Trehalose Induced Modifications in the Solvation Pattern of N

Article pubs.acs.org/JPCB

Trehalose Induced Modifications in the Solvation Pattern of N‑Methylacetamide Subrata Paul and Sandip Paul* Department of Chemistry, Indian Institute of Technology, Guwahati, Assam, India-781039 S Supporting Information *

ABSTRACT: We have carried out molecular dynamics simulation to investigate the role of trehalose molecules on the change in the structural and dynamical properties of aqueous N-methylacetamide (NMA) solution. In this study, we considered six different trehalose concentrations ranging from 0 to 66%. Results are discussed in the framework of hydrophobic interactions between different methyl groups of NMA, structure of the solutions, and hydrogen bonding interactions between different solution species. We observe that the propensity of hydrophobic association through the methyl groups of NMA is essentially insensitive to trehalose concentration except for higher trehalose concentration where the hydrophobic interactions between the hydrophobic methyl groups are getting reduced. Also observed are (i) trehalose induced slight collapse of the second hydration shell of water, (ii) presence of excess water molecules near NMA, and (iii) exclusion of trehalose from NMA. Our NMA−water radial distribution function analyses followed by average number of hydrogen bonds per NMA calculations reveal that, in the hydration of NMA molecules, its carbonyl group oxygen (over amide hydrogen) is predominantly involved. As trehalose is added, we observe, in accordance with the water replacement hypothesis, the replacement of water−NMA hydrogen bonds by NMA−trehalose hydrogen bonds, keeping the average number of hydrogen bonds formed by a single NMA with different solution species essentially unchanged. Our hydrogen bond calculations further reveal that addition of trehalose replaces water−NMA hydrogen bonds by water−trehalose hydrogen bonds. And as a result, we find that the average number of hydrogen bonds formed by a water molecule remain unchanged. We also find that addition of trehalose decreases the translational motion of all the solution species sharply.

I. INTRODUCTION

In order to understand the molecular mechanisms which make trehalose a superior bioprotecting agent, three hypotheses are proposed: (1) Mechanical entrapment (vitrification): This mechanism suggests that trehalose molecules form a highly viscous glassy matrix to keep biomolecules fixed. Formation of this glassy matrix protects the biomolecule in its biological conformation like an insect trapped in amber.10 (2) Water replacement hypothesis: According to this hypothesis, trehalose molecules form direct hydrogen bonds with protein and they (trehalose) replace water molecules from the protein hydration shell.11 (3) Water entrapment hypothesis: This hypothesis tells that water molecules are trapped in the intermediate layer between trehalose and biomolecule. Moreover, trehalose molecules help to concentrate the residual water molecules around the biomolecule. This mechanism can also be termed as the preferential hydration hypothesis of a biomolecule in dilute sugar solution.12 Despite various experimental and theoretical works,13−20 the properties that make trehalose an effective bioprotective agent are still yet to be understood completely and none of the above three hypotheses can explain the

In living systems, the biological activity of a protein depends on its three-dimensional native structure. Under extreme conditions such as high and low temperature, high pressure, desiccation, etc., a protein molecule may denature and loses its activity. The aqueous solutions of simple carbohydrates are normally used to protect the proteins, and they help to retain the proteins’ activity. It has been reported that, under cold and heat stress, organisms accumulate a wide variety of this class of compounds to protect their cells from possible damage.1,2 Among several carbohydrates, trehalose, a nonreducing disaccharide, is recognized as a very effective cryoprotectant for its interesting bioprotecting abilities.3,4 It is composed of two glucopyranosyl units linked together through an α,α-(1 → 1) glycosidic oxygen linkage between their anomeric carbon atoms (see Figure 1). A wide variety of organisms such as bacteria, yeast, fungi, insects, invertebrates, as well as vascular plants and a few flowering plants contain trehalose.5 Besides this, it is also often widely used as an additive for long-term preservation of therapeutic proteins, foods, and cosmetics in industries.6−8 Moreover, in a recent study, it has been reported that trehalose can also act as a stabilizer to stabilize human blood platelet cells.9 © 2014 American Chemical Society

Received: August 4, 2013 Revised: January 10, 2014 Published: January 14, 2014 1052

dx.doi.org/10.1021/jp407782x | J. Phys. Chem. B 2014, 118, 1052−1063

The Journal of Physical Chemistry B

Article

Note that we consider the dilute NMA solution in order to have a sufficient number of hydration sites and to avoid possible NMA−NMA hydrogen bonding. Thus, our systems represent a typical solvent exposed state of protein in which the protein backbone can interact freely with the different solution species. Our main focus is to study the structural properties involving different solution species and the influence of trehalose onto it. We further explored the trehalose induced change in the hydrogen bond properties and translational motion of NMA, water, and trehalose and 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 We have performed classical molecular dynamics (MD) simulations for aqueous solutions of NMA and trehalose with different trehalose concentrations. The six different systems with varying trehalose concentrations considered here are summarized in Table 1. The initial configurations of our 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.

