Importance of Protein Conformational Motions and Electrostatic

Article pubs.acs.org/Langmuir

Importance of Protein Conformational Motions and Electrostatic Anchoring Sites on the Dynamics and Hydrogen Bond Properties of Hydration Water Somedatta Pal and Sanjoy Bandyopadhyay* Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur-721302, India ABSTRACT: The microscopic dynamic properties of water molecules present in the vicinity of a protein are expected to be sensitive to its local conformational motions and the presence of polar and charged groups at the surface capable of anchoring water molecules through hydrogen bonds. In this work, we attempt to understand such sensitivity by performing detailed molecular dynamics simulations of the globular protein barstar solvated in aqueous medium. Our calculations demonstrate that enhanced confinement at the protein surface on freezing its local motions leads to increasingly restricted water mobility with long residence times around the secondary structures. It is found that the inability of the surface water molecules to bind with the protein residues by hydrogen bonds in the absence of protein−water (PW) electrostatic interactions is compensated by enhanced water−water hydrogen bonds around the protein with uniform bulklike behaviors. Importantly, it is further noticed that in contrast to the PW hydrogen bond relaxation time scale, the kinetics of the breaking and formation of such bonds are not affected on freezing the protein’s conformational motions.

1. INTRODUCTION The influence of protein−water (PW) interaction on the dynamics of water molecules at the surface of the protein is a long-standing problem in chemical biology.1−4 Often, controversy exists in interpreting experimental findings, as different methods measure different quantities over a wide range of time scales.5,6 Besides, it is often nontrivial to separately identify the contributions originating from different components in dynamical response of a solvated protein. As a result, despite significant efforts, a microscopic picture of the dynamical correlations that exist between a protein and the water molecules hydrating its surface is yet to emerge. Over the years, time-resolved fluorescence spectroscopy with both intrinsic and extrinsic probes has become an important tool to characterize the dynamics of proteins in different forms and their solvation time scales.3,4,7−13 In general, most of these studies reveal bimodal distribution of solvation time scales with a fast component occurring within a few picoseconds and a slower one in the range of tens of picoseconds or more. It is believed that the faster component originates due to the presence of free bulklike water molecules near the protein, whereas the slow long-time component corresponds to restricted mobility of surface water molecules coupled with the local conformational motions of the residue side chains. It is observed that the slower component is sensitive to the location of the probe molecules in the protein.8,10,12,13 The conclusions reached from these time-resolved studies are found to be consistent with recent neutron scattering14,15 and early nuclear magnetic relaxation (NMR) studies.16,17 Interestingly, recent NMR studies by Halle and co-workers18,19 predicted that although the protein hydration water exhibits sluggish © 2013 American Chemical Society

dynamics, the degree of retardation is much less compared to that observed in fluorescence studies. They suggested that the residue side chain motions rather than the restricted water mobility are responsible for observing the slower component in fluorescence experiments. In recent times, several neutron scattering studies on hydration water have been reported.14,20−23 Nonexponential relaxation behavior of hydration water molecules and their amorphous icelike characteristics have been revealed from such studies.14,22 The collective dynamics of solvated proteins have also been studied from terahertz (THz) spectroscopy.24−26 Only recently, Lipps et al.27 investigated the hydration dependent picosecond dynamics of proteins at low temperatures using THz spectroscopy. Infrared (IR) spectroscopy has also been employed to study the structure and dynamics of proteins and their hydration water.28,29 Using ultrafast 2D-IR vibrational echo spectroscopy, Fayer and co-workers30 reported how solvent viscosity alters the dynamics of small peptides. Dielectric relaxation spectroscopy (DRS) has been used recently by Panagopoulou and coworkers31 to probe the dynamics of the protein bovine serum albumin (BSA) with different water contents. Various relaxation processes associated with the hydrated protein were characterized. Computer simulation in general and molecular dynamics (MD) in particular are powerful alternative methods to explore PW interactions and the dynamic correlations that exist around solvated proteins. The major advantages of MD simulation are Received: October 5, 2012 Revised: December 14, 2012 Published: January 5, 2013 1162

dx.doi.org/10.1021/la303959m | Langmuir 2013, 29, 1162−1173

Langmuir

Article

the other the PW electrostatic interactions were turned off along with the frozen protein matrix. The calculations revealed that while freezing of the protein’s degrees of freedom does not modify the heterogeneous arrangement of surrounding water molecules, on eliminating the PW electrostatic interactions water arrangement becomes near-uniform. In this work, we probe how the local motions of the protein segments and the PW electrostatic contributions influence the microscopic dynamics and hydrogen bond properties of the hydration layer water molecules. The rest of the article is organized as follows. In Section 2, we provide a brief description of the setup of the systems and the simulation methods employed. The primary sequence of barstar and its secondary structural arrangements are also highlighted in this section. The results obtained from our investigations are presented and discussed in the following section (Section 3). The important findings from our study and the conclusions reached therefrom are highlighted in Section 4.

that one can directly measure the contributions originating from different components on protein hydration and also obtain microscopic information by varying the time and length scale resolutions. In general, the simulation studies predict retarded translational and rotational motions of hydration water.32−37 In an important contribution, Pizzitutti et al.38 demonstrated that both conformational and energetic heterogeneities at the surface of a protein play important roles in restricting water motions around it. It is shown that the anomalous behaviors of surface water molecules reduce in the presence of conformational oscillations of the protein. Presence of electrostatic anchoring sites in a protein leads to the formation of PW hydrogen bonds spanning the protein surface. This results in breaking of regular water−water (WW) hydrogen bond network and its rearrangement in an aqueous protein solution. The time scale associated with redistribution of hydrogen bonds at the protein surface is expected to control the dynamic coupling and exchange of water molecules between the bulk and the hydration layer. In an important study, Xu and Berne39 showed that compared to the bulk state, the kinetics of WW hydrogen bonds around proteins slow down significantly. In recent times, it has been shown that the time scale of formation and breaking of PW hydrogen bonds affects the low-frequency intermolecular vibrational density of states of water and its density fluctuations.40,41 In an important series of works, Tarek and Tobias42 showed that the structural relaxation of a protein is sensitive to the dynamics of PW hydrogen bonds. Only recently, Laage and co-workers43 made an attempt to obtain a microscopic description of protein hydration water dynamics in terms of water reorientational motion and the hydrogen bond properties around the protein. They demonstrated that the water reorientation at the protein surface occurs through large angular jumps similar to that noticed in pure bulk water. Influence of the surface topography of proteins on the structural arrangement of surrounding water molecules has also been demonstrated from simulation studies.44−46 Recently, Chakravarty and co-workers47 characterized the ordering and energetics of water molecules around peptides. It is further shown that the locally heterogeneous structure of a protein results in nonuniform ordering of solvent around it.48 In an important effort, THz spectroscopy in combination with MD simulations has been used recently to study the dynamics of water in protein solvaton shells.49 It is shown that the THz absorption of the protein solvation shell is correlated with the blue shift observed in water vibrational modes in MD simulations. In a related work, Perera et al.50 reported the existence of dangling OH bonds in the hydration layers of nonpolar solutes from vibrational Raman spectroscopy and MD simulations. Garde and co-workers51,52 extensively used molecular simulations to study the hydration properties of hydrophobic solutes including proteins. They showed that the compressibility and fluctuations in hydraton shells around hydrophobic solutes depend on the solute size. Importantly, it is demonstrated that hydration water fluctuations can be used as a tool to probe the hydrophobicity of a protein surface. Recently, we studied the influence of the heterogeneous conformational motions and distribution of electrostatic anchoring sites at the surface of the protein barstar on the local structure and ordering of nearby water molecules.53 The results obtained from the simulation of the flexible protein were compared with two other simulations, in one of which the conformational motions of the protein were kept frozen and in

