Article pubs.acs.org/JPCB
Effects of Protein Conformational Flexibilities and Electrostatic Interactions on the Low-Frequency Vibrational Spectrum of Hydration Water Somedatta Pal and Sanjoy Bandyopadhyay* Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India S Supporting Information *
ABSTRACT: The conformational flexibility of a protein and its ability to form hydrogen bonds with water are expected to influence the microscopic properties of water layer hydrating the protein. Detailed molecular dynamics simulations with an aqueous solution of the globular protein barstar have been carried out to explore such influence on the lowfrequency vibrational spectrum of the hydration water molecules. The calculations reveal that enhanced degree of confinement at the protein surface on freezing its local motions leads to increasingly restricted oscillatory motions of the hydration water molecules as evident from larger blue shifts of the corresponding band. Interestingly, conformational fluctuations of the protein and electrostatic component of its interaction with the solvent have been found to affect the transverse and longitudinal oscillations of hydration water molecules in a nonuniform manner. It is further noticed that the distributions of the lowfrequency modes for the water molecules hydrogen bonded to the residues of different segments of the protein are heterogeneously altered. The effect is more around the frozen protein matrix and agrees well with slower protein−water hydrogen bond relaxations. the fluorescence results contradict with recent NMR dispersion (NMRD) experiments.18,19 The NMRD studies predict much smaller reardation of protein hydration water motions as compared to that obtained from fluorescence measurements. These studies conclude that the slower component observed in fluorescence studies originate from the side chain motions of protein residues rather than from the coupled slow dynamics of surface water. Terahertz (THz) spectroscopy has also been used recently to probe the collective dynamics of solvated protein systems.20−23 Several recent neutron scattering studies have revealed nonexponential relaxation behavior and the amorphous nature of protein hydration water.17,24,25 Infrared (IR) spectroscopy is another important tool that has been used to explore the structure and dynamics of proteins at different hydration levels.26,27 In an important work, ultrafast optical Kerr-effect spectroscopy has been used by Wynne and coworkers28 to understand the influence of PW hydrogen bonds on the intermolecular low-frequency modes of water molecules hydrating the protein surface. Inelastic neutron scattering (INS) study revealed that such low-frequency modes of protein hydration water are similar to that of amorphous ice.29 Computer simulation methods in general and molecular dynamics (MD) in particular have become increasingly powerful tools in recent times to explore PW interactions and their effects on microscopic properties of solvated proteins at different time resolutions. Simulation studies in general
1. INTRODUCTION Protein−water (PW) interaction is one of the most important and perhaps least understood problems in chemical biology.1−3 Water around proteins exhibits properties different from that in the bulk liquid state and is often termed as “biological water”,1 as it plays vital roles in controlling the three-dimensional structures and dynamics of proteins, and hence their functions. As the time scales associated with various dynamical events involving a solvated protein generally vary over wide ranges, it is often a challenge to analyze experimental data. Different experiments measuring different quantities over widely separated time domains further created confusions in interpreting their findings.4,5 As a result, despite significant efforts, a unified microscopic picture of PW interaction in hydrated proteins is yet to emerge. The dynamics of solvated proteins have been extensively studied in recent times using time-resolved fluorescence spectroscopy.6−12 These experiments have high degree of spatial resolutions, and therefore can provide precise local dynamical picture around the solvated probe. In general, bimodal distribution of time scales has been revealed from such studies. The initial component within a few picoseconds has been attributed to the librational/reorientational motions of free bulklike water molecules present near the protein. The relatively slower second component in the range of tens of picoseconds has been identified as the retarded motions of the hydration water molecules that are dynamically coupled with the protein’s local conformational motions. These findings are in agreement with early nuclear Overhauser effect (NOE) NMR13,14 and recent neutron scattering15−17 studies. However, © 2013 American Chemical Society
Received: March 17, 2013 Revised: April 19, 2013 Published: April 19, 2013 5848
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description of the setup of the systems and the methodology employed. The results are presented and discussed in section 3. The important findings and the conclusions reached from our study are highlighted in the last section (section 4).