Table 1. Number of NMA (NNMA), Trehalose (Ntre), and Water (Nwat) Molecules and Weight Percentage of Trehalose (wtre%) for the above System

molecular mechanism of trehalose fully. For example, it has been reported that sugar molecules form hydrogen bonds with the polar group of membranes and proteins by replacing the water molecules present in their hydration shell. As a result of this, the three-dimensional structure of protein is preserved even at low water content and protein does not lose its activity. This observation is in accordance with the water replacement hypothesis11,19,21 In a Raman spectroscopy study, it has been observed that the protein hydration layer is preserved even in low water content and the water molecules present in the hydration layer are trapped in the glassy matrix formed by trehalose molecules, and this helps the protein to retain its native structure and biological activity, supporting the water entrapment hypothesis.12 As mentioned, a good deal of effort has been directed toward understanding the molecular mechanism of trehalose induced protection of biomolecules, but there is still no definitive generally accepted answer to this question and it remains a subject of active research. In view of this, it would be interesting to examine more closely the structural, dynamical, and hydrogen bond properties of aqueous trehalose solutions and this is the purpose of this paper. Since the interactions between protein, solvent water, and trehalose would be very complex in nature, the present molecular dynamics simulations are carried out with N-methylacetamide (NMA, Figure 1) in pure water as well as in trehalose solutions of varying concentration. Since it is a well-known fact that hydrophobic interactions and hydrogen bonding play an important role in protein stability, we mimic protein by NMA because of the fact that NMA is the simplest molecule which contains hydrophobic methyl groups as well as hydrophilic amide (peptide linkage) groups. The advantages of using NMA (over protein) are twofold. (i) The complexity of our systems is now reduced, and (ii) the hydrogen bonding interactions between different solution species and the role of trehalose onto it can also be explored.

system

NNMA

Ntre

Nwat

volume (nm3)

wtre%

S0 S1 S2 S3 S4 S5

20 20 20 20 20 20

0 10 20 50 75 100

1000 1000 1000 1000 1000 1000

32.15 35.29 39.30 49.83 58.86 67.97

0 16 28 49 59 66

systems are prepared using the Packmol program.22 For the trehalose molecule, we have used the GLYCAM06 force field,23 the popular SPC/E24 model was used for water, and for NMA we have adopted the all atom force field AMBER94 model where methyl hydrogens are considered explicitly.25 Classical MD simulations for all systems are carried out in a cubic box at 300 K using the AMBER1226 suite of programs. To obtain a reasonable initial structure, the systems are energy minimized for 5000 steps with the first 2500 steps in the steepest descent method followed by the same number of steps in the conjugate gradient method. This is followed by heating up the system slowly from 0 to 300 K for 100 ps in the canonical (NVT) ensemble, and then, the systems are equilibrated in the isothermal−isobaric (NPT) ensemble for 2 ns. For calculating different structural and hydrogen bond properties, all production runs are performed in the NPT ensemble for 40 ns and one atmospheric pressure. Periodic boundary conditions were applied for all the simulations, and the temperature was controlled by the Langevin dynamics method with a collision frequency of 1 ps−1 and a time step of 2 fs used in all simulations. For all nonbonding interactions, a cutoff radius of 10 Å was applied and the long-range electrostatic interactions were treated using the particle mesh Ewald method. Bonds involving hydrogen are constrained by applying the SHAKE algorithm.27 In order to maintain the physical pressure, the Berendsen barostat was used with a pressure relaxation time of 2 ps.28 Finally, in order to calculate the diffusion coefficients of different solution species, the simulations were, further, 1053



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continued for another 15 ns simulation run in the microcanonical ensemble (NVE). We have used the ptraj program of the AMBER12 toolkit to analyze the trajectories obtained from production runs, and the Visual Molecular Dynamics (VMD)29 package was used to analyze the hydrogen bonding of the systems. The GLYCAM06 force field parameters are mostly used for the simulations of monosaccharides and oligosaccharides. In this force field, the 1−4 electrostatic and nonbonded scaling factors are set to unity. Furthermore, in AMBER12 (and AMBER11), the mixed scaling of scee and scnb (electrostatic and nonbonded scaling factors) can be used for the simulations of carbohydrates and proteins. A comparison between different disaccharide force fields reveals that the GLYCAM06 force field is in excellent agreement with DFT and experimental results.30,31 For example, the self-diffusion constant of α-Disomaltose computed with the GLYCAM06 force field is in agreement with the experimental value.31 Further, as mentioned in the Introduction section, trehalose molecules form a highly viscous glassy matrix at high concentration. As a result, the dynamics of both water and trehalose molecules are greatly affected. A comparison of different water models shows that, when compared to the experimental results, the SPC/E water model produces better bulk dynamics and structure over the SPC and TIP3P model.32 In this regard, it is worth noting that Verde et al.,33 in a recent study, have used the GLYCAM06 force field for trehalose and kojiboise combined with SPC/E water for their study.

Figure 2. Site−site radial distribution functions involving hydrophobic groups in NMA. The systems S0, S1, S2, S3, S4, and S5 are represented as black, red, green, blue, magenta, and brown, respectively.

The higher the value of Ka, the higher will be the association of the hydrophobic methyl groups of NMA molecules. Consequently, any perturbation that decreases the Ka value will favor the dissolution of the hydrophobic moieties. We find that the value of Ka is essentially insensitive to the change in trehalose concentration except for system S5, where a modest drop in its value is observed (see Table 1 in the Supporting Information). B. Interaction of NMA with Different Solution Species. Since hydrophobic association of a hydrophobic moiety and its solvation is intimately related with its interaction with different solution species, in order to investigate NMA interactions with different solution constituents, we first computed selected site− site rdf’s involving NMA and different solution species, and these are shown in Figures 3−5. Figure 3 displays the rdf’s involving the hydrophobic methyl groups of NMA and water oxygen (Ow). Focusing on the hydration of Me1 and Me2 of NMA in the absence of trehalose first, we find that the Me1−Ow rdf starts to rise at 2.75 Å and reaches the bulk density (where g(r) = 1) at 3.65 Å. This suggests that there is actually exclusion of water molecules from the solvation shell of Me1 below 3.65 Å. The outer limit of the first solvation shell (i.e., first minimum) appears at 5.45 Å. The Me2−Ow rdf shows similar hydration characteristics as that of the Me1−Ow rdf except for the fact that in the case of former the hydration is a little more defined as revealed by a slight increase in the peak height and an inward movement of the first minimum. Further, the hydration of these methyl groups in pure water shows the typical behavior of hydrophobic hydration.35,36 In this context, we note that the water distribution around these two methyl groups is less narrow as compared to that for methane.35 Furthermore, as observed before, we also find that the water structure around the hydrophobic region is perturbed in the presence of other groups.37,38 For example, Pratt and Chandler reported that the solvent density around a methyl group in ethane is somewhat less structured than that of methane.37 Similarly, it has also been shown that the water peaks are narrower and sharper for