2. SYSTEM SETUP AND SIMULATION PROTOCOLS Barstar is a 89-residue protein54 that acts as an inhibitor of potentially lethal ribonuclease barnase in bacterium bacillus amyloliquefacien.55 It contains three parallel α-helices packed onto a three-stranded parallel β-sheet. There is an additional αhelix linking the second central strand and the fourth α-helix. The amino acid sequence of the protein is K(1)KAVINGEQIRSISDLHQTLKKELALPEYYGENLDALWDCLTGWVEYPLVLEWRQFEQSKQLTENGAESVLQVFREAKAEGCDITIILS(89), with the N-terminus residue K(1) and the C-terminus residue S(89). We denote the individual segments as helix-1 (Ser-14 to Ala-25), helix-2 (Asn33 to Gly-43), helix-3 (Phe-56 to Thr-63), helix-4 (Glu-68 to Gly-81), and β-sheet (Lys-1 to Asn-6, Leu-49 to Arg-54, and Asp-83 to Ser-89). The loops interconnecting these segments are denoted as loop-1 (Gly-7 to Ile-13), loop-2 (Leu-26 to Glu32), loop-3 (Trp-44 to Pro-48), and loop-4 (Glu-64 to Ala-67). It is believed that barstar inhibits the function of barnase by sterically blocking its active site by helix-2 and the loop connecting it with helix-1 (loop-2).55 Three simulations (S1, S2, and S3) were carried out with the aqueous solution of barstar. The fully flexible protein molecule in equilibrium with the solvent was studied in simulation S1. In simulation S2, the protein molecule was kept frozen, whereas in S3, the electrostatic interactions between the protein and the water molecules were turned off in addition to freezing the protein matrix. In all cases, the initial coordinates of the protein molecule were taken from that reported from NMR studies (PDB code: 1BTA).54 The two end residues (Lys-1 and Ser89) were taken as ammonium and carboxylate ionic forms, and the whole protein was immersed in a large cubic box containing equilibrated water molecules. To avoid unfavorable contacts between the protein and the solvent, the insertion process was carried out by carefully removing those water molecules that were found within 2 Å from the protein atoms. The overall charge of the system (simulations S1 and S2) was neutralized by adding 6 Na+ ions. The final system contained the protein solvated in a cubic box with 60 Å initial edge length containing 5389 water molecules and 6 Na+ counterions. Note that there was no counterion in system S3, and the protein behaves like a nonpolar solute. The simulations were carried out with the NAMD code.56 The systems were first minimized using the conjugate gradient energy minimization method.56 The temperature of each of the 1163


Langmuir

Article

simulations S1 and S2 can provide a microscopic understanding of the influence of the energetic disorder at the protein surface on water dynamics originating from nonuniform electrostatic interactions between the segment residues and the water molecules. 3.1. Water Translational Motion. Water translational motion is monitored by tracking the hydration layer water molecules along the simulated trajectories and measuring their mean square displacements (MSD) (⟨Δr2⟩) with respect to time. This is a standard practice, and ⟨Δr2⟩ is defined as

systems was then increased gradually to room tempetarure (300 K) within a short MD run of about 100 ps under isothermal−isobaric ensemble (NPT) conditions at a constant pressure of 1 atm. The systems were then equilibrated at 300 K under NPT ensemble conditions for 3 ns duration each. The temperatures of the systems were controlled by the Langevin dynamics method with a friction constant of 1 ps−1, and the pressures were controlled by the Nosé−Hoover Langevin piston method.57 The cell volumes were allowed to fluctuate isotropically during this period. At this stage, the cell volumes were found to attain steady values with edge lengths of 55.7, 55.5, and 55.8 Å, for systems S1, S2, and S3, respectively. The cell volumes were then fixed and the simulation conditions changed from that of NPT to NVT ensemble (constant volume and temperature). The NVT equilibration runs were continued for another 20 ns duration at 300 K for each case. After this, the simulation conditions were changed to that of constant energy in microcanonical ensemble (NVE). Long NVE trajectories were then generated for about 57 ns duration for each of the three systems. The average temperatures as obtained from the NVE trajectories were found to be 302.5 (±0.9), 301.9 (±1.1), and 300.1 (±1.0) K, for systems S1, S2, and S3, respectively. All the three simulations were carried out with a time step of 1 fs, while the trajectories were stored with a time resolution of 500 fs for analysis. The minimum image convention58 was employed to calculate the short-range Lennard-Jones interactions using a spherical cutoff distance of 12 Å with a switch distance of 10 Å. The long-range electrostatic interactions were calculated using the particle-mesh Ewald (PME) method.59 We have employed the all-atom CHARMM22 force field and potential parameters for proteins,60 and the TIP3P model61 was used for water in the calculations.

⟨Δr 2⟩ = ⟨|ri(t ) − ri(0)|2 ⟩

(1)

Here, ri(t) and ri(0) denote oxygen atom position vectors of the ith hydration layer water molecule around a particular segment of the protein at time t and at t = 0, respectively. The calculations corresponding to different segments are carried out by averaging over all the tagged water molecules and over different time origins. The results are presented in Figure 1. As

3. RESULTS AND DISCUSSION Recently, we studied the roles played by differential local conformational motions and nonuniform PW electrostatic interactions in modulating the structural organization of water molecules present in the hydration layer spanning the surface of the protein barstar. In this work, we explore how the nonuniform distribution of electrostatic anchoring sites at the protein surface and the local motions of the secondary structures influence the microscopic dynamics and hydrogen bond properties of the hydration water molecules. The calculations are carried out by considering those water molecules that are present within a distance of 5 Å from the surface of different segments of barstar (α-helices, β-sheet, and the interconnecting loops). This is consistent with earlier reports62,63 where it is shown that the effects of a protein on water dynamical properties are primarily restricted to those that are present in its first hydration layer (within ∼5 Å). The results reported are based on calculations over the equilibrated trajectories as obtained from simulations S1, S2, and S3. It may be noted that in all the calculations presented here, the reentries of the tagged water molecules into the first hydration layers of the protein segments over times are considered. In other words, only the durations of the trajectory of a tagged water molecule when it is found to reside within 5 Å of a segment are included in the calculations. It is important to note at this stage that comparison of results between simulations S1 and S2 allows us to study the influence of heterogeneous local motions of the protein segments on the dynamical properties of surrounding water molecules. On the other hand, comparison of the results obtained from simulation S3 with that from

Figure 1. Mean square displacement (MSD) of water molecules present in the first hydration layers of different segments of barstar for (a) the flexible protein system (simulation S1), (b) the frozen protein system (simulation S2), and (c) the frozen protein system with PW electrostatic interactions turned off (simulation S3).

a reference, the corresponding result for pure bulk water as obtained from a separate room temperature MD simulation of TIP3P water is included in the figure. It is evident that the first hydration layer water molecules around the protein segments exhibit restricted translational motions as compared to water in pure bulk state. Such restricted motions of water at the surface of biomolecules are well-known.32−34,36,38 The relative heterogeneity in water motions around different segments of the protein as observed in Figure 1a is consistent with nonuniform local motions of the segments and their interactions with solvent, as reported earlier.53 The present results show that the restricted mobility of hydration water occurs irrespective of whether the protein degrees of freedom are kept frozen and/or the PW electrostatic interactions are turned off. Interestingly, the degree of restriction in water motions and their heterogeneous nature around the segments as observed in Figure 1a are found to be significantly influenced in the absence of the protein’s local conformational motions and nonuniform PW electrostatic contributions (Figure 1b and c). A comparison between Figure 1a and b shows that with PW electrostatic interactions on, the hydration water molecules exhibit much restricted mobility on freezing the protein’s 1164


