Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.     

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.     

Dielectric studies of the binding of water to lysozyme

Dielectric dispersion measurements as a function of hydration are reported for lysozyme powder. The dispersion that occurs in the frequency range 10 kHz to 10 GHz can be analysed in terms of bound water molecules that form single or multiple hydrogen bonds, and the numbers found in these two categories agree well with recent X-ray data for lysozyme crystals. The dielectric data also indicate that at 20% ($\text{w}\text{w}$) hydration the bound water acts as a plasticizer to increase the vibrational freedom of the protein structure, and that this may be of relevance to the fact that the onset of enzymatic activity occurs at this hydration level. Also, a sudden transition in the polarizability of the protein-water system is found to occur at 7% ($\text{w}\text{w}$) hydration.     

Hydration Water, Charge Transport and Protein Dynamics

The hydration water of proteins is essential to biological activity but its properties are not yet fully understood. A recent study of dielectric relaxation of hydrated proteins [A. Levstik et al., Phys. Rev E.60 7604 (1999)] has found a behavior typical of a proton glass, with a glass transition of about 268 K. In order to analyze these results, we investigate the statistical mechanics and dynamics of a model of `two-dimensional water' which describes the hydrogen bonding scheme of bounded water molecules. We discuss the connection between the dynamics of bound water and charge transport on the protein surface as observed in the dielectric measurements.     

Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents

A comprehensive study of the hydration mechanism of an enzyme in nonaqueous media was done using molecular dynamics simulations in five organic solvents with different polarities, namely, hexane, 3-pentanone, diisopropyl ether, ethanol, and acetonitrile. In these solvents, the serine protease cutinase from Fusarium solani pisi was increasingly hydrated with 12 different hydration levels ranging from 5% to 100% (w/w) (weight of water/weight of protein). The ability of organic solvents to 'strip off' water from the enzyme surface was clearly dependent on the nature of the organic solvent. The rmsd of the enzyme from the crystal structure was shown to be lower at specific hydration levels, depending on the organic solvent used. It was also shown that organic solvents determine the structure and dynamics of water at the enzyme surface. Nonpolar solvents enhance the formation of large clusters of water that are tightly bound to the enzyme, whereas water in polar organic solvents is fragmented in small clusters loosely bound to the enzyme surface. Ions seem to play an important role in the stabilization of exposed charged residues, mainly at low hydration levels. A common feature is found for the preferential localization of water molecules at particular regions of the enzyme surface in all organic solvents: water seems to be localized at equivalent regions of the enzyme surface independently of the organic solvent employed.     

Water in keratin. Piezoelectric, dielectric, and elastic experiments.

To investigate actions of water in keratin, the piezoelectric, dielectric, and elastic constants are measured at 10 Hz, at temperatures between -160 and 150 degrees C, and at various hydration levels. From changes in the piezoelectric, dielectric, and dynamic mechanical parameters with moisture content (m.c.), we have identified three regimes (I, II, and III) in the hydration of water for keratin. At high hydration (21% m.c.) around 0 degree C, the piezoelectric constants for keratin steeply decrease with increasing temperature. This may be attributed to interfacial polarization which is strongly related to self-associated water molecules (particularly regime III water) just around crystalline helical regions which can exhibit the stress-induced, i.e., piezoelectric, polarization and may be attributed to electrode polarization induced by the increase of mobile ions in the amorphous matrix region, some of which would be released from their trapped states just around the piezoelectric phase by the regime III water. With increasing hydration, the elastic constants for keratin are found to increase below -70 degrees C and decrease above -70 degrees C. This suggests a viscoelastic transition of the keratin structure due to bound water (regime II water). The piezoelectric, dielectric, and elastic loss peaks are found at around -120 degrees C for hydrated keratin, believed to be due to tightly bound water (regime I water), which acts only to stiffen the keratin structure. The adsorption regions of water in keratin are discussed by a piezoelectric two-phase model, which consists of piezoelectric and nonpiezoelectric phases. It is proposed that water molecule would at least adsorb in the nonpiezoelectric phase.     

