Active-site hydration and water diffusion in cytochrome P450cam: a highly dynamic process

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.     

Low-frequency Raman spectra of lysozyme crystals and oriented DNA films: dynamics of crystal water.

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.     

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

The dynamics of water molecules near the protein surface are different from those of bulk water and influence the structure and dynamics of the protein itself. To elucidate the temperature dependence hydration dynamics of water molecules, we present results from the molecular dynamic simulation of the water molecules surrounding two proteins (Carboxypeptidase inhibitor and Ovomucoid) at seven different temperatures (T=273 to 303 K, in increments of 5 K). Translational diffusion coefficients of the surface water and bulk water molecules were estimated from 2 ns molecular dynamics simulation trajectories. Temperature dependence of the estimated bulk water diffusion closely reflects the experimental values, while hydration water diffusion is retarded significantly due to the protein. Protein surface induced scaling of translational dynamics of the hydration waters is uniform over the temperature range studied, suggesting the importance protein-water interactions.     

Solvent effects on protein motion and protein effects on solvent motion. Dynamics of the active site region of lysozyme

The stochastic boundary molecular dynamics methodology is applied to the active site of the enzyme lysozyme. A comparison is made of in vacuo dynamics results from the stochastic boundary method and a full conventional molecular dynamics simulation of lysozyme. Excellent agreement between the two approaches is obtained. The influence of solvent on the residues in the active site region is explored and it is shown that both the structure and dynamics are affected. Of particular importance for the structure of the protein is the solvation of polar residues and the stabilization of like-charged ion pairs. The magnitude of the fluctuations is only slightly altered by the solvent; the overall increase in the root-mean-square fluctuations, relative to the vacuum run, is 11%. The solvent effect on dynamical properties is found not to be simply related to the solvent viscosity. Both the solvent exposure and dynamic aspects of protein-solvent interactions, including the relative time scales of the motions, are shown to play a role. The effects of the protein on solvent dynamics and structure are also observed to be significant. The solvent molecules around atoms in charged, polar and apolar side-chains show markedly different diffusion coefficients as well as exhibiting different solvation structures. One key example is the water around apolar groups, which is much less mobile than bulk water, or water solvating polar groups.     

C-phycocyanin hydration water dynamics in the presence of trehalose: an incoherent elastic neutron scattering study at different energy resolutions

We present a study of C-phycocyanin hydration water dynamics in the presence of trehalose by incoherent elastic neutron scattering. By combining data from two backscattering spectrometers with a 10-fold difference in energy resolution we extract a scattering law S(Q,omega) from the Q-dependence of the elastic intensities without sampling the quasielastic range. The hydration water is described by two dynamically different populations--one diffusing inside a sphere and the other diffusing quasifreely--with a population ratio that depends on temperature. The scattering law derived describes the experimental data from both instruments excellently over a large temperature range (235-320 K). The effective diffusion coefficient extracted is reduced by a factor of 10-15 with respect to bulk water at corresponding temperatures. Our approach demonstrates the benefits and the efficiency of using different energy resolutions in incoherent elastic neutron scattering over a large angular range for the study of biological macromolecules and hydration water.     

Permeability of lysozyme tetragonal crystals to water

Diffusion of water within cross-linked tetragonal crystals of hen egg-white lysozyme has been measured and simulated on a computer using the X-ray structure of water-filled channels within the crystal lattice. Relative to the self-diffusion coefficient of bulk water molecules, the experimental diffusion coefficient of water within the crystal was found to be 13 times reduced in the (001) crystallographic plane and 5 times reduced in the [001] direction. Comparison of the experimental and computer simulated diffusion coefficients shows that steric limitations for water diffusion are mostly responsible for this reduction of the water diffusion in the crystal, with the self-diffusion coefficient of intracrystalline water reduced by no more than 30–40% as compared to that of bulk water.     

Influence of water clustering on the dynamics of hydration water at the surface of a lysozyme

Dynamics of hydration water at the surface of a lysozyme molecule is studied by computer simulations at various hydration levels in relation with water clustering and percolation transition. Increase of the translational mobility of water molecules at the surface of a rigid lysozyme molecule upon hydration is governed by the water-water interactions. Lysozyme dynamics strongly affect translational motions of water and this dynamic coupling is maximal at hydration levels, corresponding to the formation of a spanning water network. Anomalous diffusion of hydration water does not depend on hydration level up to monolayer coverage and reflects spatial disorder. Rotational dynamics of water molecules show stretched exponential decay at low hydrations. With increasing hydration, we observe appearance of weakly bound water molecules with bulklike rotational dynamics, whose fraction achieves 20-25% at the percolation threshold.     

Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.     

Dynamics of hydration in hen egg white lysozyme

We investigate the hydration dynamics of a small globular protein, hen egg-white lysozyme. Extensive simulations (two trajectories of 9 ns each) were carried out to identify the time-scales and mechanism of water attachment to this protein. The location of the surface and integral water molecules in lysozyme was also investigated. Three peculiar temporal scales of the hydration dynamics can be discerned: two among these, with sub-nanosecond mean residence time, tau(w), are characteristic of surface hydration water; the slower time-scale (tau(w) approximately 2/3 ns) is associated with buried water molecules in hydrophilic pores and in superficial clefts. The computed tau(w) values in the two independent runs fall in a similar range and are consistent with each other, thus adding extra weight to our result. The tau(w) of surface water obtained from the two independent trajectories is 20 and 24 ps. In both simulations only three water molecules are bound to lysozyme for the entire length of the trajectories, in agreement with nuclear magnetic relaxation dispersion estimates. Locations other than those identified in the protein crystal are found to be possible for these long-residing water molecules. The dynamics of the hydration water molecules observed in our simulations implies that each water molecule visits a multitude of residues during the lifetime of its bound with the protein. The number of residues seen by a single water molecule increases with the time-scale of its residence time and, on average, is equal to one only for the water molecules with shorter residence time. Thus, tau(w) values obtained from inelastic neutron scattering and based on jump-diffusion models are likely not to account for the contribution of water molecules with longer residence time.     

Characterisation by triple-quantum filtered 17O-NMR of water molecules buried in lysozyme and trapped in a lysozyme-inhibitor complex.

Triple-quantum filtering NMR sequences were used to study the multiexponential relaxation behaviour of H2 17O in the presence of hen egg white lysozyme. By this means, the fraction and the correlation time of water were determined in slow motion, as well as the relaxation time of water in the extreme narrowing limit. The small number of water molecules in slow motion, which is between four and five per lysozyme, seems to correspond to the 'integral' water, buried or in the cleft inside the protein, whereas water in fast motion corresponds to all other water molecules, interacting or not with the macromolecules. The same experiment was performed after addition of the inhibitor tri-N-acetylglucosamine (NAG)3. For solutions of sufficient viscosity, there were approximately three supplementary water molecules in slow motion per lysozyme, probably trapped between the protein and the inhibitor. The correlation time of these water molecules was estimated at 2 ns, which should correspond to their residence time in the complex.     

Bound Water and Cryptobiosis:Thermodynamic Properties of Water at Biopolymer Surfaces

The molecular mechanisms underlying the adaptations to water loss developed in several tardigrade species remain poorly understood. It seems, however, that the binding of the disaccharide trehalose to membranes and other cellular components at low water contents is important for the tolerance to extreme drought. Trehalose is thus thought to replace interfacial- or “bound” water and enhance the conformational stability of labile macromolecules. To gain further insight into this we investigate here thermodynamic properties of water bound to the protein lysozyme at low water content (<100 water molecules pr. protein). It appears that this surface water has a higher enthalpy and higher entropy than the bulk liquid. These observations call for re-evaluation of the term “bound water” since “bound” carries the connotation of a low-energy, ordered (i.e. low-entropy) state.

To rationalize these observations it is suggested that — in addition to the self-evident energetic contribution from biopolymer-water contacts — the properties of interfacial water are dominated by two effects. These are i) the ability of water to facilitate fast movements of individual parts of biopolymers and ii) the high molecular cohesion in the aqueous bulk. Thus, the hydration of a surface leads to enhanced flexibility in the biopolymer and breakage in the network of hydrogen bonding in the liquid bulk, and these effects collectively increase the enthalpy and entropy of the system. As a result, the thermodynamic parameters of hydration of lysozyme carry the thermodynamic hallmarks of an order → disorder process, even for the first hundred (i.e. most strongly associated) water molecules. We discuss these data for protein hydration together with some recent, very similar observations for the hydration of lipid bilayer membranes.     