Langmuir

Article

averaged over all the tagged water molecules and over different time origins. As already mentioned, the calculations are carried out for the water molecules present in the first hydration layers of the protein segments. The relaxations of Cμ(*+t) as obtained from the three systems are shown in Figure 2. The result for pure

degrees of freedom. This is true for water around both the rigid secondary structures (α-helices and the β-sheet) and the flexible loops. In absence of local motions (Figure 1b), the protein acts as a heterogeneous rigid wall with greater degree of confinement at the surface. This results in enhanced restricted dynamics of the surface water molecules, thereby leading to increased rigidity of the first hydration layers around the protein segments. On the other hand, protein conformational motions increase the available dimension at the surface allowing the hydration water molecules to undergo slightly less restricted dynamics (relatively less rigid hydration layer) as evident from Figure 1a. We notice from Figure 1c that the degree of retardation in water translational motions at the protein surface diminishes significantly in the absence of PW electrostatic interactions. Several simulation studies have shown that water molecules present near the polar and charged groups of proteins and peptides are capable of forming strong hydrogen bonds with them.36,39 The present result shows that without such hydrogen bond anchoring sites at the protein surface on eliminating PW electrostatic contributions (system S3), the hydration layer water molecules exhibit slower mobility with respect to bulk water, but they diffuse on a time scale significantly faster than that observed for systems S1 and S2. Interestingly, it is further noticed that the water molecules around different segments of the protein exhibit nearhomogeneous dynamics in system S3. Thus, the present results prove that the heterogeneous distribution of the polar and charged groups at the surface is primarily responsible for differential dynamics of water hydrating the protein segments. To explore how the sublinear anomalous diffusion patterns of the first hydration layer water molecules are affected by the conformational flexibility of the protein and their electrostatic interactions with the residues, we have fitted the MSD curves with a power law as ⟨Δr 2⟩ ∼ t α

Figure 2. Reorientational time correlation function (Cμ(t)) for water molecules present in the first hydration layers of different segments of barstar for (a) the flexible protein system (simulation S1), (b) the frozen protein system (simulation S2), and (c) the frozen protein system with PW electrostatic interactions turned off (simulation S3).

bulk water is also included in the figure for comparison. It is evident that along with the translational diffusion, the reorientational motions of the first hydration layer water molecules are also restricted. Further, local conformational motions of the protein and the PW electrostatic interactions are found to have significant influence on the degree of retardation and the relative heterogeneous reorientations of water molecules hydrating the protein segments. It is clear from Figure 2b that due to reduced available dimension with consequent increased confinement around the protein on freezing its degrees of freedom, the water molecules near the surface of most of the segments (except loop-4) exhibit noticeably slower rotations as compared to that observed for the flexible protein system (Figure 2a). However, in contrast to normal expectation, the relaxation of the function for water around loop-4 (interconnecting helices 3 and 4) becomes slightly faster in the frozen form. This is an interesting observation which shows that the conformational flexibility of a protein segment can often affect the translational and rotational motions of water molecules present in its proximity in a nonuniform manner. The important role played by PW electrostatic contributions in controlling water rotations is evident from Figure 2c. We find that on turning off the electrostatic component from total PW interaction, the water molecules present near the protein segments can not only diffuse faster but are also able to reorient themselves on a much shorter time scale. Further, the fact that a heterogeneous distribution of polar and charged groups at the protein surface is responsible for nonuniform reorientational times for waters around the protein is clearly evident from homogeneous relaxation patterns of Cμ(t) in the absence of PW electrostatic component. To obtain an estimate of the effects of protein flexibility and PW electrostatic interactions on the average times (⟨τμ⟩) taken

(2)

where the value of α (0 ≤ α ≤ 1) determines the degree of anomaly. The calculated α-values averaged over the protein segments as obtained for systems S1, S2, and S3 are found to be around 0.61, 0.48, and 0.78, respectively. This clearly demonstrates that while strong PW electrostatic interaction is primarily responsible for anomalous diffusion behavior of hydration water, local conformational motions of protein on the other hand tend to reduce to some extent such anomaly. Our results are consistent with an earlier simulation study on another protein.38 3.2. Water Rotational Motion. We now investigate the effects of nonuniform conformational fluctuations of different segments of barstar and the PW electrostatic interactions on water rotational motions. As before,36 this is done by measuring the reorientational dynamics of water dipole μ⃗ , defined as the vector joining the oxygen atom of a tagged water molecule to the center of the line joining its two hydrogen atoms. The dipole−dipole time correlation function (TCF), Cμ(t), defined as Cμ(t ) =

⟨μî (t ) ·μî (0)⟩ ⟨μî (0) ·μî (0)⟩

(3)

is then calculated to monitor the time evolution of μ⃗. Here, μ̂ i(t) is the ith water molecule’s unit dipole moment vector at time t. The angular brackets indicate that the calculations are 1165


Langmuir

Article

by water molecules present in the first hydration layers around the protein segments, the Cμ(t) decay curves are fitted with biexponentials. The amplitude-weighted ⟨τμ⟩ values as obtained from the fits are listed in Table 1. Longer times taken by the

are performed for the three systems, and the results are shown in Figure 3. With PW electrostatic interactions on, the presence

Table 1. Average Reorientational Times (⟨τμ⟩) for Water Molecules Present in the First Hydration Layers of Different Segments of Barstar As Obtained from Simulations S1, S2, and S3a ⟨τμ⟩ (ps)

a

segment

S1

S2

S3

helix-1 helix-2 helix-3 helix-4 β-sheet loop-1 loop-2 loop-3 loop-4 bulk water

12.90 10.68 12.78 10.92 14.75 16.16 8.64 12.24 14.44

24.52 14.92 14.46 14.72 20.75 17.94 13.77 18.46 11.76 2.03

4.41 4.04 4.07 3.84 4.0 3.99 4.01 4.17 4.01

Figure 3. Residence time correlation function (CR(t)) for water molecules present in the first hydration layers of different segments of barstar for (a) the flexible protein system (simulation S1), (b) the frozen protein system (simulation S2), and (c) the frozen protein system with PW electrostatic interactions turned off (simulation S3).

The corresponding value for pure bulk water is listed for comparison.

surface water molecules to reorient themselves with respect to bulk water are clearly evident for all the three systems. We find that the first hydration layer water molecules around the flexible protein exhibit ⟨τμ⟩ values that are 4−8 times longer than that of pure bulk water. In comparison, the corresponding values are about 6−12 times longer in the frozen form. As discussed above, on freezing the conformational fluctuations, water around loop-4 takes about 20% less time than that in the flexible form to reorient. Near-uniform water reorientation times in the absence of PW electrostatic interactions which are only twice longer than that for bulk water are evident from the calculated data. It may be noted that water exhibits diffusive behavior in bulk state within 1−2 ps. It is evident from the above discussion that although the hydration water molecules exhibit sluggish dynamics, the effects of geometrical and energetic disorders of the protein segments on water mobility are minimal within such a short time period. However, the effects of protein−water correlations on water motions are apparent at relatively longer time scales as described in Figures 1 and 2. 3.3. Water Residence Time. The influence of conformational flexibility and PW energetic disorders on water motions as discussed in earlier sections should affect water residence times at the surface of the protein segments. We explore that by calculating the residence TCF, CR(t), defined as C R (t ) =

⟨b(0)B(t )⟩ ⟨b(0)b(0)⟩

of slower long-time components in the relaxation patterns of the function is evident from Figure 3a and b. This provides direct evidence of the presence of strongly bound water molecules close to the protein surface with relatively long residence times. The results agree well with restricted translational and rotational motions of such water molecules as discussed earlier. The curves are fitted with triexponentials to extract the average residence times (⟨τR⟩) of the first hydration layer water molecules, which are listed in Table 2. It is clear that Table 2. Average Residence Times (⟨τR⟩) of Water Molecules Present in the First Hydration Layers of Different Segments of Barstar As Obtained from Simulations S1, S2, and S3a ⟨τR⟩ (ps) segment

S1

S2

S3

helix-1 helix-2 helix-3 helix-4 β-sheet loop-1 loop-2 loop-3 loop-4

16.03 9.70 15.24 11.85 14.28 11.64 8.27 11.01 11.85

44.26 27.63 25.83 19.88 29.87 26.18 20.18 22.64 19.97

10.17 8.14 10.18 8.85 7.02 8.32 6.69 6.86 9.46

a

(4)

The corresponding value for water in pure bulk state is listed for comparison.