III. RESULTS AND DISCUSSION A. Hydrophobic Interaction. It is a well-known fact that the hydrophobic interactions among the nonpolar moieties play an important role in the stability of a protein in its native state.34 In view of this, it would be interesting to see the role of trehalose on the hydrophobic interactions between the methyl groups of NMA. The site−site radial distribution functions (rdf’s) involving methyl groups of NMA in pure water and in different trehalose solutions are shown in Figure 2. In pure water, the first peak and second peaks in the Me1−Me1 (Figure 2a) rdf appear at about 4.05 and 7.35 Å, respectively. Considering the effect of trehalose, we find that trehalose has little effect on the Me1− Me1 distribution function except for the highest trehalose concentration considered here (system S5) where a modest change in both first and second peak heights is observed, indicating trehalose induced change in the solvation of this methyl group. Me1−Me2 and Me2−Me2 distribution functions (Figure 2b and c) behave in a similar fashion as the Me1−Me1 distribution function does. For a better understanding of the hydrophobic association between different methyl groups of NMA and the influence of trehalose on it, we have calculated the association constant (Ka). It is defined as K a = 4π

∫0

ra

r 2e−W (r)/ kBT dr

(1)

where kB is the Boltzmann constant and ra is the position of the first minimum in the corresponding rdf. W(r) represents the potentials of mean force and can be calculated from the site− site rdf’s (gss(r)) by using the relation W (r ) = −kBT ln gss(r )

(2) 1054



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Table 2. Number of First Shell Water Molecules around Methyl Groups of NMA Molecules (CNMe1 and CNMe2)a system

CNMe1

CNMe2

ni

νi/w

S0 S1 S2 S3 S4 S5

17.59 16.10 (16.03) 14.70 (14.52) 12.47 (11.45) 11.50 (9.62) 9.77 (8.40)

14.95 13.72 (13.62) 13.31 (12.34) 10.76 (9.73) 9.93 (8.18) 8.27 (7.14)

0.62 0.58 0.47 0.39 0.37 0.22

0.76 0.80 0.60 0.56 0.61 0.13

The numbers given in parentheses represent the first shell coordination number if the only change with added trehalose came through the number density change. ni and νi/w are the number of first shell Me1 and the preferential interaction parameter, respectively. a

presented in Table 2 in parentheses. It is apparent that the water coordination numbers increase slightly more than “expected” from the water density change, and this change is becoming pronounced at higher trehalose concentration. In order to probe the hydrophobic hydration more closely, we, further, calculated the preferential binding parameter, νi/w, by using the relation40

Figure 3. Solid lines represent the site−site distribution function between NMA methyl groups and the water oxygen atom. Dashed lines are for NMA methyl−water hydrogen rdf’s for system S0. Different lines correspond to the same convention as that in Figure 2.

spherical molecules (e.,g., methane, neopentane, etc.) than those of nonspherical molecules.38 We see that the addition of trehalose does nothing to decrease the peak heights; rather, it appears to be enhanced by trehalose. Furthermore, the information about the orientation of water molecules near the hydrophobic methyl groups of NMA can be obtained by comparing the peak positions of NMA methyl−water oxygen and NMA methyl−water hydrogen rdf’s (see Figure 3). In the rdf profiles, since the positions of the peaks for these two rdf’s appear at the similar locations, surface parallel orientations of water molecules near the methyl groups can be suggested. The tail of water hydrogen density at shorter distances reflects a closer approach of water hydrogens toward the methyl surface. We also notice that the general behavior of these distribution functions is qualitatively similar for the two NMA methyl groups. Further insight into the hydration of NMA can be obtained by computing the average number of water molecules in the first solvation shell of Me1 and Me2. The average number of species β in the first shell of a reference molecule α can be calculated from the corresponding rdf, gαβ(r), using the relation39 CN = 4πρβ

∫0

rc

r 2gαβ (r ) dr

νi / w =

ni N × w −1 nw Ni

(4)

where ni is the number of Me1 in the first solvation shell of Me1 and nw represents the number of water molecules in the first solvation shell of Me1. Ni and Nw correspond to the total number of Me1 and water molecules present in the system. A positive value of νi/w suggests that Me1 interacts preferentially with Me1 of another NMA molecule, and a negative value indicates preferential hydration of the hydrophobic Me1 group of NMA. Note that since the different methyl groups show similar hydrophobic and hydration characteristics as revealed by the corresponding rdf’s, in the preferential interaction parameter calculations, we concentrate on the behavior of the Me1 group of NMA in the context of hydrophobic interactions involving another Me1 and its hydration pattern involving water. The different values of νi/w for different systems are tabulated in Table 2. We find that the value of νi/w is positive for all systems, and as trehalose is added, its value fluctuates around the value for the pure water system except for system S5 where there is a sharp drop in the νi/w observed, suggesting less hydrophobic interaction and more hydration of the NMA Me1 group. Due to the presence of hydrophilic carbonyl oxygen and amide hydrogen atom, NMA molecules are expected to participate in hydrogen bonding interaction with both water and trehalose molecules. These particular interactions are demonstrated in Figures 4 and 5. For pure water, the first peaks involving NMA oxygen−water hydrogen (O−Hw) and NMA oxygen−water oxygen (O−Ow) rdf profiles (see Figure 4), which characterize the first neighbor, appear at about 1.75 and 2.75 Å, respectively, indicating CO···HwOw hydrogen bonding interaction.41 On the other hand, the rdf involving NMA hydrogen−water oxygen (H−Ow) represents the hydrogen bonding interactions between NMA and water where NMA acts as a donor and water as an acceptor and the contact peak of this rdf appears at about 2.05 Å. In pure water, the height of the first peak of the H−Ow rdf is less than 1, suggesting relatively weaker hydration of the amide hydrogen atom by water molecules when compared to the hydration of the

