Charge density-dependent strength of hydration and biological structure.

Small ions of high charge density (kosmotropes) bind water molecules strongly, whereas large monovalent ions of low charge density (chaotropes) bind water molecules weakly relative to the strength of water-water interactions in bulk solution. The standard heat of solution of a crystalline alkali halide is shown here to be negative (exothermic) only when one ion is a kosmotrope and the ion of opposite charge is a chaotrope; this standard heat of solution is known to become proportionally more positive as the difference between the absolute heats of hydration of the corresponding gaseous anion and cation decreases. This suggests that inner sphere ion pairs are preferentially formed between oppositely charged ions with matching absolute enthalpies of hydration, and that biological organization arises from the noncovalent association of moieties with matching absolute free energies of solution, except where free energy is expended to keep them apart. The major intracellular anions (phosphates and carboxylates) are kosmotropes, whereas the major intracellular monovalent cations (K+; arg, his, and lys side chains) are chaotropes; together they form highly soluble, solvent-separated ion pairs that keep the contents of the cell in solution.     

Differences in hydration structure near hydrophobic and hydrophilic amino acids.

We use molecular dynamics to simulate recent neutron scattering experiments on aqueous solutions of N-acetyl-leucine-amide and N-acetyl-glutamine-amide, and break down the total scattering function into contributions from solute-solute, solute-water, water-water, and intramolecular correlations. We show that the shift of the main diffraction peak to smaller angle that is observed for leucine, but not for glutamine, is attributable primarily to alterations in water-water correlations relative to bulk. The perturbation of the water hydrogen-bonded network extends roughly two solvation layers from the hydrophobic side chain surface, and is characterized by a distribution of hydrogen bonded ring sizes that are more planar and are dominated by pentagons in particular than those near the hydrophilic side chain. The different structural organization of water near the hydrophobic solute that gives rise to the inward shift in the main neutron diffraction peak under ambient conditions may also provide insight into the same directional shift for pure liquid water as it is cooled and supercooled.     

Water in protein hydration and ligand recognition

This review describes selected basics of water in biomolecular recognition. We focus on a qualitative understanding of the most important physical aspects, how these change in magnitude between bulk water and protein environment, and how the roles that water plays for proteins arise from them. These roles include mechanical support, thermal coupling, dielectric screening, mass and charge transport, and the competition with a ligand for the occupation of a binding site. The presence or absence of water has ramifications that range from the thermodynamic binding signature of a single ligand up to cellular survival. The large inhomogeneity in water density, polarity and mobility around a solute is hard to assess in experiment. This is a source of many difficulties in the solvation of protein models and computational studies that attempt to elucidate or predict ligand recognition. The influence of water in a protein binding site on the experimental enthalpic and entropic signature of ligand binding is still a point of much debate. The strong water‐water interaction in enthalpic terms is counteracted by a water molecule's high mobility in entropic terms. The complete arrest of a water molecule's mobility sets a limit on the entropic contribution of a water displacement process, while the solvent environment sets limits on ligand reactivity.     

C-C-A-G-G-C-m5C-T-G-G. Helical fine structure, hydration, and comparison with C-C-A-G-G-C-C-T-G-G.

The self-complementary DNA duplex C-C-A-G-G-C-m5C-T-G-G has been refined against 1.75-A x-ray diffraction data to an R value of 17.4%. In the crystal of space group P6, 10-base pair DNA fragments with characteristic sequence-related fine structure stack end to end to form long antiparallel B-type double helices. As shown by a structure analysis at lower resolution (Heinemann, U., and Alings, C. (1991) EMBO J. 10, 35-43), the overall geometry of C-C-A-G-G-C-m5C-T-G-G is similar to that of the unmethylated analog C-C-A-G-G-C-C-T-G-G despite a different crystal environment. The present high resolution structure analysis permits a detailed comparison of the two duplexes and their hydration spheres. Helical parameters are significantly correlated between both molecules, with the exception of the base pair propeller. Sugar pucker and backbone torsion angles alpha, gamma, delta, and chi show similar mean values, but their individual values deviate significantly between duplexes. In contrast, torsion angles beta, epsilon, and zeta change along the strands of both duplexes in much the same way. The effect of single-site methylation on DNA conformation appears to be small and limited to the base pairs directly involved. Methylation tends to push base pairs toward the minor groove of the helix. A regular minor groove hydration pattern involves dual hydrogen bonding of water molecules to O-4' and base atoms of C-C-A-G-G-C-m5C-T-G-G.     