the variable b(0) can be either 1 or 0 depending on whether a particular water molecule is in the first hydration layer of a protein segment at time t = 0 or not. On the other hand, the variable B(t) is 1 when the tagged water molecule remains continuously in the first hydration layer of a segment from time t = 0 to a later time t. As mentioned earlier, the angular brackets signify that the calculations are carried out by averaging over all such tagged water molecules and over different time origins. By definition, CR(t) should provide an estimation of residence times of the surface water molecules. As before, the calculations

due to increased confinement on freezing the protein’s conformational motions, the surface water molecules experience even stronger electric fields from the charged and polar residue groups and hence reside longer in the first hydration layers around the protein segments. A degree of heterogeneity in the relaxation patterns of the function indicating nonuniform water residence times around the rigid secondary structures (αhelices and β-sheet) has also been observed. The effect is found to be more for the frozen protein system (Figure 3b). It is 1166


Langmuir

Article

hydrogen bonds formed at different reference initial times. According to the definition, C(t) is independent of possible breaking and reformation of hydrogen bonds within the time 0 and t. In other words, C(t) takes into account recrossing of the barrier between the hydrogen-bonded bound and free states, and the long-time diffusive behavior. Thus, the overall relaxation time of a particular type of hydrogen bond can be extracted from the decay pattern of C(t). We now attempt to study how the local conformational motions of the protein segments and the electrostatic component of their interactions with water molecules alter the dynamics of hydrogen bonds at the surface. It may be noted that in the absence of PW electrostatic contributions in system S3, the water molecules can no longer be anchored by the protein residues, thereby resulting in the disappearance of the formation of PW hydrogen bond connectivity at the surface. However, lack of PW hydrogen bond formation in system S3 may modify the characteristics of WW hydrogen bonds formed by the surface water molecules differently than that in the presence of anchored water at the surface in systems S1 and S2. Thus, in the subsequent discussions, we analyze and compare PW hydrogen bond dynamics between systems S1 and S2, while the properties of WW hydrogen bonds involving the first hydration layer water molecules are compared between all the three systems. The relaxation patterns of the intermittent hydrogen bond TCF, CPW(t), for the PW hydrogen bonds formed by different segments of barstar in its flexible and frozen forms as obtained from simulations S1 and S2 are shown in Figure 4. As a

further evident that in the absence of electrostatic anchoring sites at the protein surface (Figure 3c), the water molecules present in the first hydration layers exhibit shorter residence times. Therefore, it is clear that both conformational fluctuations of the protein and PW electrostatic interactions affect water residence times near the surface. A comparison between the ⟨τR⟩ values reveals that while inclusion of protein flexibility leads to 1.7−2.8 times reduction in water residence times, neglect of PW electrostatic contributions results in 3−4 times shorter water residence times around most of the protein segments. This demonstrates that compared to the protein’s conformational fluctuations, the local fields generated by the polar and charged residues have relatively greater effects in altering water residence times at the surface. Such behavior of surface water molecules for the three systems agrees well with their translational and rotational motions as discussed in the previous section. 3.4. Hydrogen Bonds around the Protein. It is known that the presence of a protein in an aqueous medium alters the regular WW hydrogen bond network with the formation of PW hydrogen bonds at the surface.33,36 The time scales associated with the formation and breaking of such hydrogen bonds anchored at the protein surface should be correlated with reduced mobility of surface water molecules. MD simulations can provide quantitative information on the dynamics of hydrogen bonds through calculation of different time correlation functions, as suggested first by Stillinger64 and developed later by Luzar and Chandler.65,66 In this section, we study the hydrogen bond dynamics around the protein segments in the three simulation systems. To obtain information on hydrogen bonds from simulated trajectories, it is necessary to define those first. Generally, one uses either a geometry-based33,67,68 or an energy-based69,70 criterion to define a hydrogen bond. Here, we have employed criteria based on geometrical arrangements of appropriate atoms to identify both PW68 and WW hydrogen bonds.65,71,72 According to the criteria adopted, the first condition for an acceptor or donor atom of the protein to form a hydrogen bond with a water molecule is that the distance between the protein atom and the oxygen atom of the tagged water be within 3.3 Å. The second condition for a protein acceptor atom to form a PW hydrogen bond is that the angle between one of the O−H bond vectors of the water and the vector connecting the hydrogen atom and the acceptor atom be within 30°. On the other hand, for a protein donor atom the angle between one of the O−H bond vectors of the water molecule and the vector connecting the water oxygen atom and the hydrogen atom attached with the donor atom should be within 80°−140° for forming a PW hydrogen bond.68 Besides, a hydrogen bond is said to have formed between a pair of water molecules if the interoxygen distance is less than 3.5 Å and the oxygen− oxygen−hydrogen angle is less than 30°.65,71,72 We have characterized the dynamics of hydrogen bonds by calculating the intermittent hydrogen bond TCF (C(t)),64,73 defined as

C(t ) =

⟨h(0)h(t )⟩ ⟨h(0)h(0)⟩

Figure 4. Intermittent time correlation function (CPW(t)) for the PW hydrogen bonds formed between the water molecules and the residues of different segments of barstar for (a) the flexible protein system (simulation S1) and (b) the frozen protein system (simulation S2). As a reference, the function CWW(t) for pure bulk water is included in the inset.

reference, the decay of CWW(t) for water in pure bulk state is included in the inset. It is clear that the overall relaxation of PW hydrogen bonds occurs on a longer time scale as compared to that for WW hydrogen bonds in pure bulk water. Such slower PW hydrogen bond relaxation is consistent with restricted translational and rotational motions of water molecules present at the surface of the protein segments as described before (see Sections 3.1 and 3.2). To probe the origin of such behavior, we have calculated the strengths of the PW hydrogen bonds

(5)

where h(t) is a hydrogen bond population variable that can be either 1 or 0. If a pair of sites are hydrogen bonded at a particular time t, then h(t) = 1, otherwise it is 0. The angular brackets denote that the calculations are averaged over all the 1167


Langmuir

Article

formed at the interface. The PW hydrogen bond strength is defined as the interaction energy between the amino acid residue and the water molecule hydrogen-bonded to it. On average, this energy is found to be within −8 to −11 kcal mol−1 for different segments of the protein, which is much lower than the regular hydrogen-bonded pair interaction energy of −3.9 kcal mol−1 in pure bulk water. This shows that water molecules at the protein surface would prefer to anchor with the residues by stronger hydrogen bonds. The presence of such strongly bound water molecules at the surface results in slower structural relaxation of PW hydrogen-bonded moieties. Further, it is found that increased confinement of surface water molecules on freezing the protein’s conformational fluctuations leads to an even longer time scale of PW hydrogen bond relaxation (see Figure 4b). This results in increased rigidity of the protein hydration layer, as discussed in earlier sections. In addition, differential degree of confinement around the protein segments is evident from nonuniform relaxation of CPW(t) around the protein in its frozen form. This is particularly noticeable at the surfaces of the α-helices and β-sheet. We have fitted each of the decay curves with a sum of three exponentials to obtain the amplitude-weighted PW hydrogen bond relaxation times, ⟨τPW C ⟩, as listed in Table 3. It can be seen that compared to

Figure 5. Intermittent time correlation function (CWW(t)) for the WW hydrogen bonds formed by the water molecules present in the first hydration layers of different segments of barstar for (a) the flexible protein system (simulation S1), (b) the frozen protein system (simulation S2), and (c) the frozen protein system with PW electrostatic interactions turned off (simulation S3).