Water under the BAR

Many cellular processes require the generation of highly curved regions of cell membranes by interfacial membrane proteins. A number of such proteins are now known, and several mechanisms of curvature generation have been suggested, but so far a quantitative understanding of the importance of the various potential mechanisms remains elusive. Following previous theoretical work, we consider the electrostatic attraction that underlies the scaffold mechanism of membrane bending in the context of the N-BAR domain of amphiphysin. Analysis of atomistic molecular dynamics simulations reveals considerable water between the membrane and the positively charged concave face of the BAR, even when it is tightly bound to highly curved membranes. This results in significant screening of electrostatic interactions, suggesting that electrostatic attraction is not the main driving force behind curvature sensing, supporting recent experimental work. These results also emphasize the need for care when building coarse-grained models of protein-membrane interactions. These results are emphasized by simulations of oligomerized amphiphysin N-BARs at the atomistic and coarse-grained level. In the coarse-grained simulations, we find a strong dependence of the induced curvature on the dielectric screening.     

Conformational transitions in eosinophil cationic protein: a molecular dynamics study in aqueous environment

Extensive molecular dynamics simulations have been performed on eosinophil cationic protein (ECP). The two structures found in the crystallographic dimer (ECPA and ECPB) have been independently simulated. A small difference in the pattern of the sidechain hydrogen bonds in the starting structure has resulted in interesting differences in the conformations accessed during the simulations. In one simulation (ECPB), a stable equilibrium conformation was obtained and in the other (ECPA), conformational transitions at the level of sidechain interactions were observed. The conformational transitions exhibit the involvement of the solvent (water) molecules with a pore-like construct in the equilibrium conformation and an opening for a large number of water molecules during the transition phase. The details of these transitions are examined in terms of intra-protein hydrogen bonds, protein-water networks and the residence times of water molecules on the polar atoms of the protein. These properties show some significant differences in the region between the N-terminal helix and the loop before the C-terminal strand as a function of different conformations accessed during the simulations. However, the stable hydrogen bonds, the protein-water networks, and the hydration patterns in most part of the protein including the active site are very much similar in both the simulations, indicating the fact that these are intrinsic properties of proteins.     

Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.     

Protein structure and dynamics in nonaqueous solvents: insights from molecular dynamics simulation studies

Protein structure and dynamics in nonaqueous solvents are here investigated using molecular dynamics simulation studies, by considering two model proteins (ubiquitin and cutinase) in hexane, under varying hydration conditions. Ionization of the protein groups is treated assuming "pH memory," i.e., using the ionization states characteristic of aqueous solution. Neutralization of charged groups by counterions is done by considering a counterion for each charged group that cannot be made neutral by establishing a salt bridge with another charged group; this treatment is more physically reasonable for the nonaqueous situation, contrasting with the usual procedures. Our studies show that hydration has a profound effect on protein stability and flexibility in nonaqueous solvents. The structure becomes more nativelike with increasing values of hydration, up to a certain point, when further increases render it unstable and unfolding starts to occur. There is an optimal amount of water, approximately 10% (w/w), where the protein structure and flexibility are closer to the ones found in aqueous solution. This behavior can explain the experimentally known bell-shaped dependence of enzyme catalysis on hydration, and the molecular reasons for it are examined here. Water and counterions play a fundamental and dynamic role on protein stabilization, but they also seem to be important for protein unfolding at high percentages of bound water.     

Hydration of the dTn.dAn x dTn parallel triple helix: a Fourier transform infrared and gravimetric study correlated with molecular dynamics simulations.

We present a comparative analysis of the water organization around the dTn.dAn x dTn triple helix and the Watson-Crick double helix dTn.dAn respectively by means of gravimetric measurements, infrared spectroscopy and molecular dynamics simulations. The hydration per nucleotide determined by gravimetric and spectroscopic methods correlated with the molecular dynamics simulations shows that at high relative humidity (98% RH) the triple helix is less solvated than the duplex (17 +/- 2 water molecules per nucleotide instead of 21 +/-1). The experimental desorption curves are different for both structures and indicate that below 81% RH the triplex becomes more hydrated than the duplex. At this RH the FTIR spectra show the emergence of N-type sugars in the adenosine strand of the triplex. When the third strand is bound in the major groove of the Watson-Crick duplex molecular dynamics simulations show the formation of a spine of water molecules between the two thymidine strands.     

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.     

