected. Therefore, these act as corroborative evidence that the protection of protein molecules (by trehalose) does not come from the removal of urea molecules (by trehalose molecules) from the protein surface. Thus, what trehalose does is it dehydrates the protein surface by removing water molecules from it and forms hydrogen bonds with protein by direct interactions. F. Trehalose Clusters and Diffusion Coefficients. As reported by several studies, the presence of hydrophilic hydroxyl groups of trehalose helps to form hydrogen bonds with the like molecules and as a result of this formation an extended trehalose hydrogen bond network takes place.78,79 Due to the formation of this extended sugar−sugar hydrogen bond network, the dynamical properties of the solution species are greatly affected.80 As reported earlier, the concentrated aqueous trehalose solution has a very high glass transition temperature and forms a highly viscous glassy matrix to keep the biomolecules fixed. Therefore, trehalose molecules help in protecting the conformation of a biological molecule like an insect trapped in amber.28 Furthermore, it is also found that the aqueous solution of trehalose protects the biomolecules by slowing down the dynamics of the water molecules that surround protein.81,82 Therefore, to observe the self-aggregation propensity of trehalose molecules and how the dynamical properties of different molecules are affected in the presence of osmolytes, in this section, we focus on the quantification of trehalose clusters (for systems NT and NUT) and the estimation of diffusion coefficients of NMA and different solution species. The self-aggregation of trehalose molecules is calculated by estimating the mean trehalose cluster size for binary and ternary systems of trehalose. A trehalose cluster can be defined as an assembly of like molecules that are connected to each other by at least one hydrogen bond. The mean trehalose cluster size, ⟨ntre⟩, is defined as

donor. The hydrogen bonding interaction between urea and water increases modestly for the ternary system NUT, as is evident from the enhanced peak height. In order to probe the effects of urea and trehalose on water structure, we examine water−water rdf’s for different systems and these are shown in Figure 12. The first peak of the Ow−Ow

Figure 12. Site−site radial distribution functions involving water oxygen and water oxygen.

distribution function characterizes the first hydrogen bond neighbor, and its second peak represents the tetrahedrally located second neighbor. They appear at 2.8 and 4.5 Å, respectively. The locations of these peaks are consistent with the previously reported results.61,77 Considering the effect of urea on this distribution function alone (system NU), we notice that the magnitude of the first peak is slightly enhanced, keeping its location unchanged. However, urea makes the first valley shallower and the second peak becomes less pronounced, suggesting a modest second shell collapse in the water structure. The influence of trehalose on water structure in solutions containing trehalose is much more pronounced. Specifically, trehalose makes the first peak height even stronger and trehalose induced collapsing of water structure is much more prominent in systems NT and NUT than that of binary urea solution. Further, insights into the hydrogen bonding interactions between different solution species can be obtained by estimating the average number of hydrogen bonds formed by water, trehalose, and urea molecules with other like or different solution species. In Table 4, we have shown the calculated values of these hydrogen bonds for different systems. As can be seen, the average number of water−water hydrogen bonds reduces in aqueous osmolyte solutions. This is due to the formation of hydrogen bonds between water and the hydrogen bond active sites of the osmolytes urea and trehalose. As a result of this, the average number of hydrogen bonds formed by

⟨ntre⟩ =

Table 4. HB Represents the Average Hydrogen Bond Number (with Respect to First Species)a system

NW

NU

NT

NUT

HBW−W HBU−W HBU−U HBT−W HBT−T HBU−T

3.37

2.48 3.76 1.31

2.78

1.81 3.03 1.20 7.33 1.43 0.81

10.00 2.45

∑ ntrePtre

(6)

where ntre and Ptre correspond to the number of trehalose molecules in a given cluster and its probability of formation, respectively. The diffusion coefficients of different molecules are calculated using the popular Einstein relation. We calculate the diffusion coefficients (D) from the long time slope of the mean square displacement (MSD). lim ⟨|r(t ) − r(0)|2 ⟩

D=

a

Subscripts W−W, U−W, U−U, T−W, T−T, and U−T refer to water−water, urea−water, urea−urea, trehalose−water, trehalose− trehalose, and urea−trehalose, respectively.