Table 3. Average Relaxation Times (⟨τPW C ⟩) Calculated From CPW(t) for PW Hydrogen Bonds between Different Segments of Barstar and Water As Obtained From Simulations S1 and S2a ⟨τPW C ⟩ (ps)

⟨τWW C ⟩ (ps)

segment

S1

S2

S1

S2

S3

helix-1 helix-2 helix-3 helix-4 β-sheet loop-1 loop-2 loop-3 loop-4 bulk water

26.48 24.62 37.87 31.43 35.95 33.71 31.26 29.83 27.18

51.51 34.25 44.46 40.04 82.29 32.29 42.72 33.35 31.89

4.26 4.48 4.76 8.40 3.74 3.54 3.40 3.80 3.15

5.78 5.42 4.84 3.86 6.53 4.58 4.76 4.48 4.11 2.70

3.39 3.19 3.24 3.13 3.38 3.06 2.89 3.16 3.18

−

is flexible or not. Nonuniform relaxations of the function around the α-helices and β-sheet further indicate water molecules experiencing heterogeneous environments around them. In contrast, near-uniform WW hydrogen bond dynamics suggest identical environments around the loops. It can be seen from Figure 5c that in the absence of electrostatic anchoring sites at the surface, the water molecules surrounding the protein tend to exhibit uniform bulklike hydrogen bond dynamics. It is found that on average the number of WW hydrogen bonds per hydration layer water molecule is ∼2.5 for the systems S1 and S2 and ∼3 for S3. This shows that the inability of the surface water molecules to form hydrogen bonds with the protein residues in the absence of PW electrostatic interactions is compensated by enhanced WW hydrogen bonding. Such behavior is consistent with increased fraction of WW hydrogen bonds at a hydrocarbon−water interface as reported in an earlier work by Garde and co-workers.74 Following the procedure as described above, we have calculated the average WW hydrogen bond relaxation times (⟨τWW C ⟩) from the decay curves, and the values are listed in Table 3. Note that in the presence of strong PW electrostatic contributions, the WW hydrogen bonds formed by the first hydration layer water molecules relax on a time scale noticeably longer (often 2−3 times) than that of water in pure bulk state. However, on switching off the PW electrostatic contributions, the relaxation times of WW hydrogen bonds become only marginally longer than bulk water. The above results indicate enhanced correlations between water molecules at the protein surface in the absence of PW electrostatic interactions, a signature of increased hydrophobicity. It is demonstrated recently that water number fluctuations increase near hydrophobic interfaces.52Consistent with such studies, our preliminary analysis reveals relatively faster time scale of water number fluctuations around the increasingly hydrophobic protein molecule in the absence of PW electrostatic contributions. However, further studies are necessary to correlate the microscopic properties of water molecules hydrating a heterogeneous protein surface with such molecular measures of hydrophobicity.

The corresponding relaxation times (⟨τWW C ⟩) calculated from CWW(t) for WW hydrogen bonds involving the first hydration layer water molecules around the protein segments as obtained from simulations S1, S2, and S3 along with the data for pure bulk water are also listed.

a

WW hydrogen bonds in pure bulk water, the PW hydrogen bonds involving most of the protein segments take an order of magnitude longer time to relax. Due to increased confinement at the protein surface on freezing its local motions, the PW hydrogen bonds require even longer durations to relax. The effect can often be significant as evident from more than twice longer ⟨τPW C ⟩ values involving helix-1 and β-sheet residues in the frozen protein as compared to that in the flexible form. In Figure 5, the relaxation patterns of the function CWW(t) for WW hydrogen bonds formed by the first hydration layer water molecules around the protein segments are displayed for the three systems. It is clear that with respect to water in pure bulk state, formation of strong PW hydrogen bonds in systems S1 and S2 results in relatively slower relaxation of WW hydrogen bonds near the protein surface. The effect is found to be more for water around the regular secondary structures (αhelices and β-sheet) and is independent of whether the protein 1168


Langmuir

Article

3.5. Water Diffusion and Hydrogen Bond Dynamics. As already pointed out, the dynamics of hydrogen bonds formed between a pair of molecules is correlated with their diffusions.65,66,76 Slower diffusion allows reformation of broken hydrogen bonds, which in turn results in long-time hydrogen bond relaxation and vice versa. Such coupling is considered to be the origin of the nonexponential relaxation behavior of hydrogen bond TCFs.65 As the mass of a biomolecule is orders of magnitude more than that of a water molecule, the dynamics of hydrogen bonds at its surface is expected to be correlated with the diffusion time scale of the surface water molecules. The results showed in Figures 4 and 5 reveal the existence of such correlation for both PW and WW hydrogen bonds at the protein surface. Here, we attempt to eliminate the contributions from water motions on hydrogen bond dynamics. This is done by calculating the TCF, N(t), defined as65,66,76,77 N (t ) =

⟨h(0)(1 − h(t ))H′(t )⟩ ⟨h(0)h(0)⟩

layer of bound water molecules, which contributes to retarded water motions and the long-time scale hydrogen bond relaxations at the protein surface, as discussed earlier. Reduced dimension of the protein hydration layer with increased confinement in the absence of local conformational motions further enhances the rigidity at the surface, as evident from Figure 6b. Consistent with our earlier findings, the effect is higher around the α-helices and the β-sheet of the protein. Differential effects of confinement around the secondary structures of the protein particularly in its frozen form are also evident from the figure. How the function NWW(t) for WW hydrogen bonds formed by the water molecules present in the first hydration layers around the protein segments relaxes for all the three systems is shown in Figure 7. The rigid nature of the

(6)

for both PW and WW hydrogen bonds. H′(t) is a variable which is unity if two sites are within a distance, RH (3.3 Å for PW and 3.5 Å for WW hydrogen bonds), at time t and zero otherwise. A nonzero N(t) value means the pair of sites are not hydrogen-bonded, but they remain in the vicinity of each other within RH. A value of zero, on the other hand, suggests that the two sites either exist in the bonded form or are separated by a distance larger than RH. Therefore, N(t) provides an estimate of the time-dependent probability that a particular hydrogen bond is broken at time t, but the tagged molecules have not diffused away. In the present case, N(t) can relax either due to reformation of broken hydrogen bonds or due to diffusion of water.65 In Figure 6 the relaxations of NPW(t) involving PW hydrogen bonds formed by the protein segments in both flexible and frozen forms as obtained from simulations S1 and S2 are displayed. The result for pure bulk water (NWW(t)) is also included in the inset of the figure for comparison. Slower relaxation of the function characterizes the rigidity of the first

Figure 7. Time-dependent probability that a WW hydrogen bond is broken but the two tagged water molecules remain in the vicinity of each other (i.e., within RH). The calculations are carried out for the water molecules present in the first hydration layers of different segments of barstar for (a) the flexible protein system (simulation S1), (b) the frozen protein system (simulation S2), and (c) the frozen protein system with PW electrostatic interactions turned off (simulation S3).

protein hydration layer is evident from the data. It is clear from Figure 7b that increasing degree of confinement around the protein on freezing its local motions and the presence of polar and charged residue groups anchoring the surface water molecules by strong hydrogen bonds lead to highly rigid hydration layers around the segments. Allowing the local conformational motions of the protein reduces the hydration layer rigidity to some extent as evident from Figure 7a. However, greater sensitivity of a protein’s hydration layer rigidity on PW electrostatic forces is evident from much faster relaxation of NWW(t) in system S3. 3.6. Hydrogen Bond Formation and Breaking Kinetics. In this section, we investigate the influence of the local conformational motions of the protein segments and the PW electrostatic interactions on the kinetics of hydrogen bond formation and breaking. Following the approach as suggested by Luzar and Chandler,65,66 we describe the kinetics as

Figure 6. Time-dependent probability that a PW hydrogen bond is broken but the tagged water molecule remains in the vicinity (i.e., within RH) of the particular residue (NPW(t)). The calculations are carried out for different segments of barstar for (a) the flexible protein system (simulation S1) and (b) the frozen protein system (simulation S2). As a reference, the function NWW(t) for pure bulk water is included in the inset.