reveal restricted translational and rotational motions of protein hydration water.30−34 The degree of such restriction has been found to extend beyond the first hydration layer.35 It is further demonstrated that the alignment of water molecules and their energetics around proteins are correlated with the surface topography.36−38 Recently, Chakravarty and co-workers39 studied in great detail the ordering and energetics of water molecules around peptides in aqueous media. Pettitt and coworkers40 showed from MD simulations that the water residence time at a hydration site around a protein can vary from extremely short to long times. It is shown that the kinetics of water−water (WW) hydrogen bonds slow down significantly near the surface of a protein.41 Recently, the roles played by conformational and energetic disorders of a protein in arresting water mobility at its surface have been explored by Pizzitutti et al.42 It is shown that the rearrangement of WW hydrogen bonds that occurs in an aqueous protein solution and the time scale associated with it controls the dynamic coupling that exists at the protein surface. Interestingly, the hydrogen bond dynamics around a protein have been found to be correlated with the time scale of hydration water density fluctuations.43 Using MD simulations along with quasi-elastic neutron scattering (QENS) measurements, Tarek and Tobias44 showed that the structural relaxation of a protein is controlled by PW hydrogen bond dynamics. Recently, we performed extensive MD simulations to study how the heterogeneous conformational motions and PW electrostatic interactions at the surface of the protein barstar influence the local structure and ordering,45 and the microscopic dynamics and hydrogen bond properties46 of the hydration water molecules. The calculations revealed that the enhanced confinement at the protein surface on freezing its local motions does not qualitatively alter the ordering of the surface water molecules, but it significantly restricts their mobility around the secondary structures. Besides, it is demonstrated that the inability of water molecules to form hydrogen bonds with the protein in absence of PW electrostatic contributions is compensated by enhanced WW hydrogen bonding around the protein. In this work, we probe the effects of the local conformational motions of barstar and the nonuniform PW electrostatic interactions on the low-frequency intermolecular vibrational spectrum of the surface water molecules. Barstar formed by bacterium bacillus amyloliquefacien is a 89-residue protein.47 It acts as an intracellular inhibitor of the potentially lethal ribonuclease barnase.48 The secondary structures of barstar consist of three parallel α-helices packed onto a three-stranded parallel β-sheet. Besides, there is an additional α-helix that links 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). For convenience, we denote these segments in our discussion as helix-1 (Ser-14 to Ala-25), helix-2 (Asn-33 to Gly-43), helix-3 (Phe-56 to Thr63), 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 Glu-32), loop-3 (Trp-44 to Pro48), and loop-4 (Glu-64 to Ala-67). It is believed that barstar sterically blocks the active site of barnase by helix-2 and the loop connecting it with helix-1 (loop-2).48 The rest of the article is organized as follows. In section 2, we provide a brief
2. SYSTEM SETUP AND SIMULATION DETAILS We have carried out three simulations (S1, S2, and S3) with an aqueous solution of the protein barstar at different conditions. In simulation S1, fully flexible protein molecule in equilibrium with the solvent was studied. The protein molecule was kept frozen in simulation S2, while the electrostatic interactions between the protein and the water molecules were turned off in addition to freezing the protein matrix in simulation S3. The initial coordinates of barstar as obtained from NMR studies47 were taken from the Protein Data Bank (PDB code: 1BTA). The two end residues (Lys-1 and Ser-89) were first capped appropriately and then by avoiding unfavorable contacts the whole protein molecule was immersed in a 60 Å cubic cell containing equilibrated water molecules.46 The systems S1 and S2 were neutralized by adding 6 Na+ ions. These two systems finally contained the protein solvated by 5389 water molecules and 6 Na+ ions. There was no counterion present in S3, as it corresponds to a solvated nonpolar protein molecule. The simulations were performed using the NAMD code.49 We have employed all-atom CHARMM22 force field and potential parameters for proteins50 and TIP3P model51 for water in the calculations. The systems were first minimized using the conjugate gradient energy minimization method.49 The temperature of each of the systems was then increased gradually to the 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 individual systems were then equilibrated at 300 K under NPT ensemble conditions with isotropic cell volume fluctuations for 3 ns duration. During this period, the temperatures 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.52 At this stage, the simulation cell volumes attained steady values with edge lengths of 55.7, 55.5, and 55.8 Å, for systems S1, S2, and S3, respectively. The individual cell volumes were then fixed and the simulation conditions were changed from that of NPT to NVT ensemble (constant volume and temperature). The NVT equilibration runs were then continued further for another 20 ns duration at 300 K for each case. After this, the 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. Further details of the simulation protocols can be found elsewhere.46 The low-frequency vibrational modes of water molecules hydrating the protein have been calculated by the Fourier transformation of the corresponding velocity autocorrelation function (VACF).53,54 The VACF is defined as C V (t ) =