t →∞

6t

(7)

We have presented the change in ⟨ntre⟩/Ntre (where Ntre is the total number of trehalose molecules present in a given 9830


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Table 5. DW, DT, DU, and DN Correspond to the Translational Diffusion Coefficient Values of Water, Trehalose, Urea, and NMA, Respectively; ⟨ntre⟩/Ntre Is the Normalized Mean Cluster Size; and the Standard Errors Calculated Using the Block Average over 2 ns Are Given in Parentheses system

DW (10−5 cm 2 s−1)

NW NU NT NUT

2.43 2.38 1.89 1.27

DT (10−5 cm 2 s−1)

DU (10−5 cm 2 s−1) 1.15

0.03 0.03

0.35

DN (10−5 cm 2 s−1)

⟨ntre⟩/Ntre

1.24 0.77 0.37 0.36

0.42 (±0.04) 0.25 (±0.02)

bonding interaction between NMA and water. These rdf’s also suggest the relatively weaker hydration of NMA hydrogen by water molecules. The NMA−urea site−site rdf’s indicate the direct interaction of urea and NMA in binary urea solution, and interestingly, urea density decreases modestly near NMA molecules upon addition of trehalose. Moreover, the preferential interaction parameter calculations also imply a slight depletion of urea molecules in the vicinity of NMA molecules in a ternary urea/trehalose mixture. The depletion of the urea molecules in the close proximity of NMA molecules leads to an enhancement (more positive) in the urea−NMA van der Waals interaction energy. On the other hand, preference for trehalose in the solvation shell of NMA increases in that solution. Furthermore, the calculation of the ratio of concentrations of the solvent-separated state to the contact state, which provides information about the hydrophobic interactions between NMA molecules through their hydrophobic moieties, reveals that the presence of urea and trehalose influences Me1−Me1 hydrophobic contacts profoundly when compared to Me1−Me2 and Me2−Me2 contacts. Addition of trehalose to binary urea solution causes an enhancement in the hydrophobic interactions between Me1−Me1 for the urea/ trehalose binary mixture. Investigation of hydrogen bonding properties of NMA with the solution species showed that, in the hydrogen bonding between NMA and water, the former preferably acts as an acceptor in which its carbonyl oxygen atom accepts water hydrogen. Our results also demonstrated that a large number of NMA−water hydrogen bonds are replaced by the NMA−urea hydrogen bond in binary urea solution; more specifically, the carbonyl oxygen of NMA is shared between one water and one urea molecule. Again, when compared to the pure water system, the total number of hydrogen bonds formed by a single NMA molecule with other solution species is higher in binary urea solution. These findings support the direct interaction mechanism of urea conferred protein denaturation. Just like urea, in binary solution, trehalose also replaces water molecules from the NMA surface, but this replacement is lower than that for the aqueous urea solution. Remarkably, the total number of hydrogen bonds formed by one NMA molecule with trehalose in binary trehalose solution is nearly equal to the number of NMA−water hydrogen bonds broken. This keeps the total number of hydrogen bonds formed by a single NMA molecule with the solution species essentially unchanged as one moves from system NW to system NT. However, on addition of trehalose to urea solution (system NUT), the NMA−urea hydrogen bond number decreases slightly relative to the binary urea solution (reflected in a slight increase (more positive) in NMA−urea electrostatic interaction energy in the NUT system) and trehalose compensates the NMA−water hydrogen bonds by forming NMA−trehalose hydrogen bonds. These observations direct us to propose that the counteracting effect of trehalose on urea-conferred protein denaturation comes