(3)

where CN, ρβ, and rc represent the first shell coordination number, the number density of species β, and the position of the first minimum in the distribution function, respectively. The number of first shell water molecules around Me1 and Me2 groups of NMA for different systems is shown in Table 2. Though the first peak height of the NMA methyl−water oxygen rdf increases, the number of first shell water molecules around the methyl group decreases as trehalose is added. Some decrease is of course expected, as addition of trehalose results in increasing the box volume which in turn makes the number density of water lower. In order to nullify the effect of the reduced number density of water in trehalose solution, we have, further, calculated the number of first shell water molecules assuming that the only change with added trehalose comes through the reduced number of water and these numbers are 1055



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Table 3. First Shell Coordination Number (CN) for O−Ow, O−Hw, H−Ow, and O−H rdf’s of Water and NMA Moleculea system S0 S1 S2 S3 S4 S5

CNO−Ow 2.33 2.16 2.00 1.91 1.78 1.56

(2.12) (1.91) (1.50) (1.28) (1.10)

CNO−Hw 2.21 2.05 1.89 1.67 1.55 1.36

(2.01) (1.80) (1.42) (1.21) (1.04)

CNH−Ow 0.97 0.91 0.84 0.73 0.70 0.62

(0.83) (0.79) (0.62) (0.53) (0.46)

CNO−H 0.038 0.034 0.026 0.026 0.022 0.012

(0.035) (0.031) (0.025) (0.021) (0.018)

a The numbers given in parentheses represent the first shell coordination number if the only change with added trehalose came through the number density change.

water the number density decreases, some decrease in the coordination number value is expected due to lower water number density in higher trehalose concentration. Keeping this in mind, in the same table, we also present the number of water molecules “expected” to be in the first solvation shell due to the change in water number density (see the parentheses in Table 3). As can be seen, as trehalose is added, more and more hydration of these hydrogen bonding atomic sites of NMA takes place, as evidenced from the increased difference in the actual and expected number of coordination number values. The information about the hydrogen bonding interactions between NMA and trehalose molecules can be obtained by examining the rdf’s involving amide oxygen of NMA and different hydroxyl groups of trehalose, and this is shown in Figure 5. We note that, since trehalose hydroxyl oxygens O2, O3, and O4 behave in a similar fashion as that of O6 in the context of rdf’s involving these atoms and the amide group of NMA, in Figure 5, we have shown a rdf involving O6, O1, and O5 oxygen atoms only. This figure displays the hydrogen bonding interaction between NMA and trehalose, where the latter acts as a donor. From this figure, it is apparent that the hydrogen bonding interaction between the ring oxygen atom (O5) (as well as glycosidic oxygen atom, O1) of trehalose and the amide group of NMA is very small, as evidenced from small peak heights in the corresponding rdf’s. The strong first peaks of O6−O and O6−H, rdf’s which appear at 2.75 and 1.95 Å, suggest that the amide group of NMA molecules predominantly forms hydrogen bonds with the O6 (and with O2, O3, and O4) hydroxyl group of trehalose. Further, we can have some insights into the hydrogen bonding interactions between NMA and trehalose where NMA donates its amide hydrogen atom (which is attached to the nitrogen atom) to the hydroxyl oxygen of trehalose. The number of first shell oxygen atoms of trehalose around the NMA nitrogen atom can be obtained by integrating the corresponding rdf’s (not shown) to the first minimum. Addition of trehalose has a modest influence on the rdf’s, as observed in the slight change in the peak heights; this in turn slightly changes the coordination number values (see Tables 2 and 3 in the Supporting Information) and the hydrogen bonding pattern between trehalose and NMA. From these tables, as revealed by the total number of first shell coordination numbers, it is apparent that NMA molecules prefer to accept the hydroxyl hydrogen of trehalose rather than acting as a donor. This observation is further supported by our calculated hydrogen bond property studies (discussed below). Figure 6 displays the running coordination numbers (RCNs) involving the center of mass of water and different atomic sites of NMA. In the same figure, we have also included the RCN for

Figure 4. Site−site radial distribution function of NMA oxygen and amide hydrogen with the water molecule. Different lines correspond to the same convention as that in Figure 2.

Figure 5. Site−site radial distribution function between trehalose and NMA. Different lines correspond to the same convention as that in Figure 2.

carbonyl oxygen of NMA. Addition of trehalose causes a noticeable rise in the water density around NMA atomic sites. We, further, computed the number of first shell water molecules around the hydrogen bonding atomic sites of the NMA molecule, and the same is presented in Table 3. It is apparent that the coordination number decreases as trehalose is added. As we mentioned before that on addition of trehalose 1056



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Figure 7. Site−site radial distribution function between water oxygen and trehalose oxygens. Different lines correspond to the same convention as that in Figure 2. Figure 6. Running coordination numbers for different atomic sites of NMA. Solid lines are for water and dashed lines for trehalose. For better resolution, in the insets, the RCNs for trehalose are shown. Different lines correspond to the same convention as that in Figure 2.