⟨vi⃗(t ) ·vi⃗(0)⟩ ⟨vi⃗(0) ·vi⃗(0)⟩
(1)
where vi⃗ (t) is the velocity vector of the atom of type i (O or H for a water molecule) at time t. The angular brackets denote that averaging is carried out over all the atoms of a particular type at different reference initial times. It is necessary to define the criteria used to identify PW hydrogen bonds formed at the protein surface. Generally, either 5849
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a geometry-based or an energy-based criterion is used to define hydrogen bonds. In this work, we have employed criteria based on geometrical arrangements of appropriate atoms to identify PW hydrogen bonds.55 According to the criteria adopted, the first condition for an acceptor or a donor atom of the protein to form a PW hydrogen bond 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° to form a PW hydrogen bond. Figure 2. Velocity autocorrelation function, CHV (t), for the hydrogen atoms of 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). As a reference, the data for water in pure bulk state are included in each of the panels.
3. RESULTS AND DISCUSSION We have calculated the velocity autocorrelation function (VACF) for the water molecules that are present near different secondary structural segments (within 5 Å) of barstar. This essentially corresponds to the first hydration layer water molecules around the protein. It may be noted that re-entries of the tagged water molecules into the first hydration layers of the protein segments over times are considered in the calculations presented here. 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 computations. The VACFs for water oxygen (COV (t)) and hydrogen (CHV (t)) atoms are calculated separately, and the results are displayed for all the three systems in Figures 1 and 2,
cages around them. It is clear that, with respect to bulk water, COV (t) for the hydration layer water molecules exhibits relative deeper minima, indicating enhanced rigidity of water layer hydrating the protein surface. This is particularly true for systems S1 and S2, indicating strong backscattering of surface water molecules from the polar and/or charged protein atoms. The effect seems to be more with increased confinement at the protein surface on freezing its conformational motions in system S2. On turning off PW electrostatic contributions, the surface water molecules behave noticeably similar to bulk water. This is consistent with the effect of PW electrostatics on water mobility, as reported in our earier work.46 Further, it may be noted that a small degree of heterogeneity exists in the relaxation pattern of COV (t) among water molecules around different segments of the protein. This is particularly noticeable around the loops in the frozen protein matrix (Figure 1b). For example, it is evident that water molecules hydrating loops 3 and 4 are trapped in a more confined space as compared to water surrounding other loops in the frozen system. The VACF for the hydrogen atoms (CHV (t)) of the first hydration layer water molecules around the protein segments exhibits almost identical behavior to that of water in pure bulk state in all the three simulations, as can be seen from Figure 2. Primarily, CHV (t) provides information about water’s librational motions (hindered rotations). The results show that the existence of the protein molecule either in flexible or frozen form and in the presence or absence of PW electrostatic interactions does not have significant influence on the caged librational motions of nearby water molecules. It is known that the ability of water to form PW hydrogen bonds alters the regular WW hydrogen bond network around the protein.30,34 Besides, it was shown recently that the conformational fluctuations of the protein segments and the electrostatic component of their interactions with surrounding water molecules influence the hydrogen bond properties at the interface.46 Therefore, the signature of the VACFs for the water molecules that are bound to the protein residues by PW hydrogen bonds is expected to be sensitive to the local motions of the protein and the heterogeneous electrostatic component
Figure 1. Velocity autocorrelation function, COV (t), for the oxygen atoms of 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). As a reference, the data for water in pure bulk state are included in each of the panels.
