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.     

Structural Characteristics in Protein Hydration Investigated by Cryogenic X-ray Crystal Structure Analyses

Cryogenic X-ray crystallography has heen applied to investigate thehydration structures of proteins. The amount of hydration water moleculesidentified at cryogenic temperature is more than twice those at ambienttemperature, and the structural models of proteins with a lot of hydrationwater molecules have provided much information to elucidate the static anddynamical characteristics of hydration structures of proteins. On proteinsurface, hydration water molecules distribute non-randomly and stillretain the tetrahedral hydrogen-bond geometry as well as in bulk solvent.In addition, water molecules form clathrate-like arrangements to cover thehydrophobic residues exposed to solvent. The standard interaction geometryenables the three-dimensional extension of hydrogen-bond networks aroundprotein molecules and, simultaneously, ensures the concerted reorganizationof hydration structures during the dynamical motion of proteins at work.The hydration structure analyses at cryogenic temperatures may contributeto understanding physical principles governing the dynamics of `molecularmachines' in aqueous environment.     

Structure of hydration water in proteins: a comparison of molecular dynamics simulations and database analysis

Hydration layer water molecules play important structural and functional roles in proteins. Despite being a critical component in biomolecular systems, characterizing the properties of hydration water poses a challenge for both experiments and simulations. In this context we investigate the local structure of hydration water molecules as a function of the distance from the protein and water molecules respectively in 188 high resolution protein structures and compare it with those obtained from molecular dynamics simulations. Tetrahedral order parameter of water in proteins calculated from previous and present simulation studies show that the potential of bulk water overestimates the average tetrahedral order parameter compared to those calculated from crystal structures. Hydration waters are found to be more ordered at a distance between the first and second solvation shell from the protein surface. The values of the order parameter decrease sharply when the water molecules are located very near or far away from the protein surface. At small water-water distance, the values of order parameter of water are very low. The average order parameter records a maximum value at a distance equivalent to the first solvation layer with respect to the water-water radial distribution and asymptotically approaches a constant value at large distances. Results from present analysis will help to get a better insight into structure of hydration water around proteins. The analysis will also help to improve the accuracy of water models on the protein surface.     

Water's potential role: Insights from studies of the p53 core domain

Soluble proteins with amyloidogenic propensity such as the tumor suppressor protein p53 have high proportion of incompletely desolvated backbone H bonds (HB). Such bonds are vulnerable to water attack, thus potentially leading to the misfolding of these proteins. However, it is still not clear how the surrounding solvent influences the protein native states. To address this, systematic surveys by molecular dynamics simulations and entropy analysis were performed on the p53 core domain in this work. We examined seven wild/mutant X-ray structures and observed two types of water-network hydration in three "hot hydration centers" (DNA- or small molecule- binding surfaces of the p53 core domain). The "tight" water, resulting from the local collective hydrogen-bond interactions, is probably fundamental to the protein structural stability. The second type of water is highly "dynamical" and exchanges very fast within the bulk solution, which is unambiguously assisted by the local protein motions. An entropy mapping of the solvent around the protein and a temperature perturbation analysis further present the main features of the p53 hydration network. The particular environment created by different water molecules around the p53 core domain also partly explains the structural vulnerabilities of this protein.     

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.     

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.     

Lateral proton conduction at a lipid/water interface. Effect of lipid nature and ionic content of the aqueous phase

Fast lateral proton conduction was observed along the lipid/water interface using a fluorescence technique. This conduction can be detected for a large number of lipids, both phospholipids and glycolipids. The efficiency of the proton transfer is dependent on the molecular packing of the host lipid at a given surface pressure. The proton conduction which is present in the liquid expanded state is abolished by the transition to the liquid condensed state. The proton transfer is affected slightly by the ionic content of the aqueous subphase except in the case of calcium which can inhibit the conduction along phosphatidylglyceroethanolamine. We suggest that the transfer of the protons occurs along a bidimensional hydrogen-bond network formed from the polar head groups, their water molecules of hydration and the water molecules which are intercalated between the lipid molecules.     

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.     

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.     

Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability

Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.     

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.     

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.     