Protein structure and hydration probed by SANS and osmotic stress

Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addition of these solutes, the number of protein-associated (solute-excluded) water molecules can be estimated from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with molecular weight. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titration calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomolecular systems and assemblies using neutron scattering together with osmotic stress.     

Multilamellarity, structure and hydration of extruded POPC vesicles by SANS

The small-angle neutron scattering (SANS) data of 12 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dispersions at low lipid concentration (1 mg per 100-mg heavy water) prepared by 5, 9 and 29 extrusions through filters of pores with 50, 100, 200 and 400 nm diameter are presented. They were analyzed within a theory that permits the determination of both structural and hydration parameters of the bilayers as well as the portions of multilamellar vesicles in dispersions with negligible long-range order between the vesicles. The scattering length density profile across the bilayers is approximated by assuming a central hydrocarbon core surrounded by a water-accessible coat. It is modeled by two different forms of functions. In the boat model, the scattering length density of the coat changes linearly from core to water, whereas in the strip model it is constant across the water-accessible coat. It was found that the boat model reflects the reality better than the strip model. The decrease of the multilamellar vesicle portions, either with increasing the number of extrusions at same filter size and with decreasing the filter size, was characterized quantitatively.     

Water in channel-like cavities: structure and dynamics.

Ion channels contain narrow columns of water molecules. It is of interest to compare the structure and dynamics of such intrapore water with those of the bulk solvent. Molecular dynamics simulations of modified TIP3P water molecules confined within channel-like cavities have been performed and the orientation and dynamics of the water molecules analyzed. Channels were modeled as cylindrical cavities with lengths ranging from 15 to 60 A and radii from 3 to 12 A. At the end of the molecular dynamics simulations water molecules were observed to be ordered into approximately concentric cylindrical shells. The waters of the outermost shell were oriented such that their dipoles were on average perpendicular to the normal of the wall of the cavity. Water dynamics were analyzed in terms of self-diffusion coefficients and rotational reorientation rates. For cavities of radii 3 and 6 A, water mobility was reduced relative to that of simulated bulk water. For 9- and 12-A radii confined water molecules exhibited mobilities comparable with that of the bulk solvent. If water molecules were confined within an hourglass-shaped cavity (with a central radius of 3 A increasing to 12 A at either end) a gradient of water mobility was observed along the cavity axis. Thus, water within simple models of transbilayer channels exhibits perturbations of structure and dynamics relative to bulk water. In particular the reduction of rotational reorientation rate is expected to alter the local dielectric constant within a transbilayer pore.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.     

Water state and diffusion through lipid bilayers: Effect of hydration degree

The dependences of adsorbed water state (obtained from the variations in ¹H NMR spectra with the angle between the bilayer normal and magnetic field direction) and water diffusion along the bilayer normal (measured using pulsed field gradient ¹H NMR) on hydration degree have been studied in macroscopically oriented bilayers of dioleoylphosphatidylcholine. The angle dependences of the shape of NMR spectrum are qualitatively different only for water concentrations higher and lower than that achieved by hydration from saturated vapors (χ_eq, about 23%). At concentrations lower than χ_eq, all water in the sample either makes the hydration shells of the lipid polar heads or is in fast exchange with the shell water, so the spin-echo signal from water is detected only within a narrow range of angles close to the magic angle, 54.7°. At concentration exceeding χ_eq, the spin-echo signal from water is retained at all orientations, suggesting that a portion of water between bilayers (quasi-free water) slowly exchanges with water bound to the polar heads. There is an inverse dependence of the coefficient of water self-diffusion through the bilayer system on the hydration degree, which is described in the Tanner model with account of water self-diffusion in the hydrophobic part of the bilayer. Bilayer permeability, distribution coefficient of molecules between aqueous and lipid phases, and water self-diffusion coefficient in the hydrophobic region of the bilayer are estimated.     