respectively. For comparison, the corresponding results for water in pure bulk state as obtained from a separate MD simulation of TIP3P water under identical conditions are also included in the two figures. Following an early small bump, the function COV (t) exhibits a negative dip for bulk water, as evident from Figure 1. This arises from backscattering of water oxygen atoms due to collisions with the neighbors forming molecular 5850
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with the polar and/or charged residues with PW hydrogen bonds. Such enhanced caging effects for the bound water molecules correlate well with strong PW hydrogen bonds and their slow relaxation time scale as reported earlier.46 Importantly, comparison between the results obtained for systems S1 and S2 (Figure 3, a and b) reveals further enhanced backscattering for the hydrogen-bonded bound water molecules around the protein segments in the frozen form. It is clear that the bound water molecules experience increasingly confined local environment in the absence of protein’s conformational flexibility. The results further demonstrate differential influence of the protein segments on the caging motions of the water molecules that are involved in PW hydrogen bonds. The effect is more significant than that observed in Figure 1, where all the first hydration layer water molecules (whether involved in PW hydrogen bonds or not) were included. Such heterogeneous caging motions among the water molecules hydrogen bonded to the protein segments are more evident on freezing their degrees of freedom (Figure 3b). Unlike COV (t), the function CVH(t) for the bound water molecules is relatively less influenced, as evident from Figure 4. However, compared to Figure 2, relatively more pronounced oscillations of the function for the bound water molecules, particularly in the frozen form of the protein, can be seen. This signifies that enhanced confinement at the surface on freezing the protein’s conformational motions influence to some extent the librational motions of the water molecules that are anchored by PW hydrogen bonds. However, the effect is small compared to that on the intermolecular vibrations (caging motions) of those water molecules. The influence of the conformational fluctuations of the protein and the nonuniform distribution of polar and charged sites capable of forming PW hydrogen bonds on water VACFs should also affect the low-frequency intermolecular vibrational density of states (VDOS) of the surface water molecules. We probe such influence, if any, next. Two low-frequency bands at ∼50 and ∼200 cm−1 are experimentally observed in the vibrational spectrum of water.56−59 In general, the band around 50 cm−1 is attributed to the O···O···O bending mode originatng from triplets of hydrogen-bonded water molecules, and that around 200 cm−1 is attributed to the O···O stretching mode or longitudinal oscillations between hydrogen-bonded pairs of water molecules.56 Interestingly, it is shown from MD simulations that the ∼50 cm −1 band also exists for non-hydrogen-bonded liquids.60,61 Thus it is often suggested that this band is not necessarily associated with hydrogen bonds, but rather it originates from restricted transverse oscillations of the tagged molecule along all directions in its local environment.61 However, in a liquid like water, the network of hydrogen bonds can alter the position of this frequency band. As already mentioned, rearrangement of the WW hydrogen bond network that occurs at the surface of a protein due to the formation of PW hydrogen bonds is influenced by the protein’s conformational motions and the PW electrostatic interactions.46 Such influence is expected to reflect in the lowfrequency VDOS of the surface water molecules. Here, we explore that by calculating the oxygen and hydrogen atom power spectra (SO(ω) and SH(ω)) for the water molecules present in the first hydration layers (within 5 Å) around different segments of the protein. As mentioned in section 2, this is done by the Fourier cosine transformation of the corresponding VACFs, and the results for the three simulation systems are displayed in Figures 5 and 6, respectively. Once
of the PW interaction. We explore such sensitivity by calculating the functions COV (t) and CHV (t) for only those water molecules that are bound to the protein segments by PW hydrogen bonds. The results are shown in Figures 3 and 4,
Figure 3. Velocity autocorrelation function, COV (t), for the oxygen atoms of only those water molecules that are bound to the residues of different segments of barstar by PW hydrogen bonds for (a) the flexible protein system (simulation S1) and (b) the frozen protein system (simulation S2). As a reference, the data for water in pure bulk state are included in each of the panels.