system) as a function of trehalose concentration in Table 5. Together with this, in the same table, we show the diffusion coefficient values of water (DW), trehalose (DT), urea (DU), and NMA (DN) for different systems. We find the values of ⟨ntre⟩/ Ntre for systems NT and NUT are 0.42 and 0.25, respectively. A little lower value of ⟨ntre⟩/Ntre for system NUT is due to the formation of significant trehalose−urea hydrogen bonds. We note that the value of the mean trehalose cluster size for system NT is in line with our previous simulation results.83 Moreover, we notice that, for the binary NT system, the percolation of the trehalose hydrogen bond network is yet to be achieved. This is due to the consideration of lower trehalose concentration in this study and the presence of NMA molecules in the system. We also note that the change in ⟨ntre⟩/Ntre values for systems NT and NUT is in accordance with trehalose−trehalose (per trehalose) hydrogen bond formation for these systems discussed above (see Table 4). Now, focusing on the dynamical properties, we observe a sharp decrease in the diffusion coefficient values of all solution species with the addition of osmolytes. More insight into these values reveals that trehalose induced retardation of translational motion of water and NMA molecules is significantly larger than that of urea. The presence of both osmolytes in the system (system NUT) decreases these diffusion coefficient values further. In this context, it is worth noting that the trehalose induced significant drop in the diffusion coefficient value of water molecules (due to formation of stable water−trehalose hydrogen bonds) that are present within 5.5 Å of a solute molecule was already reported elsewhere.84 The temperature dependent diffusion coefficient measurements of binary water− trehalose mixtures show that the activation energy for the diffusive motion falls in the typical hydrogen bond energy range.85 We also observe that the translational dynamics of trehalose remains essentially unchanged for both the binary NT and ternary NUT systems. On the other hand, the diffusion coefficient of urea decreases from 1.15 × 10−5 cm2 s−1 in the binary NU system to 0.35 × 10−5 cm2 s−1 in the ternary NUT system. This means that the dynamics of urea molecules is also greatly affected in the presence of trehalose.

IV. CONCLUSIONS To explore the mechanism of trehalose induced counteraction of urea conferred protein denaturation, in this Article, we have performed an all atom MD simulation study to characterize the solvation of NMA in binary and ternary solutions of urea and trehalose. The site−site rdf’s between the methyl groups of NMA and water oxygen indicates that binary urea solution has very little effect on the solvation of methyl groups of NMA as compared to the pure water. On the other hand, trehalose enhances the solvation of methyl groups of NMA in both binary trehalose and ternary urea/trehalose solution. Further, the distributions of water hydrogen and oxygen around the NMA oxygen and hydrogen suggest the presence of hydrogen 9831