B ⇌ QF

(7)

B is defined as the bound state, where a tagged water molecule can be found involved in a PW or a WW hydrogen bond, and QF is the quasi-free state, where the hydrogen bond is broken but the tagged water molecule remains within the distance RH 1169


Langmuir

Article

(first coordination shell) from the other site. Following the definitions, the functions C(t) and N(t) correspond to the populations of B and QF states that can interconvert according to eq 8. Now, if k1 and k2 are the breaking (forward) and the reformation (backward) rate constants, then a rate equation can be written as65,66 k(t ) = −

dC(t ) = k1C(t ) − k 2N (t ) dt

(8)

k(t) can attain equilibrium by transitions from state B to state QF (i.e., reactants to products). We have calculated the time evolutions of kPW(t) associated with the breaking and reformation of PW hydrogen bonds formed by the protein segments in both flexible and frozen forms as obtained from simulations S1 and S2. The calculations are done from the derivatives of the intermittent hydrogen bond TCF, CPW(t). These results are shown in Figure 8. As a

Figure 9. WW hydrogen bond reactive flux, kWW(t) (semilog plot), for the breaking and reformation of WW hydrogen bonds formed by the water molecules present in the first hydration layers of different segments of barstar for (a) the flexible protein system (simulation S1), (b) the frozen protein system (simulation S2), and (c) the frozen protein system with PW electrostatic interactions turned off (simulation S3).

Figure 8. PW hydrogen bond reactive flux, kPW(t) (semilog plot), for the breaking and reformation of PW hydrogen bonds formed between the water molecules and the residues of different segments of barstar for (a) the flexible protein system (simulation S1) and (b) the frozen protein system (simulation S2). The function kWW(t) for pure bulk water in included as a reference. As a reference, the function kWW(t) for pure bulk water is included in the inset.

presence of electrostatic anchoring sites at the protein surface, the relaxation of the function is relatively slower than that of bulk water. Expectedly, the degree of slowness is much less than that observed for the strongly bound PW hydrogen bonds (Figure 8). Heterogeneous environment around different secondary structures (particularly the α-helices and the βsheet) of the protein originating from their nonuniform local motions and distribution of polar and charged groups is evident from the Figure 9a and b. The importance of PW electrostatic contributions in controlling water properties around the protein segments is further evident from near bulklike kinetics of WW hydrogen bonds in the absence of water anchoring sites at the protein surface as observed in Figure 9c. Such behavior of WW hydrogen bonds at the surface in the absence of electrostatic forces originating from the protein is consistent with relatively faster relaxation times of hydration water molecules.

reference, the corresponding function kWW(t) for pure bulk water is shown in the inset of the figure. It can be seen that the function kPW(t) relaxes significantly slowly compared to pure bulk water. This is true for the PW hydrogen bonds formed by all the segments irrespective of whether their local conformational motions are frozen or not. Such slow relaxation of kPW(t) indicating rapid attainment of PW hydrogen bond breaking and reformation equilibria (eq 7) arises due to restricted motions of water molecules confined at the surface and the slow long-time dynamics of PW hydrogen bonds, as discussed in the earlier sections. Interestingly, the calculations reveal near uniform kinetics involving different segments which is unaffected by the protein’s conformational flexibilities. This is an important observation which shows that although the increase in confinement at the protein surface on freezing its conformational oscillations modifies the time scale of PW hydrogen bond relaxation (Figure 4) it does not affect the local kinetics of the breaking and formation of such bonds. The relaxation patterns of kWW(t) corresponding to the WW hydrogen bonds formed by the water molecules hydrating the protein segments in the three systems are shown in Figure 9. It is found that in the

4. CONCLUSIONS In this work we have performed detailed atomistic MD simulations to study the influence of the local conformational motions and the distribution of electrostatic anchoring sites at the surface of the protein barstar on the microscopic dynamical properties of the surrounding water molecules. This is done by comparing the results obtained from the simulation of the flexible protein molecule interacting with the solvent with full potential form to that obtained from two additional simulations. In one of these two simulations, the protein conformation is kept frozen, and in the other the PW electrostatic contributions are turned off in addition to the frozen protein matrix. As a reference, the results are compared with that of water in pure bulk state. The calculated results reveal that the water molecules hydrating the protein surface exhibit restricted translational and rotational motions irrespective of whether the protein degrees of freedom are kept frozen and/or the PW electrostatic interactions are turned off. It is observed that enhanced degree of confinement at the protein surface on arresting its conformational motions results in increasingly restricted 1170


Langmuir

Article

(7) Guha, S.; Sahu, K.; Roy, D.; Mondal, S. K.; Roy, S.; Bhattacharyya, K. Slow Solvation Dynamics at the Active Site of an Enzyme: Implications for Catalysis. Biochemistry 2005, 44, 8940−8947. (8) Sen, P.; Mukherjee, S.; Dutta, P.; Halder, A.; Mandal, D.; Banerjee, R.; Roy, S.; Bhattacharyya, K. Solvation Dynamics in the Molten Globule State of a Protein. J. Phys. Chem. B 2003, 107, 14563− 14568. (9) Li, T.; Hassanali, A. A.; Kao, Y.-T.; Zhong, D.; Singer, S. J. Hydration Dynamics and Time Scales of Coupled Water−Protein Fluctuations. J. Am. Chem. Soc. 2007, 129, 3376−3382. (10) Abbyad, P.; Shi, X.; Childs, W.; McAnaney, T. B.; Cohen, B. E.; Boxer, S. G. Measurement of Solvation Responses at Multiple Sites in a Globular Protein. J. Phys. Chem. B 2007, 111, 8269−8276. (11) Zhang, L.; Yang, Y.; Kao, Y. T.; Wang, L.; Zhong, D. Protein Hydration Dynamics and Molecular Mechanism of Coupled Water− Protein Fluctuations. J. Am. Chem. Soc. 2009, 131, 10677−10691. (12) Kwon, O.-H.; Yoo, T. H.; Othon, C. M.; Deventer, J. A. V.; Tirrell, D. A.; Zewail, A. H. Hydration Dynamics at Fluorinated Protein Surfaces. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 17101−17106. (13) Jha, A.; Ishii, K.; Udgaonkar, J. B.; Tahara, T.; Krishnamoorthy, G. Exploration of the Correlation between Solvation Dynamics and Internal Dynamics of a Protein. Biochemistry 2011, 50, 397−408. (14) Malardier-Jugroot, C.; Johnson, M. E.; Murarka, R. K.; HeadGordon, T. Aqueous Peptides as Experimental Models for Hydration Water Dynamics Near Protein Surfaces. Phys. Chem. Chem. Phys. 2008, 10, 4903−4908. (15) Khodadadi, S.; Pawlus, S.; Sokolov, A. P. Influence of Hydration on Protein Dynamics: Combining Dielectric and Neutron Scattering Spectroscopy Data. J. Phys. Chem. B 2008, 112, 14273−14280. (16) Otting, G.; Liepinsh, E.; Wüthrich, K. Protein Hydration in Aqueous Solution. Science 1991, 254, 974−980. (17) Wüthrich, K.; Billeter, M.; Güntert, P.; Luginbühl, P.; Riek, R.; Wider, G. NMR Studies of the Hydration of Biological Macromolecules. Faraday Discuss. 1996, 103, 245−253. (18) Mattea, C.; Qvist, J.; Halle, B. Dynamics at the Protein−Water Interface from 17O Spin Relaxation in Deeply Supercooled Solutions. Biophys. J. 2008, 95, 2951−2963. (19) Halle, B.; Nilsson, L. Does the Dynamic Stokes Shift Report on Slow Protein Hydration Dynamics? J. Phys. Chem. B 2009, 113, 8210− 8213. (20) Orecchini, A.; Paciaroni, A.; Francesco, A. D.; Petrillo, C.; Sacchetti, F. Collective Dynamics of Protein Hydration Water by Brillouin Neutron Spectroscopy. J. Am. Chem. Soc. 2009, 131, 4664− 4669. (21) Russo, D.; Teixeira, J.; Ollivier, J. The Impact of Hydration Water on the Dynamics of Side Chains of Hydrophobic Peptides: From Dry Powder to Highly Concentrated Solutions. J. Chem. Phys. 2009, 130, 235101. (22) Russo, D.; Teixeira, J.; Kneller, L.; Copley, J. R. D.; Ollivier, J.; Perticaroli, S.; Pellegrini, E.; Gonzalez, M. A. Vibrational Density of States of Hydration Water at Biomolecular Sites: Hydrophobicity Promotes Low Density Amorphous Ice Behavior. J. Am. Chem. Soc. 2011, 133, 4882−4888. (23) Combet, S.; Zanotti, J. M. Further Evidence That Interfacial Water Is the Main Driving Force of Protein Dynamics: A Neutron Scattering Study on Perdeuterated C-Phycocyanin. Phys. Chem. Chem. Phys. 2012, 14, 4927−4934. (24) Ebbinghaus, S.; Kim, S. J.; Heyden, M.; Yu, X.; Gruebele, M.; Leitner, D. M.; Havenith, M. Protein Sequence- and pH-Dependent Hydration Probed by Terahertz Spectroscopy. J. Am. Chem. Soc. 2008, 130, 2374−2375. (25) Xu, J.; Plaxco, K. W.; Allen, S. J. Collective Dynamics of Lysozyme in Water: Terahertz Absorption Spectroscopy and Comparison with Theory. J. Phys. Chem. B 2006, 110, 24255−24259. (26) Woods, K. N. Solvent-Induced Backbone Fluctuations and the Collective Librational Dynamics of Lysozyme Studied by Terahertz Spectroscopy. Phys. Rev. E 2010, 81, 031915.