Figure 4. Velocity autocorrelation function, CHV (t), for the hydrogen atoms of only those water molecules that are bound to the residues of different segments of barstar by PW hydrogen bonds for (a) the flexible protein system (simulation S1) and (b) the frozen protein system (simulation S2). As a reference, the data for water in pure bulk state are included in each of the panels.
respectively. The results for pure bulk water are again included in the figure for comparison. It may be noted that in the absence of PW electrostatic contributions in system S3, water molecules at the surface can no longer remain bound to the protein residues by PW hydrogen bonds. Thus, we analyze the bound water VACFs for systems S1 and S2 only. Once again, the data for pure bulk water are included in the figure for comparison. Compared to Figure 1, it can be seen that the COV (t) decay curves in Figure 3 exhibit significantly deeper minima with frequent oscillations. This shows increasingly rigid environment with enhanced caging effects for those water molecules that are bound at the protein surface by anchoring 5851
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molecules around the loop regions in the frozen protein matrix. The results are consistent with the relaxation patterns of CVO(t) (Figure 1) and confirm heterogeneous effects of confinement on transverse oscillations of water molecules around the loops on freezing the protein’s conformational motions. Interestingly, the low-intensity mode around 200 cm−1 corresponding to O···O stretching or longitudinal oscillations of the first hydration layer water molecules are relatively less influenced. This signifies that the transverse and longitudinal oscillations of water around the protein are influenced in a nonuniform manner. Such differential behavior of water agrees well with earlier studies on complex biomolecules30,62 and micelles,53 and also for water confined between hydrophobic walls.63 It may be noted that the diffusion coefficient of water can be obtained from the zero-frequency (ω = 0) intensity of SO(ω). Compared to bulk water, lower zerofrequency intensities for water molecules around the protein segments suggest their sluggish dynamics. The calculated diffusion coefficients of hydration water molecules (DH) around the segments as measured from the respective zero-frequency intensities are listed in Table 1. It can be seen that reduced
Figure 5. Power spectrum, SO(ω), as obtained by Fourier transformation of the velocity autocorrelation function COV (t) for the oxygen atoms of 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). As a reference, the data for water in pure bulk state are included in each of the panels.
Table 1. Diffusion Coefficients (DH) of the Water Molecules in the First Hydration Layers (R = 5 Å) of Different Segments of Barstar As Obtained from Simulations S1, S2, and S3 DH (10−5 cm2 s−1) segment
S1
S2
S3
helix-1 helix-2 helix-3 helix-4 β-sheet loop-1 loop-2 loop-3 loop-4 bulk water
4.54 4.77 4.43 5.16 4.96 5.03 4.48 5.26 4.74
3.89 3.09 3.93 3.93 3.49 4.63 3.80 4.11 2.87 6.48
5.79 7.00 5.16 5.52 5.52 5.03 6.16 6.35 5.52
dimension with consequent enhanced confinement in the hydration layer on freezing the protein’s conformational motions along with presence of electrostatic anchoring sites (system S2) results in maximum retardation in water mobility. This agrees well with the trend observed in water translational motions around the protein as described in our earlier work.46 Water librational motions can be characterized from the hydrogen atom power spectrum (SH(ω)) that peaks around 500 cm−1.57 It is evident from Figure 6 that the heterogeneous conformational motions of the protein segments and PW electrostatic interacions have minimum influence on the band position. This is consistent with the relaxation patterns of CHV (t) (Figure 2) and indicate that the librational modes of the surface water molecules are not much affacted by the local conformational motions of the protein and strong PW electrostatic interactions. However, small but noticeable differences in intensities of the band around the segments have been observed. This is particularly true for the frozen protein systems (Figure 6b,c). It shows that the fractions of water molecules around the segments are heterogeneously affected on freezing the protein’s conformational motions. We have already demonstrated (see Figures 3 and 4) that the water molecules that are bound to the protein residues by PW
Figure 6. Power spectrum, SH(ω), as obtained by Fourier transformation of the velocity autocorrelation function CHV (t) for the hydrogen atoms of 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). As a reference, the data for water in pure bulk state are included in each of the panels.