hydrogen bonding between the water molecules and hydroxyl O6 of trehalose. Since the hydration characteristics of other hydroxyl oxygen atoms (O2, O3, and O4) are very similar to that of the hydroxyl O6 oxygen atom of trehalose (as evident from the rdf’s involving these oxygen atoms and water oxygen), we show only the O6−Ow rdf. The O1−Ow and O5−Ow rdf’s (Figure 7b and c) do not have such a well-defined first solvation shell as observed in the case of O6, and therefore, these oxygen atoms are not well hydrated. This implies that water molecules cannot be positioned in the vicinity of the glycosidic oxygen of (1−1)-linked disaccharide. Our results match well with the other previous simulation studies of aqueous trehalose solution.44,46 On increasing trehalose concentration, we observe (i) the enhancement in the peak height of the O6−Ow rdf and (ii) the first valley of the O6−Ow rdf becomes shallower. The shallower first valley indicates breaking of the water−trehalose hydrogen bond network on increasing trehalose concentration. The number of first shell water molecules around the different hydroxyl oxygens of trehalose is shown in Table 4. Though we observe an increase in the peak height of these rdf’s with increasing trehalose concentration, we find that the first shell coordination number decreases and as a result we observe a decrease in the water−trehalose per trehalose average hydrogen bond number (discussed below). We attribute this to the fact that addition of trehalose makes the water number density lower. We note that our calculated number of first shell water molecules around trehalose oxygens is in agreement with that reported in the literature considering the fact that different trehalose concentrations and different methods were adopted in this study in calculating the first shell coordination number when compared to other studies.42,47,48 To evaluate the effects of trehalose on changing water structure, we examine water−water rdf’s for different systems. These include Ow−Ow and Ow−Hw rdf’s and are shown in Figure 8. The first peak of the Ow−Ow distribution function (Figure 8a) characterizes the H-bonded first neighbor, and its

the trehalose center of mass. Since a trehalose molecule is much larger in size than a water molecule, the exclusion of trehalose molecules is expected from the certain volume shell around the atomic sites of NMA is expected from purely geometric reason. This fact is reflected in the larger radius of exclusion for trehalose. Considering the trehalose center of mass and the NMA Me1 RCN for system S1, we find that the value of the RCN reaches 1 at 7.75 Å. Now, if we consider a solvation shell of radius 7.75 Å around NMA Me1, the values of RCN for water we find are 57 and 52 for systems S0 (no trehalose system) and S1, respectively. This suggests the replacement of five water molecules by a trehalose molecule in the solvation shell of radius 7.75 Å of Me1. Due to the exclusion of water molecules, the total number of solution species in the NMA solvation shell is much higher in pure water compared to that of trehalose solution. C. Interactions between Solution Species. A trehalose molecule possesses eight hydroxyl groups. These hydroxyl groups can interact strongly with water and NMA molecules through hydrogen bonding interactions. Thus, it is important to examine how the trehalose molecule interacts with the solution species, viz., water and NMA, and its (trehalose) possible impact on the hydrogen bond network of water. Figure 7 displays the rdf’s involving O6 (hydroxylic oxygen), O1 (glycosidic oxygen), and O5 (acetalic ring oxygen) atoms of trehalose and Ow (water oxygen). As reported earlier, we also observe that hydroxyl groups of trehalose are well solvated by water molecules with well-defined nearest neighbor peaks in the corresponding trehalose oxygen−water oxygen rdf’s.42−45 In the rdf’s involving O6 and Ow (Figure 7a), a sharp peak of intensity 1.5, which appears at 2.75 Å, indicates the presence of 1057



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distribution functions for different trehalose concentrations, and the same are shown in Figure 1 of the Supporting Information. Though we find that the change in the O−H rdf is insensitive for lower trehalose concentration, the noticeable change in this rdf is observed for the highest trehalose concentration considered in this study (i.e., system S5). Specifically, the heights of the sharp first peak and the broad second peak are getting reduced remarkably for this system. The reduced first peak height of this distribution function for system S5 is complimented by sharp changes in the first shell coordination number presented in Table 3. By comparing the number of expected coordination number values due to the change in the number density of NMA (see the parentheses in Table 3) vs the actual coordination value (obtained using eq 3) for different systems, we find that the difference between these two values is much more prominent for system S5 as compared to other systems. D. Hydrogen Bond Properties. For further characterization of NMA solvation, applying a geometric definition of a hydrogen bond,50 we investigated the average number of hydrogen bonds formed by different solution species. According to the geometric definition of a hydrogen bond, formation of a hydrogen bond is considered if the following three conditions are satisfied simultaneously:

Table 4. Number of First Shell Water Molecules around the Different Hydroxyl Hydrogens of Trehalose system coordination number (CN) 1GA