Distinguishing thermodynamic and kinetic views of the preferential hydration of protein surfaces

Motivated by a quasi-chemical view of protein hydration, we define specific hydration sites on the surface of globular proteins in terms of the local water density at each site relative to bulk water density. The corresponding kinetic definition invokes the average residence time for a water molecule at each site and the average time that site remains unoccupied. Bound waters are identified by high site occupancies using either definition. In agreement with previous molecular dynamics simulation studies, we find only a weak correlation between local water densities and water residence times for hydration sites on the surface of two globular proteins, lysozyme and staphylococcal nuclease. However, a strong correlation is obtained when both the average residence and vacancy times are appropriately taken into account. In addition, two distinct kinetic regimes are observed for hydration sites with high occupancies: long residence times relative to vacancy times for a single water molecule, and short residence times with high turnover involving multiple water molecules. We also correlate water dynamics, characterized by average occupancy and vacancy times, with local heterogeneities in surface charge and surface roughness, and show that both features are necessary to obtain sites corresponding to kinetically bound waters.     

The dynamics of protein hydration water: a quantitative comparison of molecular dynamics simulations and neutron-scattering experiments

We present results from an extensive molecular dynamics simulation study of water hydrating the protein Ribonuclease A, at a series of temperatures in cluster, crystal, and powder environments. The dynamics of protein hydration water appear to be very similar in crystal and powder environments at moderate to high hydration levels. Thus, we contend that experiments performed on powder samples are appropriate for discussing hydration water dynamics in native protein environments. Our analysis reveals that simulations performed on cluster models consisting of proteins surrounded by a finite water shell with free boundaries are not appropriate for the study of the solvent dynamics. Detailed comparison to available x-ray diffraction and inelastic neutron-scattering data shows that current generation force fields are capable of accurately reproducing the structural and dynamical observables. On the time scale of tens of picoseconds, at room temperature and high hydration, significant water translational diffusion and rotational motion occur. At low hydration, the water molecules are translationally confined but display appreciable rotational motion. Below the protein dynamical transition temperature, both translational and rotational motions of the water molecules are essentially arrested. Taken together, these results suggest that water translational motion is necessary for the structural relaxation that permits anharmonic and diffusive motions in proteins. Furthermore, it appears that the exchange of protein-water hydrogen bonds by water rotational/librational motion is not sufficient to permit protein structural relaxation. Rather, the complete exchange of protein-bound water molecules by translational displacement seems to be required.     

Anomalous temperature dependence of the thermal expansion of proteins

The temperature dependence of the apparent expansibility of lysozyme and ovalbumin in solution has been measured as a function of pH. This temperature dependence is explained in terms of suppressed fluctuations in bound water due to the protein. It is shown that the thermal expansion coefficient of bound water is different from bulk water. The pH dependence can be explained by increased hydration of side chains at lower pH. The amount in volume of hydration water in a typical protein-water system varies from 0.16 to 0.7. How the intrinsic thermal expansion coefficient of proteins can be derived from the apparent quantity is discussed. Intrinsic values of the thermal expansion coefficient for lysozyme at room temperature are between 1.7 and 4.4 x 10(-4) K-1 for a 10% solution.     

Dipole–dipole interactions between tryptophan side chains and hydration water molecules dominate the observed dynamic stokes shift of lysozyme

The fluorescence intensity of tryptophan residues in hen egg-white lysozyme was measured up to 500 ps after the excitation by irradiation pulses at 290 nm. From the time-dependent variation of fluorescence intensity in a wavelength range of 320–370 nm, the energy relaxation in the dynamic Stokes shift was reconstructed as the temporal variation in wavenumber of the estimated fluorescence maximum. The relaxation was approximated by two exponential curves with decay constants of 1.2 and 26.7 ps. To interpret the relaxation, a molecular dynamics simulation of 75 ns was conducted for lysozyme immersed in a water box. From the simulation, the energy relaxation in the electrostatic interactions of each tryptophan residue was evaluated by using a scheme derived from the linear response theory. Dipole–dipole interactions between each of the Trp62 and Trp123 residues and hydration water molecules displayed an energy relaxation similar to that experimentally observed regarding time constants and magnitudes. The side chains of these residues were partly or fully exposed to the solvent. In addition, by inspecting the variation in dipole moments of the hydration water molecules around lysozyme, it was suggested that the observed relaxation could be attributed to the orientational relaxation of hydration water molecules participating in the hydrogen-bond network formed around each of the two tryptophan residues.     

