Modeling the Hydration Layer around Proteins: HyPred

Protein hydration plays an integral role in determining protein function and stability. We develop a simple method with atomic level precision for predicting the solvent density near the surface of a protein. A set of proximal radial distribution functions are defined and calculated for a series of different atom types in proteins using all-atom, explicit solvent molecular dynamic simulations for three globular proteins. A major improvement in predicting the hydration layer is found when the protein is held immobile during the simulations. The distribution functions are used to develop a model for predicting the hydration layer with sub-1-Ångstrom resolution without the need for additional simulations. The model and the distribution functions for a given protein are tested in their ability to reproduce the hydration layer from the simulations for that protein, as well as those for other proteins and for simulations in which the protein atoms are mobile. Predictions for the density of water in the hydration shells are then compared with high occupancy sites observed in crystal structures. The accuracy of both tests demonstrates that the solvation model provides a basis for quantitatively understanding protein solvation and thereby predicting the hydration layer without additional simulations.     

Analysis of thermodynamic non-ideality in terms of protein solvation.

The effects of thermodynamic non-ideality on the forms of sedimentation equilibrium distributions for several isoelectric proteins have been analysed on the statistical-mechanical basis of excluded volume to obtain an estimate of the extent of protein solvation. Values of the effective solvation parameter delta are reported for ellipsoidal as well as spherical models of the proteins, taken to be rigid, impenetrable macromolecular structures. The dependence of the effective solvated radius upon protein molecular mass exhibits reasonable agreement with the relationship calculated for a model in which the unsolvated protein molecule is surrounded by a 0.52-nm solvation shell. Although the observation that this shell thickness corresponds to a double layer of water molecules may be of questionable relevance to mechanistic interpretation of protein hydration, it augurs well for the assignment of magnitudes to the second virial coefficients of putative complexes in the quantitative characterization of protein-protein interactions under conditions where effects of thermodynamic non-ideality cannot justifiably be neglected.     

A unified picture of protein hydration: prediction of hydrodynamic properties from known structures.

Hydration is essential for the structural and functional integrity of globular proteins. How much hydration water is required for that integrity? A number of techniques such as X-ray diffraction, nuclear magnetic resonance (NMR) spectroscopy, calorimetry, infrared spectroscopy, and molecular dynamics (MD) simulations indicate that the hydration level is 0.3-0.5 g of water per gram of protein for medium sized proteins. Hydrodynamic properties, when accounted for by modeling proteins as ellipsoids, appear to give a wide range of hydration levels. In this paper we describe an alternative numerical technique for hydrodynamic calculations that takes account of the detailed protein structures. This is made possible by relating hydrodynamic properties (translational and rotational diffusion constants and intrinsic viscosity) to electrostatic properties (capacitance and polarizability). We show that the use of detailed protein structures in predicting hydrodynamic properties leads to hydration levels in agreement with other techniques. A unified picture of protein hydration emerges. There are preferred hydration sites around a protein surface. These sites are occupied nearly all the time, but by different water molecules at different times. Thus, though a given water molecule may have a very short residence time (approximately 100-500 ps from NMR spectroscopy and MD simulations) in a particular site, the site appears fully occupied in experiments in which time-averaged properties are measured.     

Large-scale networks of hydration water molecules around proteins investigated by cryogenic X-ray crystallography.

The structures at protein-water interface, i.e. the hydration structure of proteins, have been investigated by cryogenic X-ray crystal structure analyses. Hydration structures appeared far clearer at cryogenic temperature than at ambient temperature, presumably because the motions of hydration water molecules were quenched by cooling. Based on the structural models obtained, the hydration structures were systematically analyzed with respect to the amount of water molecules, the interaction modes between water molecules and proteins, the local and the global distribution of them on the surface of proteins. The standard tetrahedral interaction geometry of water in bulk retained at the interface and enabled the three-dimensional chain connection of hydrogen bonds between hydration water molecules and polar protein atoms. Large-scale networks of hydrogen bonds covering the entire surface of proteins were quite flexible to accommodate to the large-scale conformational changes of proteins and seemed to have great influences on the dynamics and function of proteins. The present observation may provide a new concept for discussing the dynamics of proteins in aqueous solution.     

Monte-Carlo simulation of hydration of nucleic acid fragments

Monte-Carlo simulation of the systems containing a stack of 6 complementary base pairs and 180 water molecules has been performed. Characteristic of the hydration shell structure in major and minor grooves has been found for the stacks of repeating A : U and G : C base pairs as well as alternating (A : U, U : A) and (G : C, C : G) ones. Probabilities of the formation of bridges, formed by 1, 2 and 3 water molecules, between hydrophilic centres of the bases have been estimated. One water molecule forms an H-bonded bridge between two adjacent hydrophilic centres with high probability if N...N, N...O or O...O distance between these centres is close to 4.3 A. Hydration shell structure was found to depend significantly on the stack sequence and configuration, while global hydration characteristics (average energy, the number of water-water and water-base H-bonds) are only slightly dependent on the stack sequence and configuration. For the stacks in A conformation the number of water molecules forming more than one H-bonds with the bases is greater in comparison with the stacks in B-like conformation. This result is discussed in connection with the concept of hydration economy during B to A transition.     