mobility of water molecules (longer residence times) present in the first hydration layers around the protein segments. On the other hand, in the absence of electrostatic anchoring sites at the surface, the hydration layer water molecules are found to exhibit homogeneous dynamics with time scales of their translational and rotational motions approaching that of pure bulk water. The calculations demonstrate further that the ability of the water molecules to form stronger PW hydrogen bonds leads to their relaxations on a time scale significantly longer (often by an order of magnitude) than that for WW hydrogen bonds around the protein segments in their hydration layers and in the bulk state. The PW hydrogen bonds take even longer times to relax due to increased confinement with consequent enhanced rigidity of water layer at the surface on freezing the protein’s conformational degrees of freedom. It is shown that the retardation in hydration water dynamics originates from such water molecules that are bound at the surface by strong PW hydrogen bonds. Importantly, it is demonstrated that the inability of the surface water molecules to form hydrogen bonds with the protein segments in the absence of PW electrostatic interactions is compensated by enhanced WW hydrogen bonding around them with uniform bulklike relaxations. Besides, it is found that the rigidity of the protein’s hydration layer is highly sensitive to PW electrostatic contributions. Further, our calculations reveal for the first time that although the reduced dimension of the protein hydration layer on freezing its local motions modifies the time scale of PW hydrogen bond relaxation the local kinetics of the breaking and formation of such bonds are not altered as such. We believe that these findings can provide valuable insights in understanding the role played by water in different processes involving proteins and other large biomolecules.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported in part by grants from the Department of Science and Technology (DST), Government of India. Part of the work was carried out using the computational facility created under DST-FIST programme (SR/FST/CSII-011/2005). S.P. thanks the Council for Scientific and Industrial Research (CSIR), New Delhi for providing a scholarship.

■

REFERENCES

(1) Protein-Solvent Interaction; Gregory, R. B., Ed.; Marcel Dekker: New York, 1995. (2) Nandi, N.; Bagchi, B. Dielectric Relaxation of Biological Water. J. Phys. Chem. B 1997, 101, 10954−10961. (3) Pal, S. K.; Peon, J.; Bagchi, B.; Zewail, A. H. Biological Water: Femtosecond Dynamics of Macromolecular Hydration. J. Phys. Chem. B 2002, 106, 12376−12395. (4) Bhattacharyya, K. Nature of Biological Water: A Femtosecond Study. Chem. Commun. 2008, 2850, 2848−2857. (5) Zhong, D.; Pal, S. K.; Zewail, A. H. Biological Water: A Critique. Chem. Phys. Lett. 2011, 503, 1−11. (6) Bagchi, B. From Anomalies in Neat Liquid to Structure, Dynamics and Function in the Biological World. Chem. Phys. Lett. 2012, 529, 1−9. 1171


Langmuir

Article

(27) Lipps, F.; Levy, S.; Markelz, A. G. Hydration and Temperature Interdependence of Protein Picosecond Dynamics. Phys. Chem. Chem. Phys. 2012, 14, 6375−6381. (28) Bryan, M. A.; Brauner, J. W.; Anderle, G.; Flach, C. R.; Brodsky, B.; Mendelsohn, R. FTIR Studies of Collagen Model Peptides: Complementary Experimental and Simulation Approaches to Conformation and Unfolding. J. Am. Chem. Soc. 2007, 129, 7877−7884. (29) Liltorp, K.; Maréchal, Y. Hydration of Lysozyme As Observed by Infrared Spectrometry. Biopolymers 2005, 79, 185−196. Maréchal, Y. Observing the Water Molecule in Macromolecules Using Infrared Spectrometry: Structure of the Hydrogen Bond Network and Hydration Mechanism. J. Mol. Struct. 2004, 700, 217−223. (30) Bagchi, S.; Nebgen, B. T.; Loring, R. F.; Fayer, M. D. Dynamics of a Myoglobin Mutant Enzyme: 2D IR Vibrational Echo Experiments and Simulations. J. Am. Chem. Soc. 2010, 132, 18367−18376. Bagchi, S.; Thorpe, D. G.; Thorpe, I. F.; Voth, G. A.; Fayer, M. D. Conformational Switching between Protein Substates Studied with 2D IR Vibrational Echo Spectroscopy and Molecular Dynamics Simulations. J. Phys. Chem B. 2010, 114, 17187−17193. (31) Panagopoulou, A.; Kyritsis, A.; Shinyashiki, N.; Pissis, P. Protein and Water Dynamics in Bovine Serum Albumin−Water Mixtures over Wide Ranges of Composition. J. Phys. Chem. B 2012, 116, 4593−4602. (32) Rocchi, C.; Bizzarri, A. R.; Cannistraro, S. Water Dynamical Anomalies Evidenced by Molecular-Dynamics Simulations at the Solvent−Protein Interface. Phys. Rev. E 1998, 57, 3315−3325. (33) Bizzarri, A. R.; Cannistraro, S. Molecular Dynamics of Water at the Protein Solvent Interface. J. Phys. Chem. B 2002, 106, 6617−6633. (34) Marchi, M.; Sterpone, F.; Ceccarelli, M. Water Rotational Relaxation and Diffusion in Hydrated Lysozyme. J. Am. Chem. Soc. 2002, 124, 6787−6791. (35) Wong, V.; Case, D. A. Evaluating Rotational Diffusion from Protein MD Simulations. J. Phys. Chem. B 2008, 112, 6013−6024. (36) Sinha, S. K.; Bandyopadhyay, S. Local Heterogeneous Dynamics of Water Around Lysozyme: A Computer Simulation Study. Phys. Chem. Chem. Phys. 2012, 14, 899−913. (37) Sinha, S. K.; Bandyopadhyay, S. Polar Solvation Dynamics of Lysozyme from Molecular Dynamics Studies. J. Chem. Phys. 2012, 136, 185102. (38) Pizzitutti, F.; Marchi, M.; Sterpone, F.; Rossky, P. J. How Protein Surfaces Induce Anomalous Dynamics of Hydration Water. J. Phys. Chem. B 2007, 111, 7584−7590. (39) Xu, H.; Berne, B. J. Hydrogen-Bond Kinetics in the Solvation Shell of a Polypeptide. J. Phys. Chem. B 2001, 105, 11929−11932. (40) Chakraborty, S.; Sinha, S. K.; Bandyopadhyay, S. LowFrequency Vibrational Spectrum of Water in the Hydration Layer of a Protein: A Molecular Dynamics Simulation Study. J. Phys. Chem. B 2007, 111, 13626−13631. (41) Chakraborty, S.; Bandyopadhyay, S. Correlation between the Dynamics of Hydrogen Bonds and the Local Density Reorganization in the Protein Hydration Layer. J. Phys. Chem. B 2007, 111, 7626− 7630. (42) Tarek, M.; Tobias, D. J. The Dynamics of Protein Hydration Water: A Quantitative Comparison of Molecular Dynamics Simulations and Neutron-Scattering Experiments. Biophys. J. 2000, 79, 3244−3257. Tarek, M.; Tobias, D. J. Role of Protein−Water Hydrogen Bond Dynamics in the Protein Dynamical Transition. Phys. Rev. Lett. 2002, 88, 138101. (43) Sterpone, F.; Stirnemann, G.; Laage, D. Magnitude and Molecular Origin of Water Slowdown Next to a Protein. J. Am. Chem. Soc. 2012, 134, 4116−4119. (44) Merzel, F.; Smith, J. C. Is the First Hydration Shell of Lysozyme of Higher Density than Bulk Water? Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5378−5383. (45) Merzel, F.; Smith, J. C. High-Density Hydration Layer of Lysozymes: Molecular Dynamics Decomposition of Solution Scattering Data. J. Chem. Inf. Model. 2005, 45, 1593−1599. (46) Smolin, N.; Winter, R. Molecular Dynamics Simulations of Staphylococcal Nuclease: Properties of Water at the Protein Surface. J. Phys. Chem. B 2004, 108, 15928−15937.