again, to compare the results, the data for pure bulk water are included in the two figures. It can be seen from Figure 5 that compared to bulk water there is a noticeable shift to higher frequency (blue shift) for the band corresponding to O···O···O bending or restricted transverse oscillations of water molecules around the protein segments. With PW electrostatic interactions on, the effect is maximum (blue shift of ∼40 cm−1) around the frozen form of the protein (Figure 5b). However, near-bulklike behavior of surface water molecules in the absence of PW electrostatic contributions is evident from the minimum shift (by ∼15 cm−1) in the peak position in Figure 5c. Interestingly, noticeable differences in intensities of the band have been noticed, particularly among water 5852
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and 8, respectively. Comparison of the results shown in Figure 7 with that in Figure 5 indicates that the band corresponding to O···O···O bending or restricted transverse oscillations are further blue-shifted for the bound water molecules. With respect to bulk water, the shifts occur by 40−50 cm−1 around the flexible form of the protein (system S1). Consistent with our earlier discussion, on freezing the protein conformational motions (system S2), the transverse oscillations of the water molecules anchored at the surface are further restricted as evident from even higher shifts in the band position (Figure 7b). Importantly, compared to Figure 5, a significant heterogeneous distribution of the band position and intensity has been observed among the water molecules bound to different segments of the protein. Due to differential flexibility of the loops,45 the heterogeneity is more for water bound to them as compared to water bound to the helices and the βsheet in system S1. Such heterogeneity increases further in the frozen form of the protein (Figure 7b). In agreement with our earlier discussion, it is clear that with PW electrostatic interactions on, freezing the protein’s degrees of freedom alters the confined environment around the bound water molecules in a highly nonuniform manner. Although the formation of PW hydrogen bonds does not have much influence on the position of the O···O stretching band, the band intensity is affected heterogeneously around different segments. The results demonstrate that larger nonuniform fractions of water molecules anchored with the polar and/or charged groups of different segments exhibit restricted longitudinal oscillations. Again, the effect and the extent of heterogeneity are more significant around the frozen protein (Figure 7b). It may be noted that the heterogeneous influence of protein conformational flexibilities and PW electrostatic contributions on the two low-frequency modes of the bound water molecules agree well with nonuniform influence of protein segments on the time scale of formation and breaking of PW hydrogen bonds, as reported earlier.46 Further lowering of zero-frequency intensities for the bound water molecules shows severely retarded motions of such water molecules. Unlike the result shown in Figure 6, the band corresponding to the librational modes for the water molecules that are bound by PW hydrogen bonds are significantly affected, as evident from Figure 8. Thus, due to the formation of strong PW hydrogen bonds and their slow relaxations,46 the librations of the bound water molecules are noticeably restricted. Further, the position of the band around 500 cm−1 is found to be heterogeneously influenced with blue shifts of 50−100 cm−1 for the bound water molecules around the protein segments. In agreement with our earlier discussion, the effect is more around the frozen protein, indicating that the restriction on the librational motions is more with increase in confinement at the surface in the frozen form of the protein.