Molecular Mechanisms of Tight Binding through Fuzzy Interactions

Many intrinsically disordered proteins (IDPs) form fuzzy complexes upon binding to their targets. Although many IDPs are weakly bound in fuzzy complexes, some IDPs form high-affinity complexes. One example is the nonstructural protein 1 (NS1) of the 1918 Spanish influenza A virus, which hijacks cellular CRKII through the strong binding affinity (K_d ～10 nM) of its proline-rich motif (PRM^NS1) to the N-terminal Src-homology 3 domain of CRKII. However, its molecular mechanism remains elusive. Here, we examine the interplay between structural disorder of a bound PRM^NS1 and its long-range electrostatic interactions. Using x-ray crystallography and NMR spectroscopy, we found that PRM^NS1 retains substantial conformational flexibility in the bound state. Moreover, molecular dynamics simulations showed that structural disorder of the bound PRM^NS1 increases the number of electrostatic interactions and decreases the mean distances between the positively charged residues in PRM^NS1 and the acidic residues in the N-terminal Src-homology 3 domain. These results are analyzed using a polyelectrostatic model. Our results provide an insight into the molecular recognition mechanism for a high-affinity fuzzy complex.     

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.     

The molecular stoichiometric hydration model (SHM) as applied to tendon/collagen, globular proteins and cells

This report describes and documents the presence of multiple water-of-hydration fractions on proteins and in cells. Initial studies of hydration fractions in g of water/g of DM (dry mass) for tendon/collagen led to the development of the molecular SHM (stoichiometric hydration model) and the development of methods for calculating the size of hydration fractions on a number of different proteins of known amino acid composition. The water fractions have differences in molecular motion and other physical properties due to electrostatic interactions of polar water molecules with electric fields generated by covalently bound pairs of opposite partial charge on the protein backbone. The methods allow calculation of the size of four hydration fractions: single water bridges, double water bridges, dielectric water clusters over polar-hydrophilic surfaces and water clusters over hydrophobic surfaces. These four fractions provide monolayer water coverage. The predicted SHM hydration fractions match closely measured hydration fraction values for collagen and for globular proteins. This report also presents water sorption findings that support the SHM. The SHM is applicable for cell systems where it has been studied. In seven cell systems studied, more than half of all of the cell water had properties unlike those of bulk water. The SHM predicts and explains the commonly cited and measured bound water fraction of 0.2-0.4 g of water/g of DM on proteins. The commonly accepted concept that water beyond this bound water fraction can be considered bulk-like water in its physical properties is unwarranted.     

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.     

Ab initio molecular dynamics study of the hydration of the formohydroxamate anion

We apply ab initio molecular dynamics (AIMD) to study the hydration structures and electronic properties of the formohydroxamate anion in liquid water. We consider the cis- nitrogen-deprotonated, cis- oxygen-deprotonated, and trans- oxygen-deprotonated formohydroxamate tautomers. They form an average of 6.3, 6.9, and 6.0 hydrogen bonds with water molecules, respectively. The predicted pair correlation functions and time dependence of the hydration numbers suggest that water is highly structured around the nominally negatively charged oxime oxygen in O-deprotonated tautomers but significantly less so around the nitrogen atom in the N-deprotonated species. Wannier function analysis suggests that, in the O-deprotonated anions, the negative charge is concentrated on the oxime oxygen, while in the N-deprotonated case, it is partially delocalized between the nitrogen and the adjoining oxime oxygen atom.     

Myosin-induced volume increase of the hyper-mobile water surrounding actin filaments

Microwave dielectric spectroscopy can measure the rotational mobility of water molecules that hydrate proteins and the hydration-shell volume. Using this technique, we have recently shown that apart from typical hydrating water molecules with lowered mobility there are other water molecules around the actin filaments (F-actin) which have a much higher mobility than that of bulk water [Biophys. J. 85 (2003) 3154]. We report here that the volume of this water component (hyper-mobile water) markedly increases without significant change of the volume of the ordinary hydration shell when the myosin motor-domain (S1, myosin subfragment-1) binds to F-actin. No hyper-mobile component was found in the hydration shell of S1 itself. The present results strongly suggest that the solvent space around S1 bound to F-actin is diffusionally asymmetric, which supports our model of force generation by actomyosin proposed previously [op. cit.].