Molecular dynamics simulations of Nafion and sulfonated polyether sulfone membranes. I. Effect of hydration on aqueous phase structure

We measured the water uptakes and proton conductivities of a Nafion membrane and three sulfonated polyether sulfone membranes (SPESs) with different values of ion-exchange capacity (IEC = 0.75, 1.0 and 1.4 meq/g) in relation to relative humidity in order to apply the findings to polymer electrolyte membrane fuel cells. The number of water molecules per sulfonic acid group λ at each humidity level was independent of the relative humidity for all membranes, but the proton conductivities of the SPESs were inferior to that of Nafion for the same λ value. Classical molecular dynamics simulations for the same membranes were carried out using a consistent force field at λ = 3, 6, 9, 12 and 15. The structural properties of water molecules and hydronium ions at a molecular level were estimated from radial distribution functions and cluster size distributions of water. We found that the radial distribution function of S(sulfonic acid)–S(sulfonic acid) of Nafion at λ = 3 indicated a significant correlation between the S–S pair, due to water channels, while the S–S pair of the SPESs showed a poor correlation. The cluster size distribution of water was also calculated in order to estimate the connectivity of the water channel. It is clear that some water is present in the SPESs as small, isolated clusters, especially when the water content is low.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

The structure and hydration of the A-DNA fragment d(GGGTACCC) at room temperature and low temperature.

The DNA fragment d(GGGTACCC) was crystallized as an A-DNA duplex in the hexagonal space group P6(1). The structure was analyzed at room temperature and low temperature (100K) at a resolution of 2.5 A. The helical conformations at the two temperatures are similar but the low-temperature structure is more economically hydrated than the room-temperature one. The structure of d(GGGTACCC) is compared to those of d(GGGTGCCC) and d(GGGCGCCC). This series of molecules, which consists of a mismatched duplex and its two Watson-Crick analogues, exhibits three conformational variants of the A-form of DNA, which are correlated with the specific intermolecular interactions observed in the various crystals. The largest differences in local conformation are displayed by the stacking geometries of the central pyrimidine-purine and the flanking purine-pyrimidine sites in each of the three duplexes. Stacking energy calculations performed on the crystal structures show that the mismatched duplex is destabilized with respect to each of the error-free duplexes, in accordance with helix-coil transition measurements.     

Responding phospholipid membranes--interplay between hydration and permeability.

Osmotic forces are important in regulating a number of physiological membrane processes. The effect of osmotic pressure on lipid phase behavior is of utmost importance for the extracellular lipids in stratum corneum (the outer part of human skin), due to the large gradient in water chemical potential between the water-rich tissue on the inside, and the relative dry environment on the outside of the body. We present a theoretical model for molecular diffusional transport over an oriented stack of two-component lipid bilayers in the presence of a gradient in osmotic pressure. This gradient serves as the driving force for diffusional motion of water. It also causes a gradient in swelling and phase transformations, which profoundly affect the molecular environment and thus the local diffusion properties. This feedback mechanism generates a nonlinear transport behavior, which we illustrate by calculations of the flux of water and solute (nicotine) through the bilayer stack. The calculated water flux shows qualitative agreement with experimental findings for water flux through stratum corneum. We also present a physical basis for the occlusion effect. Phase behavior of binary phospholipid mixtures at varying osmotic pressures is modeled from the known interlamellar forces and the regular solution theory. A first-order phase transformation from a gel to a liquid--crystalline phase can be induced by an increase in the osmotic pressure. In the bilayer stack, a transition can be induced along the gradient. The boundary conditions in water chemical potential can thus act as a switch for the membrane permeability.     