0GA

S1

S2

S3

S4

S5

O1Ow O2Ow O3Ow O4Ow O5Ow O6Ow O2Ow O3Ow O4Ow O5Ow O6Ow

S0

0.88 2.05 2.92 2.10 0.76 2.42 2.14 2.62 1.92 0.71 2.07

0.73 2.12 2.51 1.92 0.59 2.12 2.12 2.67 1.83 0.61 2.24

0.74 2.11 2.14 1.57 0.54 2.00 2.01 2.32 1.65 0.64 1.92

0.81 1.92 2.42 1.74 0.54 2.02 1.93 2.28 1.58 0.64 1.90

0.87 1.96 2.15 1.56 0.59 1.86 1.86 2.19 1.68 0.54 1.79

total

20.59

19.46

17.64

17.78

17.05

rDA ≤ r1cut

rAH ≤ r2cut angle D−H−A ≥ 120°

where A and D represent acceptor and donor atoms, respectively, and H represents hydrogen attached to the donor atom D. rDA and rAH are the distances between donor and acceptor atoms and between an acceptor atom and a hydrogen of another molecule, respectively. The cutoff cut distances rcut 1 and r2 are obtained from the positions of the first minimum from the corresponding rdf’s, and these distances are presented in Table 5 of the Supporting Information. Note that six different types of hydrogen bonds are possible involving different solution species out of which NMA is involved in three different types, namely, NMA−water, NMA−trehalose, and NMA−NMA. Besides this, we also computed hydrogen bonds between water−water, water− trehalose, and trehalose−trehalose. In order to verify the geometric criteria used here for defining hydrogen bonds between the solution species, we have shown the probability contour plots as a function of D−H−A angle and A−H distance for system S5 (see Figures 2 and 3 in the Supporting Information). We find that the criteria used in this study consider the region of high probability density for the formation of strong hydrogen bonds. Note that different types of hydrogen bonds in this study are abbreviated as Ix−Jy, where I and J can be N (for NMA), W (for water), and T (for trehalose) and x and y can be a (acceptor) and d (donor). For example, the hydrogen bonds of type H···Ow are abbreviated as Nd−Wa, where NMA is a donor and water acts as an acceptor and so on. The results for the average number of hydrogen bonds are shown in Tables 5 and 6. Note that the average number of hydrogen bonds shown in Tables 5 and 6 is with respect to the first species. In agreement with the previous observations40,51,52 and our coordination number calculation analyses discussed above, we observe that, in the formation of NMA−water and NMA−trehalose hydrogen bonds, NMA acts

Figure 8. Site−site radial distribution function between water and water. Different lines correspond to the same convention as that in Figure 2.

second peak represents the tetrahedrally located second neighbor. The positions of these peaks, which appear at about 2.8 and 4.5 Å, respectively, are in agreement with those already reported earlier.36,49 We observe that, as trehalose is added, the first peak height is getting enhanced, keeping its location essentially unchanged. However, the addition of trehalose makes the first valley shallower and the second peak less pronounced, suggesting a slight second shell collapse in the water structure. The Ow−Hw rdf’s for different systems are shown in Figure 8b. The information about water−water hydrogen bonds can be obtained from this rdf. To get insight into the nature of water−water hydrogen bonding, we have estimated the first shell coordination numbers for different systems (see Table 4 in the Supporting Information). We observe that the number of water−water hydrogen bonds decreases with increasing trehalose concentration. This is expected due to the destructuring of the water hydrogen bond network by trehalose and the strong hydrogen bond formation ability with water molecules by large trehalose molecules. The insight into the effect of trehalose concentration on the hydrogen bonding interaction between NMA molecules can be obtained by calculating NMA oxygen−NMA hydrogen 1058



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trehalose hydrogen bonds. This reduction of the average number of hydrogen bonds between water−water in the presence of trehalose compares well with the results of Dinnamaria et al.43 reported earlier. Further, we find the average number of trehalose−water (per trehalose) hydrogen bonds is 10.94 in the case of system S1. However, previous MD simulations have demonstrated that, with the same hydrogen bonding criterion, the number of hydrogen bonds to the trehalose is found to be 22.4 for the TIP3P model of water in most diluted solution.54 This deviation in hydrogen bond number might be due to the presence of solute NMA molecules and the higher trehalose concentration used in our study. The average number of water−trehalose (per trehalose) hydrogen bonds decreases as trehalose is added. A closer look into the total number of different types of hydrogen bonds formed by a single water molecule with different solution species (including other water molecules) reveals that the total number of hydrogen bonds (formed by a water molecule) is essentially unchanged. For example, in system S0 (where no trehalose is present), a single water molecule is involved in 0.05 hydrogen bonds with NMA and 3.31 hydrogen bonds with other water molecules (in total 3.36 hydrogen bonds per water molecule). Now, for system S5, a single water molecule forms on average 3.27 hydrogen bonds (2.33 with water, 0.03 with NMA, and 0.91 with trehalose). This fact suggests that, as trehalose is added, some of the water−water and water−NMA hydrogen bonds are broken and they are replaced by newly formed water−trehalose hydrogen bonds, keeping the average number of hydrogen bonds formed by a single water molecule unchanged. E. Trehalose Clusters. It has already been reported that, with increasing concentration, due to enhancement in the trehalose−trehalose hydrogen bonding interactions, association of trehalose molecules takes place.55,56 Further, the dynamical properties are influenced strongly due to the formation of an extended sugar−sugar hydrogen bond network.57 In order to see the effect of trehalose concentration on its self-aggregation, we estimated the mean trehalose cluster size for all systems. We define a cluster as an assembly of trehalose molecules that are connected to each other by at least one hydrogen bond. The mean trehalose cluster size, ⟨ntre⟩, is defined as

Table 5. Average Number of Hydrogen Bonds between NMA−Water and NMA−Trehalose per Trehalosea systems

Na−Wd

Nd−Wa

totalNW

S0

1.78

0.52

S1

1.64

0.49

S2

1.52

0.45

S3

1.35

0.40

S4

1.21

0.37

S5

1.14

0.35

2.30 (0.050) 2.13 (0.041) 1.97 (0.040) 1.75 (0.035) 1.58 (0.032) 1.49 (0.030)

Na−Td

Nd−Ta

totalNT

total 2.30

0.11

0.05

0.16

2.29

0.20

0.10

0.30

2.27

0.33

0.17

0.50

2.25

0.36

0.21

0.57

2.15

0.50

0.28

0.78

2.27

a

Different hydrogen bond types are defined in the text. TotalNW and TotalNT represent the total number of NMA−water and NMA− trehalose bonds per NMA molecule, and Total is the sum of TotalNW and TotalNT. In the parentheses, the average number of water−NMA hydrogen bonds per water is shown.

preferably as an acceptor where its carbonyl oxygen accepts hydrogens of water and trehalose hydroxyl groups (see Table 5). In this context, it is worth noting that, in NMA−water hydrogen bonds, it has been reported that the hydrogen bond energies of both Nd−Wa and Na−Wd types are isoenergic.40,53 Though addition of trehalose decreases the average number of NMA−water hydrogen bonds and increases the NMA− trehalose hydrogen bonds, the most striking effect that we observe is the average number of hydrogen bonds formed by a single NMA molecule with the solution species water and trehalose is essentially unchanged (see the last column of Table 5). In Table 6, we have shown the average number of water− water, trehalose−water (per trehalose), trehalose−trehalose, and NMA−NMA hydrogen bonds for different systems. Considering the average number of NMA−NMA hydrogen bonds first, we find a NMA molecule forms a negligible number of hydrogen bonds with another like molecule. This is expected as we consider systems with dilute NMA solution and as we mentioned above that these systems represent a typical solvent exposed state of proteins where the protein backbone can form hydrogen bonds with different solution species. As trehalose is added, we observe a decrease in the average number of water− water hydrogen bonds and enhancement of trehalose−

⟨ntre⟩ =

∑ ntrePtre

(5)

where ntre and Ptre represent the number of trehalose molecules in a given cluster and its probability of formation, respectively.