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(8) O’Brien, E. P.; Dima, R. I.; Brooks, B.; Thirumalai, D. Interactions Between Hydrophobic and Ionic Solutes in Aqueous Guanidinium Chloride and Urea Solutions: Lessons for Protein Denaturation Mechanism. J. Am. Chem. Soc. 2007, 129, 7346−7353. (9) Finer, E. G.; Franks, F.; Tait, M. J. Nuclear Magnetic Resonance Studies of Aqueous Urea Solutions. J. Am. Chem. Soc. 1972, 94, 4424− 4429. (10) Das, A.; Mukhopadhyay, C. Urea−Mediated Protein Denaturation: A Consensus View. J. Phys. Chem. B 2009, 113, 12816−12824. (11) Almarza, J.; Rincon, L.; Bahsas, A.; Brito, F. Molecular Mechanism for the Denaturation of Proteins by Urea. Biochemistry 2009, 48, 7608−7613. (12) Vanzi, F.; Madan, B.; Sharp, K. Effect of the Protein Denaturants Urea and Guanidinium on Water Structure: A Structural and Thermodynamic Study. J. Am. Chem. Soc. 1998, 120, 10748− 10753. (13) Hua, L.; Zhou, R.; Thirumalai, D.; Berne, B. J. Urea Denaturation by Stronger Dispersion Interactions with Proteins than Water Implies a 2-Stage Unfolding. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 16928−16933. (14) Caballero-Herrera, A.; Nordstrand, K.; Berndt, K. D.; Nilsson, L. Effect of Urea on Peptide Conformation in Water: Molecular Dynamics and Experimental Characterization. Biophys. J. 2005, 89, 842−857. (15) Daggett, V. Protein Folding-Simulation. Chem. Rev. 2006, 106, 1898−1916. (16) Bennion, B. J.; Daggett, V. The Molecular Basis for the Chemical Denaturation of Proteins by Urea. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 5142−5147. (17) Ou, W. B.; Park, Y. D.; Zhou, H. M. Molecular Mechanism for Osmolyte Protection of Creatine Kinase Against Guanidine Denaturation. Eur. J. Biochem. 2001, 268, 5901−5911. (18) Crowe, J. H.; Carpenter, J. F.; Crowe, L. M. The Role of Vitrification in Anhydrobiosis. Annu. Rev. Physiol. 1998, 60, 73−103. (19) Crowe, J. H.; Crowe, L. M.; Jackson, S. A. Preservation of Structural and Functional Activity in Lyophilized Sarcoplasmic Reticulum. Arch. Biochem. Biophys. 1983, 220, 477−484. (20) Crowe, J. H.; Crowe, L. M.; Chapman, D. Preservation of Membranes in Anhydrobiotic Organisms: The Role of Trehalose. Science 1984, 223, 701−703. (21) Crowe, L. M. Lessons From Nature: The Role of Sugars in Anhydrobiosis. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2002, 131, 505−513. (22) Patist, A.; Zoerb, H. Preservation Mechanisms of Trehalose in Food and Biosystems. Colloids Surf., B 2005, 40, 107−113. (23) Watanabe, M. Anhydrobiosis in Invertebrates. Appl. Entomol. Zool. 2006, 41, 15−31. (24) Carpenter, J. F.; Pikal, M. J.; Chang, B. S.; Randolph, T. W. Rational Design of Stable Lyophilized Protein Formulation: Some Practical Advice. Pharm. Res. 1997, 14, 969−975. (25) Roser, B. Trehalose, A New Approach to Premium Dried Foods. Trends Food Sci. Technol. 1991, 2, 166−169. (26) Higashiyama, T. Novel Functions and Applications of Trehalose. Pure Appl. Chem. 2002, 74, 1263−1269. (27) Carpenter, J. F.; Crowe, J. H. An Infrared Spectroscopic Study of the Interactions of Carbohydrates with Dried Proteins. Biochemistry 1989, 28, 3916−3922. (28) Green, J. L.; Angell, C. A. Phase Relations and Vitrification in Saccharide-Water Solutions and the Trehalose Anomaly. J. Phys. Chem. 1989, 93, 2880−2882. (29) Belton, P. S.; Gil, A. M. IR and Raman Spectroscopic Studies of the Interaction of Trehalose with Hen Egg White Lysozyme. Biopolymers 1994, 34, 957−961. (30) Zou, Q.; Bennion, B. J.; Daggett, V.; Murphy, K. P. The Molecular Mechanism of Stabilization of Proteins by TMAO and Its Ability to Counteract the Effects of Urea. J. Am. Chem. Soc. 2002, 124, 1192−1202. (31) Wang, A.; Bolen, D. W. A Naturally Occurring Protective System in Urea-Rich Cells: Mechanism of Osmolyte Protection of

from the replacement of protein−water hydrogen bonds by protein−trehalose hydrogen bonds (lending support from the water replacement hypothesis). This essentially reduces the availability of water molecules (at the protein surface) to form hydrogen bonds with the protein backbone, and as a result, the protein native structure is preserved. Furthermore, the formation of a large number of trehalose−water hydrogen bonds also suggests the preferential solvation of trehalose molecules by water molecules, which indirectly reduces the availability of water molecules further. The cluster structure analysis shows that the trehalose cluster size decreases from binary trehalose solution to ternary trehalose solution. The value of the mean cluster size for different trehalose containing systems suggests that the percolation of the trehalose hydrogen bond network has not been achieved at this concentration of trehalose. The calculation of translational diffusion coefficients shows a trehalose induced slowing down of translational motion of all solution species and the effect is more pronounced for NMA and water molecules. Therefore, the present study explicitly elucidates the counteracting effects of trehalose on urea induced unfolding of protein and also explains the underlying mechanism of the counteracting effect of trehalose at high urea concentration. Finally, these conclusions may further improve our understanding of compatibility and counteracting effects of osmolytes on proteins in protein science.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The financial support of Board of Research in Nuclear Sciences (BRNS), Government of India, is gratefully acknowledged. Part of the research was enabled by using the computational facility of C-DAC, Pune, India.

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