Aliphatics vs. aromatics hydration thermodynamics

By comparing the hydration thermodynamics of benzene with that of a hypothetical aliphatic hydrocarbon having the same accessible surface area (ASA) of benzene, Makhatadze and Privalov concluded that the whole difference is due to the weak H-bonds that water forms with the aromatic ring. The formation of such H-bonds would be characterized by a negative Gibbs energy change, slightly increasing in magnitude with temperature, and a positive entropy change over a large temperature range. The latter thermodynamic feature is not physically reliable for the formation of H-bonds. In the present article, by using a statistical mechanical dissection scheme of hydration, a microscopic interpretation for the numbers obtained by Makhatadze and Privalov is proposed. The difference in hydration Gibbs energy should be attributed to the different strength of van der Waals interactions that benzene can do with water, owing to the larger polarizability of the aromatic ring with respect to an aliphatic hydrocarbon of equal size. In addition, the difference in hydration entropy should account for the different extent of H-bond reorganization upon the insertion of benzene and the corresponding aliphatic hydrocarbon in water.     

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.     

Relation between structure and the degree of hydration for cobalt (II) bonded to nucleoside 5'-monophosphates.

The variation of coordination geometry with degree of hydration has been studied for cobalt(II) bonded to 5′-AMP, 5′-GMP, 5′-IMP, 5′-UMP, and 5′-CMP. Pink compounds with octahedral coordination about the metal ions were isolated but partial dehydration results in conversion to blue compounds containing tetrahedral cobalt. The ease of conversion is markedly dependent on the identity of the nucleotide. In the case of Co^II-5′-AMP warming in water at 34°C is sufficient to cause the change to the tetrahedral form. Spectral results are given and some possible implications to the behaviour of cobalt-containing metalloenzymes, such as Co-RNA polymerase from E. coli, are briefly discussed.     

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.     

Thermodynamic functions of biopolymer hydration. II. Enthalpy–entropy compensation in hydrophilic hydration processes

The thermodynamic functions of biopolymer hydration were investigated by multitemperature vapor pressure studies. Desorption measurements were performed that allowed determination of reversible isotherms in the hydration range of 0.1 to 0.3–0.5 g H₂O/g dry polymer. These isotherms are accessible to thermodynamic interpretation and are relevant to the interaction of water with biopolymers in their solution conformation. The results obtained on a series of different biopolymers (lysozyme, α-chymotrypsin, apo-lactoferrin, and desoxyribonucleic acid), show the following common features of interest: (1) The differential excess enthalpies (ΔH^e ) and entropies (ΔS^e ) are negative, and exhibit pronounced anomalies in a well-defined low-humidity range (approx. 0.1 g H₂O/g dry polymer). These initial extrema are interpretable by structural changes, induced in the native biopolymer structures by water removal below a critical degree of hydration. (2) The ΔH^e and ΔS^e terms exhibit statistically significant linear enthalpy–entropy compensation effects in all biopolymer–water systems investigated. The compensation temperatures \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta = \overline {\Delta H} ^e /\overline {\Delta S} ^e $\end{document} are approximately identical for all biopolymers, ranging from 360 to 500 K. The compensation effects are attributable to phase transitions of water molecules between the bulk liquid and the inner-sphere hydration shell of native biopolymers. (3) The negative excess free energies (ΔG^e ) decrease monotonically with increasing water content and are close to zero at 0.3 to 0.5 g H₂O/g polymer. This result indicates that only transitions between the bulk liquid and the inner-sphere hydration shell are associated with significant net free energy effects. The outer-sphere hydration water is thermodynamically comparable to bulk water. The importance of the proportionality factor \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta $\end{document} in the control of the free energy term is discussed.