Table 6. Average Hydrogen Bond Number between Water−Water, Trehalose−Water (per Trehalose), Trehalose−Trehalose, and NMA−NMA Moleculesa

a

system

water−water

S0 S1

3.31 3.17

S2

3.08

S3

2.79

S4

2.53

S5

2.33

trehalose−water

trehalose−trehalose

NMA−NMA

10.94 (0.11) 10.90 (0.49) 9.81 (0.49) 9.62 (0.72) 9.05 (0.91)

1.76

0.02 0.02

1.70

0.01

2.50

0.01

2.48

0.01

2.76

0.01

In the parentheses, the average number of water−trehalose hydrogen bonds per water is shown. 1059



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In Figure 9, we have shown the change in ⟨ntre⟩/Ntre (where Ntre is the number of trehalose molecules in a system) as a

effect of trehalose on the dynamical properties of different solution species (in particular for NMA), using Einstein’s relation, we have computed diffusion coefficients (D) from the long time slope of the mean square displacement (MSD). lim ⟨|r(t ) − r(0)|2 ⟩

D=

t →∞

(6)

6t

A representative table showing the diffusion coefficient values of water (Dw), trehalose (DT), and NMA (DN) for different trehalose concentrations is presented in Table 7. We find that Table 7. Diffusion Coefficients of Water (Dw), Trehalose (DT), and NMA (DN)

Figure 9. The normalized mean trehalose cluster size, ⟨ntre⟩/Ntre, for different systems. The standard errors are calculated using the block average over 2 ns.

function of trehalose concentration. Notwithstanding the error bars, we find that, for systems S1 and S2, the values of ⟨ntre⟩/ Ntre remain practically unchanged and they appear at around 0.3. Addition of more trehalose molecules increases the value of ⟨ntre⟩/Ntre, suggesting growth of trehalose clusters. The value of ⟨ntre⟩/Ntre reaches 0.7 for the highest trehalose concentration considered here, suggesting that the percolation of a trehalose hydrogen bond network has not been achieved. In this context, it is worth noting that the percolation of a trehalose hydrogen bond network at 66 wt % trehalose concentration (in absence of NMA) has been reported earlier.56 This discrepancy is due to the formation of a large number of NMA−trehalose hydrogen bonds which increases with increasing trehalose concentration (see Table 5), which affects the formation of the trehalose−trehalose hydrogen bond network. We also note that the change in ⟨ntre⟩/Ntre values with trehalose concentration is in accordance with trehalose−trehalose (per trehalose) hydrogen bond formation discussed above (see Table 6). In order to probe the involvement of NMA molecules in the trehalose cluster, we have calculated the fraction of NMA molecules (Xn) that engage in n number of hydrogen bond formation with trehalose molecules in the cluster of size ntre > 1 (see Figure 4 in the Supporting Information). We observe that with increasing trehalose concentration more and more NMA molecules are engaged in hydrogen bonding interactions with trehalose molecules in the cluster. Moreover, the fraction of NMA molecules in which each NMA molecule forms more than one hydrogen bond with the trehalose molecules present in the cluster increases with increasing trehalose concentration. Furthermore, trehalose−trehalose self-association can be probed by spatial density distribution of the trehalose pair correlation function. In Figure 5 of the Supporting Information, considering the C1−O1−C1 reference coordinate system, we show a projection of the spatial distribution function of trehalose−trehalose interaction for system S3. It can be seen that there is overlap between the high probability density region for directly hydrogen bonded trehalose molecules and the high density region of up to 10 Å, around the reference trehalose molecule. This suggests the formation of hydrogen bond mediated trehalose clusters. F. Diffusion Coefficient. It has already been reported that concentrated aqueous trehalose solution has a very high glass transition temperature and forms a highly viscous glassy matrix to keep the biomolecules fixed. This glassy matrix helps in protecting the biomolecule in its biological conformation like an insect trapped in amber.10 In view of this, to evaluate the

system

Dw (10−5 cm2 s−1)

DT (10−5 cm2 s−1)

DN (10−5 cm2 s−1)