The origin and impact of bound water around intrinsically disordered proteins

Proteins and water couple dynamically over a wide range of time scales. Motivated by their central role in protein function, protein-water dynamics and thermodynamics have been extensively studied for structured proteins, where correspondence to structural features has been made. However, properties controlling intrinsically disordered protein (IDP)-water dynamics are not yet known. We report results of megahertz-to-terahertz dielectric spectroscopy and molecular dynamics simulations of a group of IDPs with varying charge content along with structured proteins of similar size. Hydration water around IDPs is found to exhibit more heterogeneous rotational and translational dynamics compared with water around structured proteins of similar size, yielding on average more restricted dynamics around individual residues of IDPs, charged or neutral, compared with structured proteins. The on-average slower water dynamics is found to arise from excess tightly bound water in the first hydration layer, which is related to greater exposure to charged groups. The more tightly bound water to IDPs correlates with the smaller hydration shell found experimentally, and affects entropy associated with protein-water interactions, the contribution of which we estimate based on the dielectric measurements and simulations. Water-IDP dynamic coupling at terahertz frequencies is characterized by the dielectric measurements and simulations.     

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.     

Calculation of translational friction and intrinsic viscosity. II. Application to globular proteins.

The translational friction coefficients and intrinsic viscosities of four proteins (ribonuclease A, lysozyme, myoglobin, and chymotrypsinogen A) are calculated using atomic-level structural details. Inclusion of a 0.9-A-thick hydration shell allows calculated results for both hydrodynamic properties of each protein to reproduce experimental data. The use of detailed protein structures is made possible by relating translational friction and intrinsic viscosity to capacitance and polarizability, which can be calculated easily. The 0.9-A hydration shell corresponds to a hydration level of 0.3-0.4 g water/g protein. Hydration levels within this narrow range are also found by a number of other techniques such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, calorimetry, and computer simulation. The use of detailed protein structures in predicting hydrodynamic properties thus allows hydrodynamic measurement to join the other techniques in leading to a unified picture of protein hydration. In contrast, earlier interpretations of hydrodynamic data based on modeling proteins as ellipsoids gave hydration levels that varied widely from protein to protein and thus challenged the existence of a unified picture of protein hydration.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Role of flexibility and polarity as determinants of the hydration of internal cavities and pockets in proteins

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized. This might explain differences in the patterns of hydration observed in crystal structures obtained at cryogenic and room temperature conditions. The ability of molecular dynamics simulations to determine the most likely sites of water binding in internal pockets and cavities depends on its efficiency in sampling the hydration of internal sites and alternative protein and water conformations. This can be enhanced significantly by performing multiple molecular dynamics simulations as well as simulations started from different initial hydration states.     

The mechanism of the type III antifreeze protein action: a computational study

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.     

Dynamic properties of water at phosphatidylcholine lipid-bilayer surfaces as seen by deuterium and pulsed field gradient proton NMR

The dynamic properties of water in phosphatidylcholine lipid/water dispersions have been studied, applying a combination of ²H-NMR techniques (quadrupole splitting and spin-lattice relaxation time) and self-diffusion measurements using pulsed field gradient (PFG) ¹H-NMR. The hydration properties of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) were compared with those of DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) and EYL (egg yolk phosphatidylcholine (lecithin)). A model is presented that assumes an exponentially decaying influence of the bilayer surface on water dynamics as well as on water orientation with increasing hydration. This assumption is based on an exponentially decaying hydration potential which results from direct lipid-water and water-water interactions. The model describes successfully the experimental data for a large water concentration range, especially at low hydration, where other models failed. With the exception of a small fraction of water which is significantly influenced by the surface in slowing down the mobility, the interbilayer water has isotropic, free water characteristics in terms of correlation times and molecular order. Hydration properties of POPC are comparable with those of EYL but differ from DOPC. At very low water content the correlation times of headgroup segmental reorientation and water are similar, indicating a strong coupling of this water to the lipid lattice. The hydration properties of the three lipids studied are explained in terms of slightly different headgroup conformations due to different lateral packing of the molecules by their fatty-acid chain composition.     

Strong repulsive forces between protein and oligo (ethylene glycol) self-assembled monolayers: a molecular simulation study

Restrained molecular dynamics simulations were performed to study the interaction forces of a protein with the self-assembled monolayers (SAMs) of S(CH2)4(EG)4OH, S(CH2)11OH, and S(CH2)11CH3 in the presence of water molecules. The force-distance curves were calculated by fixing the center of mass of the protein at several separation distances from the SAM surface. Simulation results show that the relative strength of repulsive force acting on the protein is in the decreasing order of OEG-SAMs > OH-SAMs > CH3-SAMs. The force contributions from SAMs and water molecules, the structural and dynamic behavior of hydration water, and the flexibility and conformation state of SAMs were also examined to study how water structure at the interface and SAM flexibility affect the forces exerted on the protein. Results show that a tightly bound water layer adjacent to the OEG-SAMs is mainly responsible for the large repulsive hydration force.     