Modeling the hydration of proteins: prediction of structural and hydrodynamic parameters from X-ray diffraction and scattering data

The implications of protein-water interactions are of importance for understanding the solution behavior of proteins and for analyzing the fine structure of proteins in aqueous solution. Starting from the atomic coordinates, by bead modeling the scattering and hydrodynamic properties of proteins can be predicted reliably (Debye modeling, program HYDRO). By advanced modeling techniques the hydration can be taken into account appropriately: by some kind of rescaling procedures, by modeling a water shell, by iterative comparisons to experimental scattering curves (ab initio modeling) or by special hydration algorithms. In the latter case, the surface topography of proteins is visualized in terms of dot surface points, and the normal vectors to these points are used to construct starting points for placing water molecules in definite positions on the protein envelope. Bead modeling may then be used for shaping the individual atomic or amino acid residues and also for individual water molecules. Among the tuning parameters, the choice of the scaling factor for amino acid hydration and of the molecular volume of bound water turned out to be crucial. The number and position of bound water molecules created by our hydration modeling program HYDCRYST were compared with those derived from X-ray crystallography, and the capability to predict hydration, structural and hydrodynamic parameters (hydrated volume, radius of gyration, translational diffusion and sedimentation coefficients) was compared with the findings generated by the water-shell approach CRYSOL. If the atomic coordinates are unknown, ab initio modeling approaches based on experimental scattering curves can provide model structures for hydrodynamic predictions.     

Hydration in proteins observed by high-resolution neutron crystallography

It is well known that water molecules surrounding a protein play important roles in maintaining its structural stability. Water molecules are known to participate in several physiological processes through the formation of hydrogen bonds. However, the hydration structures of most proteins are not known well at an atomic level at present because X-ray protein crystallography has difficulties to localize hydrogen atoms. In contrast, neutron crystallography has no problem in determining the position of hydrogens with high accuracy.1 In this article, the hydration structures of three proteins are described- myoglobin, wild-type rubredoxin, and a mutant rubredoxin-the structures of which were solved at 1.5- or 1.6-A resolution by neutron structure determination. These hydration patterns show fascinating features and the water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen bond formation and dynamics of hydration. Our results further show that there are strong relationships between these shapes and the water environments.     

Crystallographic study of hydration of an internal cavity in engineered proteins with buried polar or ionizable groups

Although internal water molecules are essential for the structure and function of many proteins, the structural and physical factors that govern internal hydration are poorly understood. We have examined the molecular determinants of internal hydration systematically, by solving the crystal structures of variants of staphylococcal nuclease with Gln-66, Asn-66, and Tyr-66 at cryo (100 K) and room (298 K) temperatures, and comparing them with existing cryo and room temperature structures of variants with Glu-66, Asp-66, Lys-66, Glu-92 or Lys-92 obtained under conditions of pH where the internal ionizable groups are in the neutral state. At cryogenic temperatures the polar moieties of all these internal side chains are hydrated except in the cases of Lys-66 and Lys-92. At room temperature the internal water molecules were observed only in variants with Glu-66 and Tyr-66; water molecules in the other variants are probably present but they are disordered and therefore undetectable crystallographically. Each internal water molecule establishes between 3 and 5 hydrogen bonds with the protein or with other internal water molecules. The strength of interactions between internal polar side chains and water molecules seems to decrease from carboxylic acids to amides to amines. Low temperature, low cavity volume, and the presence of oxygen atoms in the cavity increase the positional stability of internal water molecules. This set of structures and the physical insight they contribute into internal hydration will be useful for the development and benchmarking of computational methods for artificial hydration of pockets, cavities, and active sites in proteins.     

How thick is the layer of thermal volume surrounding the protein?

Investigation on the volume properties of protein hydration layers is reported. Presented results are based on combination of Monte Carlo modeling and available experimental data. Six globular proteins with known data are chosen for analysis. Analyzing the model and the experimental results we found that water molecules bound to proteins by hydrogen bond are preferentially located at the places with local depressions on the protein surface. Consequently, the hydration level is not strictly proportional to the area of charged and polar surfaces, but also depends on the shape of the molecular surface. The thickness of the thermal volume layer as calculated in the framework of the scaled particle theory is 0.6-0.65 A for chosen proteins. The obtained value is significantly lower than that presented for proteins in earlier papers (where proportionality between the hydration level and the area of charged and polar surfaces was assumed), but is close to the value published for small solute molecules. Discussion including the influence of protein size and the thermal motion of the surface is presented.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

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.     

Non‐interacting surface solvation and dynamics in protein–protein interactions

Protein–protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure‐based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein–protein binding, even for simple lock‐and‐key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein–protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein–protein surfaces. We compare properties of the interface, rim, and non‐interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non‐interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non‐interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein–protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein–protein complexes from unintended protein–protein interactions. Proteins 2015; 83:445–458. © 2014 Wiley Periodicals, Inc.