S0 S1 S2 S3 S4 S5

2.43 1.74 1.75 0.99 0.48 0.27

0.13 0.04 0.009 0.004 0.003

1.24 0.72 0.32 0.13 0.09 0.03

there is a sharp decrease in the diffusion coefficient values of all solution species with increasing trehalose concentration and the decrease is more pronounced for trehalose and NMA when compared to that of water. For example, changing the trehalose concentration from 16 to 66%, the diffusion coefficient of water decreases from 1.74 × 10−5 to 0.27 × 10−5 cm2 s−1 (6.4 times decrease), whereas for trehalose and NMA the decrease in the diffusion coefficients is 43 and 24 times, respectively. In other words, the ratio of Dw and DT increases sharply with increasing trehalose concentration. For example, the value of Dw:DT for system S1 is approximately 13:1 and that for system S5 is 90:1. The decrease of Dw and DT values is due to the formation of a hydrogen bonded complex between different solution species. We hereby note that trehalose induced a significant decrease in the diffusion coefficient of water molecules that are present near 5.5 Å of a solute molecule and formation of more stable water−trehalose (over water−water) hydrogen bonds reported earlier.58 Trehalose induced retardation of translational and rotational motion of surrounding water molecules was also observed in recent terahertz absorption measurements.59 As shown previously in the temperature dependent diffusion coefficient measurements of a water−trehalose mixture in the absence of NMA,60 the activation energy for the diffusive motion falls in the typical hydrogen bond energy range. It was, further, suggested that the translational diffusion coefficients of water and trehalose are driven by breaking and formation of intermolecular hydrogen bonds. We also note that the sharp decrease in the diffusion coefficient values for water and trehalose observed in our study is in agreement with the experimental diffusion coefficient reported earlier.61 The sharp decrease of D T , observed mainly at higher trehalose concentration, is in good agreement with the sharp increase of trehalose viscosity values.62 Further, it has been hypothesized that the trehalose induced bioprotection comes because of the retardation of the surrounding water due to the presence of trehalose molecules.63,64 Since protein−water dynamics are coupled, trehalose induced retardation in surrounding water dynamics influences protein dynamics. 1060



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IV. CONCLUSIONS To provide a molecular level understanding of the influence of trehalose on protein stability, by employing a classical molecular dynamics simulation technique, we investigated the solvation characteristics of NMA molecules in different trehalose concentrations. Computations of different site−site rdf’s involving hydrophobic methyl groups of NMA showed that trehalose has a negligible influence on the hydrophobic interactions except for a very high trehalose concentration where a modest decrease in the first and second peak heights in the methyl−methyl rdf is observed. This observation is further supported by association constant values for different systems. From the calculations of first shell water molecules around the methyl groups, slightly more than expected water molecules is found. As revealed by the preferential interaction parameter calculations, in concentrated trehalose solution, the affinity of a NMA methyl group to interact with a methyl group of another NMA molecule (over water) is reduced. This acts as corroborative evidence of trehalose induced hydration of methyl groups of NMA at higher trehalose concentration and in accordance with the trehalose induced reduction in the hydrophobic interactions reported recently.65 We also found that, as trehalose was added, the number of water molecules present near NMA was slightly in excess when compared to its expected value. Moreover, the total coordination number of hydroxyl oxygen groups of trehalose around NMA oxygen decreases, though the number density of trehalose increases with the addition of trehalose. In this context, it is worth noting that a MD simulation study of a protein−trehalose−water mixture13 also revealed the presence of excess water and exclusion of trehalose from the protein surface, suggesting the water entrapment hypothesis.12 Investigation of the hydrogen bonding ability of NMA molecules with water and trehalose showed that NMA molecules prefer to act as an acceptor over a donor where its carbonyl oxygen atom participates predominantly. When compared to water, our running coordination number calculations suggested a larger exclusion radius of trehalose and each trehalose molecule replaces five water molecules from the NMA solvation shell having a radius of 7.75 Å. It was observed, further, that, on addition of trehalose, water−NMA hydrogen bonds were replaced by NMA−trehalose. However, interestingly, trehalose has essentially no effect on changing the total number of hydrogen bonds formed by a single NMA molecule with solution species. In the context of trehalose induced protection of protein, the above observation suggested the water replacement hypothesis which says that trehalose molecules replace water molecules from the hydration shell of a protein and form direct hydrogen bonds with the latter.11 We note IR spectra studies of a dry protein−sugar mixture also suggested that a direct sugar−protein hydrogen bond plays a major role in protein stability.19,20 Also observed are (i) a trehalose induced slight second shell collapse of the water network and (ii) replacement of water−NMA hydrogen bonds by water−trehalose hydrogen bonds keeping the number of hydrogen bonds formed for a single water molecule unchanged. Calculation of translational diffusion coefficients showed trehalose induced slowing down of translational motion of all solution species, and the effect is more pronounced for NMA and trehalose molecules than for water. On addition of 66% trehalose, the diffusion coefficient of NMA was reduced from 1.24 × 10−5 to 0.03 × 10−5 cm2 s−1. In this context, we note

that trehalose molecules form a highly viscous glassy matrix and thereby the biological molecule gets trapped and thus protects the biomolecule in its biological conformation.10 It is worth noting that our log−log plot of mean-squared displacement versus time (see Figure 6 in the Supporting Information) indicated that, even for the highest trehalose concentration considered (i.e., 66% trehalose) here, we have not observed the appearance of any so-called “boson” peak as reported earlier.45 This, further, suggested that the most concentrated trehalose solution was still above the glass transition temperature for this trehalose model.

■

ASSOCIATED CONTENT

S Supporting Information *

Tables for the association constant for methyl groups of NMA in different systems, the number of different trehalose hydroxyl oxygen atoms in the first solvation shell of the oxygen atom of NMA, the number of different trehalose oxygen atoms in the first solvation shell of the N atom of NMA, first shell water− water hydrogen bonding coordination number for all the systems, and different cutoff distances for hydrogen bondings. Site−site radial distribution function of H−O of NMA, probability distribution contour plots for NMA oxygen−water hydrogen and water oxygen−water hydrogen for system S5, probability distribution contour plots for trehalose hydroxyl O6−trehalose hydroxyl hydrogen, NMA oxygen−trehalose hydroxyl hydrogen, water oxygen−trehalose hydroxyl hydrogen, the fraction of NMA molecules that form n number of hydrogen bonds with trehalose molecules that are present in a trehalose cluster of size ntre > 1, and the projection of the spatial distribution function of trehalose−trehalose interaction for system S3. log−log plot of mean-squared displacement vs time for water and trehalose molecules for systems S1 and S5. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The financial support of Board of Research in Nuclear Sciences (BRNS), Govt. of India, is gratefully acknowledged. REFERENCES

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Trehalose Induced Modifications in the Solvation Pattern of N

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