Deducing hydration sites of a protein from molecular dynamics simulations

Invariant water molecules that are of structural or functional importance to proteins are detected from their presence in the same location in different crystal structures of the same protein or closely related proteins. In this study we have investigated the location of invariant water molecules from MD simulations of ribonuclease A, HIV1-protease and Hen egg white lysozyme. Snapshots of MD trajectories represent the structure of a dynamic protein molecule in a solvated environment as opposed to the static picture provided by crystallography. The MD results are compared to an analysis on crystal structures. A good correlation is observed between the two methods with more than half the hydration sites identified as invariant from crystal structures featuring as invariant in the MD simulations which include most of the functionally or structurally important residues. It is also seen that the propensities of occupying the various hydration sites on a protein for structures obtained from MD and crystallographic studies are different. In general MD simulations can be used to predict invariant hydration sites when there is a paucity of crystallographic data or to complement crystallographic results.     

H-bonding in protein hydration revisited

H-bonding between protein surface polar/charged groups and water is one of the key factors of protein hydration. Here, we introduce an Accessible Surface Area (ASA) model for computationally efficient estimation of a free energy of water-protein H-bonding at any given protein conformation. The free energy of water-protein H-bonds is estimated using empirical formulas describing probabilities of hydrogen bond formation that were derived from molecular dynamics simulations of water molecules at the surface of a small protein, Crambin, from the Abyssinian cabbage (Crambe abyssinica) seed. The results suggest that atomic solvation parameters (ASP) widely used in continuum hydration models might be dependent on ASA for polar/charged atoms under consideration. The predictions of the model are found to be in qualitative agreement with the available experimental data on model compounds. This model combines the computational speed of ASA potential, with the high resolution of more sophisticated solvation methods.     

Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization

Water plays an essential role in determining the structure and function of all biological systems. Recent methodological advances allow for an accurate and efficient estimation of the thermodynamic properties of water molecules at the surface of proteins. In this work, we characterize these thermodynamic properties and relate them to various structural and functional characteristics of the protein. We find that high-energy hydration sites often exist near protein motifs typically characterized as hydrophilic, such as backbone amide groups. We also find that waters around alpha helices and beta sheets tend to be less stable than waters around loops. Furthermore, we find no significant correlation between the hydration site-free energy and the solvent accessible surface area of the site. In addition, we find that the distribution of high-energy hydration sites on the protein surface can be used to identify the location of binding sites and that binding sites of druggable targets tend to have a greater density of thermodynamically unstable hydration sites. Using this information, we characterize the FKBP12 protein and show good agreement between fragment screening hit rates from NMR spectroscopy and hydration site energetics. Finally, we show that water molecules observed in crystal structures are less stable on average than bulk water as a consequence of the high degree of spatial localization, thereby resulting in a significant loss in entropy. These findings should help to better understand the characteristics of waters at the surface of proteins and are expected to lead to insights that can guide structure-based drug design efforts.     

Large-scale networks of hydration water molecules around bovine beta-trypsin revealed by cryogenic X-ray crystal structure analysis.

The hydration structure of bovine beta-trypsin was investigated in cryogenic X-ray diffraction experiments. Three crystal forms of the enzyme inhibited by benzamidine with different molecular packing were selected to deduce the hydration structure for the entire surface of the enzyme. The crystal structures in all three of the crystal forms were refined at the resolution of 1.8 A at 100 K and 293 K. The number of hydration water molecules around the enzyme at 100 K was 1.5 to two times larger than that at 293 K, indicating that the motion of hydration water was quenched by cooling. In particular, the increase in the number of hydration water molecules was prominent on flat and electrostatically neutral surface areas. The water-to-protein mass ratio and the radius of gyration of a structural model of hydrated trypsin at 100 K was consistent with the results obtained by other experimental techniques for proteins in solution. Hydration water molecules formed aggregates of various shapes and dimensions, and some of the aggregates even covered hydrophobic residues by forming oligomeric arrangements. In addition, the aggregates brought about large-scale networks of hydrogen bonds. The networks covered a large proportion of the surface of trypsin like a patchwork, and mechanically linked several secondary structures of the enzyme. By merging the hydration structures of the three crystal forms at 100 K, a distribution function of hydration water molecules was introduced to approximate the static hydration structure of trypsin in solution. The function showed that the negatively charged active site of trypsin tended to be easily exposed to bulk solvent. This result is of interest with respect to the solvent shielding effect and the recognition of a positively charged substrate by trypsin.