Acceptable protein and solvent behavior in primary hydration shell simulations of hen lysozyme

The "primary hydration shell" method in molecular dynamics simulations uses a two- to three-layer thick shell of explicitly represented water molecules as the solvent around the protein of interest. We show that despite its simplicity, this computationally cheap model is capable of predicting acceptable water and protein behavior using the CHARMM22/CMAP potential function. For protein dynamics, comparisons are made with Lipari-Szabo order parameters. These have been derived from NMR relaxation parameters for pico-nano second motions of the NH groups in the main-chain and NH(2) groups in Asn/Gln side chains in hen lysozyme. It is also shown that an even simpler, and therefore faster, water-shell model leads to results in similarly good agreement with experiments, and also compared with simulations using a full box of water with periodic boundary conditions or with an implicit solvation model. Thus, the primary hydration shell method should be useful in making larger systems accessible to extensive simulations.     

Hydration dependence of conformational dielectric relaxation of lysozyme

Dielectric response of hen egg white lysozyme is measured in the far infrared (5-65 cm-1, 0.15-1.95 THz, 0.6-8.1 meV) as a function of hydration. The frequency range is associated with collective vibrational modes of protein tertiary structure. The observed frequency dependence of the absorbance is broad and glass-like. For the entire frequency range, there is a slight increase in both the absorbance and index of refraction with increasing hydration for <0.27 h (mass of H2O per unit mass protein). At 0.27 h, the absorbance and index begin to increase more rapidly. This transition corresponds to the point where the first hydration shell is filled. The abrupt increase in dielectric response cannot be fully accounted for by the additional contribution to the dielectric response due to bulk water, suggesting that the protein has not yet achieved its fully hydrated state. The broad, glass-like response suggests that at low hydrations, the low frequency conformational hen egg white lysozyme dynamics can be described by a dielectric relaxation model where the protein relaxes to different local minima in the conformational energy landscape. However, the low frequency complex permittivity does not allow for a pure relaxational mechanism. The data can best be modeled with a single low frequency resonance (nu approximately 120 GHz=4 cm-1) and a single Debye relaxation process (tau approximately .03-.04 ps). Terahertz dielectric response is currently being considered as a possible biosensing technique and the results demonstrate the required hydration control necessary for reliable biosensor applications.     

The evaluation of standard sedimentation coefficient in band sedimentation of macromolecules

Standardization in the calculation of the sedimentation coefficient of macromolecules by means of band techniques is discussed. When a sample of macromolecules suspended in a solvent is layered on to a denser bulk solution, the macromolecules do not sediment in this solution alone, but sediment in a mixture of bulk solution and sample solvent. This is caused by the diffusion between sample solvent and bulk solution. Experimental evidence of this process is shown during band sedimentation of ribosomes when the variation of the density and the viscosity along the cell is measured. The calculation shows that in the experimental conditions frequently used, standardization made in the usual way leads to a sedimentation coefficient which is largely overestimated; while standardization yields the correct coefficient if the diffusion effect of the sample solvent in to the bulk solution is taken into account, together with possible deuteration effects. A method to calculate the standard coefficient with the aid of a computer is proposed.     

Glass Transition and Dynamics in Lysozyme�CWater Mixtures Over Wide Ranges of Composition

Differential scanning calorimetry (DSC) and two dielectric techniques, broadband dielectric relaxation spectroscopy and thermally stimulated depolarization currents (TSDC), were employed to study glass transition and water and protein dynamics in mixtures of water and a globular protein, lysozyme, in wide ranges of water content, both solutions, and hydrated solid samples. In addition, water equilibrium sorption isotherms (ESI) measurements were performed at room temperature. The main objective was to correlate results by different techniques to each other and to determine critical water contents for various processes. From ESI measurements the content of water directly bound to primary hydration sites was determined to 0.088 (grams of water per grams of dry protein), corresponding to 71 water molecules per protein molecule, and that where clustering becomes significant to about 0.25. Crystallization and melting events of water were first observed at water contents 0.270 and 0.218, respectively, and the amount of uncrystallized water was found to increase with increasing water content. Two populations of ice crystals were observed by DSC, primary and bulk ice crystals, which give rise to two separate relaxations in dielectric measurements. In addition, the relaxation of uncrystallized water was observed, superimposed on a local relaxation of polar groups on the protein surface. The glass transition temperature, determined by DSC and TSDC in rather good agreement to each other, was found to decrease significantly with increasing water content and to stabilize at about −90 °C for water contents higher than about 0.25. This is a novel result of this study with potential impact on cryoprotection and pharmaceutics.