hydrogen bonds exhibit enhanced caging motions in a relatively rigid environment. As a result, the low-frequency VDOS of such bound water molecules should also be influenced. We explore that by comparing the power spectra SO(ω) and SH(ω) for only those water molecules that are anchored at the protein surface by PW hydrogen bonds. Sensitivity of the bound water DOS distributions to the conformational motions of the protein segments and heterogeneous PW electrostatic interactions has been specifically explored. Once again, due to the nonpolar nature of the protein in system S3, the calculations are carried out for systems S1 and S2 only. The results along with that for water in pure bulk state are compared in Figures 7
Figure 7. Power spectrum, SO(ω), as obtained by Fourier transformation of the velocity autocorrelation function COV (t) for the oxygen atoms of only those water molecules that are bound to the residues of different segments of barstar by PW hydrogen bonds for (a) the flexible protein system (simulation S1) and (b) the frozen protein system (simulation S2). As a reference, the data for water in pure bulk state are included in each of the panels.
4. CONCLUSIONS We have performed atomistic MD simulations to probe the effects of the nonuniform conformational flexibilities and distribution of polar and/or charged groups of a protein capable of anchoring solvent water through PW hydrogen bonds on the low-frequency vibrational DOS of the surface water molecules. In particular, the calculations are carried out around room temperature with an aqueous solution of the 89 residue protein barstar. The results obtained from the simulation of the protein in its flexible form interacting with water with full potential are compared to that obtained from two additional simulations. In one of these two simulations, the
Figure 8. Power spectrum, SH(ω), as obtained by Fourier transformation of the velocity autocorrelation function CHV (t) for the hydrogen atoms of only those water molecules that are bound to 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 data for water in pure bulk state are included in each of the panels. 5853
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23/2007), Government of India. Part of the work was carried out using the computational facility created under DST-FIST program (SR/FST/CSII-011/2005). S.P. thanks the Council for Scientific and Industrial Research (CSIR), New Delhi, for providing a scholarship.
protein is kept frozen and in the other along with the frozen protein matrix, the PW electrostatic interactions are turned off. The calculation reveals detectable shifts to higher frequency (blue shifts) for the band corresponding to O···O···O bending or restricted transverse oscillations involving the first hydration layer water molecules around the protein segments. It is noticed that enhanced confinement near the rigid protein wall on freezing its local conformational fluctuations results in increasingly restricted oscillatory motions of the hydration water molecules as evident from further blue shifts of the band. However, the effect on water oscillatory motions reduces significantly in the absence of PW electrostatic contributions with near-bulklike distribution of the band. In contrast, the band corresponding to O···O stretching due to longitudinal oscillations of water molecules present in the first hydration layers around the protein segments is found to be less sensitive to whether the protein’s conformational motions are kept frozen and/or the PW electrostatic interactions are switched off. This is an interesting observation that shows nonuniform influence of a protein’s conformational flexibility and PW electrostatic contributions on the transverse and longitudinal degrees of freedom of water molecules hydrating the protein. Effects of the protein’s nonuniform conformational motions and PW electrostatic contributions on the low-frequency VDOS of only those water molecules that are directly anchored at the surface by PW hydrogen bonds have also been explored. Significantly larger blue shifts for the O···O···O bending mode indicating further restricted transverse oscillations have been observed for such water molecules. A heterogeneous distribution of the band around different segments of the protein has been observed, the effect being more in the frozen form. It is further demonstrated that a larger fraction of bound water molecules exhibits restricted longitudinal oscillations around the frozen protein matrix. Water librational mode too in the bound form is nonuniformly altered with increased confinement around the frozen protein, as evident from differential blue shifts of the SH(ω) band. The results agree well with heterogeneous degree of confinement and increased PW hydrogen bond relaxation times for the water molecules bound to the protein segments in the absence of their conformational fluctuations, as reported earlier.46 Raman, IR, and IINS experiments capable of identifying the low-frequency modes can be used to verify the influence of protein conformational motions and PW electrostatic interactions on them as obtained from the present study. This in turn can provide direct understanding of the correlated dynamical picture of water hydrating the surface of a protein.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported in part by grants from the Department of Science and Technology (DST) (SR/S1/PC5854
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