(47) Agarwal, M.; Kushwaha, H. R.; Chakravarty, C. Local Order, Energy, and Mobility of Water Molecules in the Hydration Shell of Small Peptides. J. Phys. Chem. B 2010, 114, 651−659. (48) Sinha, S. K.; Bandyopadhyay, S. Differential Flexibility of the Secondary Structures of Lysozyme and the Structure and Ordering of Surrounding Water Molecules. J. Chem. Phys. 2011, 134, 115101. (49) Heyden, M.; Havenith, M. Combining THz Spectroscopy and MD Simulations to Study Protein−Hydration Coupling. Methods 2010, 52, 74−83. (50) Perera, P. N.; Fega, K. R.; Lawrence, C.; Sundstrom, E. J.; Tomlinson-Phillips, J.; Ben-Amotz, D. Observation of Water Dangling OH Bonds Around Dissolved Nonpolar Groups. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 12230−12234. (51) Sarupria, S.; Garde, S. Quantifying Water Density Fluctuations and Compressibility of Hydration Shells of Hydrophobic Solutes and Proteins. Phys. Rev. Lett. 2009, 103, 037803. (52) Jamadagni, S. N.; Godawat, R.; Garde, S. Hydrophobicity of Proteins and Interfaces: Insights From Density Fluctuations. Annu. Rev. Chem. Biomol. Eng. 2011, 2, 147−171. (53) Pal, S.; Bandyopadhyay, S. Effects of Protein Conformational and Energetic Heterogeneities on Water Properties. Submitted. (54) Lubienski, M. J.; Bycroft, M.; Freund, S. M. V.; Fersht, A. R. Three-Dimensional Solution Structure and 13C Assignments of Barstar Using Nuclear Magnetic Resonance Spectroscopy. Biochemistry 1994, 33, 8866−8877. (55) Buckle, A. M.; Schreiber, G.; Fersht, A. R. Protein−Protein Recognition: Crystal Structural Analysis of a Barnase−Barstar Complex at 2.0-Å Resolution. Biochemistry 1994, 33, 8878−8889. (56) Philips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorsid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781− 1802. (57) Feller, S. E.; Zhang, Y.; Pastor, R. W.; Brooks, B. R. Constant Pressure Molecular Dynamics Simulation: The Langevin Piston Method. J. Chem. Phys. 1995, 103, 4613−4621. (58) Allen, M. P.; Tidlesley, D. J. Computer Simulation of Liquids; Clarendon: Oxford, U.K., 1987. (59) Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An Nlog(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089−10092. (60) MacKerell, A. D., Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L., Jr.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E., III; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem. B 1998, 102, 3586−3616. (61) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926−935. (62) Sinha, S. K.; Chakraborty, S.; Bandyopadhyay, S. Thickness of the Hydration Layer of a Protein From Molecular Dynamics Simulation. J. Phys. Chem. B 2008, 112, 8203−8209. (63) Sinha, S. K.; Chakraborty, S.; Bandyopadhyay, S. Effect of Unfolding on the Thickness of the Hydration Layer of a Protein. Ind. J. Phys. 2009, 83, 49−64. (64) Stillinger, F. H. Water Revisited. Science 1980, 209, 451−457. Stillinger, F. H. Theory and Molecular Models for Water. Adv. Chem. Phys. 1975, 31, 1−101. (65) Luzar, A.; Chandler, D. Hydrogen-Bond Kinetics in Liquid Water. Nature 1996, 379, 55−57. Luzar, A.; Chandler, D. Effect of Environment on Hydrogen Bond Dynamics in Liquid Water. Phys. Rev. Lett. 1996, 76, 928−931. (66) Luzar, A. Resolving the Hydrogen Bond Dynamics Conundrum. J. Chem. Phys. 2000, 113, 10663−10675. Luzar, A. Extent of InterHydrogen Bond Correlations in Water. Temperature Effect. Chem. Phys. 2000, 258, 267−276. 1172


Langmuir

Article

(67) Berendsen, H. J. C.; van Gunsteren, W. F.; Zwinderman, H. R. J.; Geurtsen, R. G. Simulations of Proteins in Water. Ann. N.Y. Acad. Sci. 1986, 482, 269−286. (68) Reddy, C. K.; Das, A.; Jayaram, B. Do Water Molecules Mediate Protein-DNA Recognition? J. Mol. Biol. 2001, 314, 619−632. (69) Mezei, M.; Beveridge, D. L. Theoretical Studies of Hydrogen Bonding in Liquid Water and Dilute Aqueous Solutions. J. Chem. Phys. 1981, 74, 622−632. (70) Stillinger, F. H.; Rahman, A. Improved Simulation of Liquid Water by Molecular Dynamics. J. Chem. Phys. 1974, 60, 1545−1557. (71) Luzar, A.; Chandler, D. Structure and Hydrogen Bond Dynamics of Water−Dimethyl Sulfoxide Mixtures by Computer Simulations. J. Chem. Phys. 1993, 98, 8160−8173. (72) Chandra, A. Effects of Ion Atmosphere on Hydrogen−Bond Dynamics in Aqueous Electrolyte Solutions. Phys. Rev. Lett. 2000, 85, 768−771. Chandra, A. Dynamical Behavior of Anion−Water and Water−Water Hydrogen Bonds in Aqueous Electrolyte Solutions: A Molecular Dynamics Study. J. Phys. Chem. B 2003, 107, 3899−3906. (73) Rapaport, D. C. Hydrogen Bonds in Water Network Organization and Lifetimes. Mol. Phys. 1983, 50, 1151−1162. (74) Patel, H. A.; Nauman, E. B.; Garde, S. Molecular Structure and Hydrophobic Solvation Thermodynamics at an Octane−Water Interface. J. Chem. Phys. 2003, 119, 9199−9206. (75) Kropman, M. F.; Bakker, H. J. Dynamics of Water Molecules in Aqueous Solvation Shells. Science 2001, 291, 2118−2120. Kropman, M. F.; Bakker, H. J. Femtosecond Mid-Infrared Spectroscopy of Aqueous Solvation Shells. J. Chem. Phys. 2001, 115, 8942−8948. (76) Xu, H.; Stern, H. A.; Berne, B. J. Can Water Polarizability Be Ignored in Hydrogen Bond Kinetics? J. Phys. Chem. B 2002, 106, 2054−2060. (77) Paul, S.; Chandra, A. Hydrogen Bond Dynamics at Vapour− Water and Metal−Water Interfaces. Chem. Phys. Lett. 2004, 386, 218− 224.

1173


Importance of Protein Conformational Motions and Electrostatic

Recommend Documents