Probing protein hydration and conformational states in solution

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.     

Role of water in protein kinase C catalysis and its binding to membranes

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.     

Watching small molecules move: interrogating ionic channels using neutral solutes

Whether they are small enough to wriggle through the current-carrying part of an ionic channel or big enough to be kept outside and thus able to exert an osmotic stress on the channel space, polymers interact with channels in several instructive ways. The osmotic stress of excluded polymers allows one to measure the number of water molecules that come out of the channel in transitions between various "open" to "closed" states. The loss of osmotic activity, due to the partial or completely unrestricted admission of small polymers becomes a measure of the transfer probabilities of polymers from solution to small cavities; it provides an opportunity to study polymer conformation in a perfectly sieved preparation. Current fluctuations due to the partial blockage by a transient polymer are converted into estimates of times of passage and diffusion constants of polymers in channels. These estimates show how a channel whose functional states last for milliseconds is able to average over the interactions with polymers, interactions that last only microseconds. One sees clearly that in this averaging, the macromolecular channel is large enough to react like a macroscopic object to the chemical potentials of the species that modulate its activity.     

Effects of hydration, ion release, and excluded volume on the melting of triplex and duplex DNA

The stability of DNA duplex and triplex structures not only depends on molecular forces such as base pairing or tripling or electrostatic interactions but also is sensitive to its aqueous environment. This paper presents data on the melting of Escherichia coli and poly(dA).poly(dT) duplex DNA and on the poly(dT).poly(dA). poly(dT) triplex in a variety of media to assess the contributions from the osmotic status and salt content of the media. The effects of volume exclusion on the stability of the DNA structures are also studied. From thermal transition measurements in the presence of low-molecular weight osmotic stressors, the number of water molecules released upon melting is found to be four waters per base pair for duplex melting and one water for the conversion of triplex to single-strand and duplex. The effects of Na+ counterion binding are also determined in ethylene glycol solutions so that the variation of counterion binding with water activity is evaluated. The data show that there is a modest decrease in the extent of counterion binding for both duplex and triplex as water activity decreases. Finally, using larger polyethylene glycol cosolutes, the effects on melting of volume exclusion by the solutes are assessed, and the results correlated with simple geometric models for the excluded volume. These results point out that DNA stability is sensitive to important conditions in the environment of the duplex or triplex, and thus, conformation and reactivity can be influenced by these solution conditions.     

Evolving ideas about osmosis and capillary fluid exchange

When a solute is dissolved in water at (T, pel), the temperature and external pressure applied to the solution, the water in the solution is altered as is pure liquid water at (T, pel - piH2Ol). The liquid water and the water in the solution are in equilibrium when piH2Ol is the osmotic pressure of the water in the solution. Every partial molar property of the water in the solution at (T, pel), including its vapor pressure, chemical potential, volume, internal energy, enthalpy and entropy, is identical with the same molar property of pure liquid water at (T, pel - piH2Ol). This elementary fact was deduced by Hulett in 1903 from a thought experiment; he concluded that the internal tension in the force bonding the water is the same in both solution and pure liquid water, in equilibrium, at these differing applied pressures. Hulett's understanding of osmosis and the means by which the water was altered by the solute were neglected and abandoned. Competing ideas included the notions that the solute attracts the water into the solution and that the solute lowers the activity (or concentration) of the water in the solution. These ideas imply that the solute acts on the solvent at the semipermeable membrane separating the solution and water. Hulett's theory of osmosis requires that the solute alter the water at the free surface of the solution where the solute exerts an internal pressure on the boundary of the solution retaining the solute. Fluid exchange across the capillary endothelium is influenced, in part, by colloidal proteins in the plasma. The role of the proteins in capillary fluid exchange must be reinterpreted based on Hulett's view, the only valid view of osmosis.     

Measured change in protein solvation with substrate binding and turnover

Osmotic stress is used to measure solvation changes that accompany the conformational changes of an active enzyme. For hexokinase both the equilibrium dissociation constant and the kinetic Michaelis-Menten constant for glucose vary linearly, and to the same extent, with the activity of water in the protein medium, as adjusted with large molecular weight (> 2000) osmolytes. The variation over the whole osmotic pressure range studied indicates that glucose binding is accompanied by the release of at least 65 +/- 10 water molecules, and this is reversed on enzyme turnover. The results indicate that near the physiological range of pressures the number may be higher. Most of this water, which behaves like an inhibitor, likely comes from the cleft which is induced to close around the substrate. Such large dehydration/rehydration reactions during turnover imply a significant contribution of solvation to the energetics of the conformational changes. Osmotic stress is a method of general applicability to probe water's contribution to functioning molecules.     

The experience of fathers of adult children with schizophrenia

A new method for calculating the total conformational free energy of proteins in water solvent is presented. The method consists of a relatively brief simulation by molecular dynamics with explicit solvent (ES) molecules to produce a set of microstates of the macroscopic conformation. Conformational energy and entropy are obtained from the simulation, the latter in the quasi-harmonic approximation by analysis of the covariance matrix. The implicit solvent (IS) dielectric continuum model is used to calculate the average solvation free energy as the sum of the free energies of creating the solute-size hydrophobic cavity, of the van der Waals solute-solvent interactions, and of the polarization of water solvent by the solute's charges. The reliability of the solvation free energy depends on a number of factors: the details of arrangement of the protein's charges, especially those near the surface; the definition of the molecular surface; and the method chosen for solving the Poisson equation. Molecular dynamics simulation in explicit solvent relaxes the protein's conformation and allows polar surface groups to assume conformations compatible with interaction with solvent, while averaging of internal energy and solvation free energy tend to enhance the precision. Two recently developed methods--SIMS, for calculation of a smooth invariant molecular surface, and FAMBE, for solution of the Poisson equation via a fast adaptive multigrid boundary element--have been employed. The SIMS and FAMBE programs scale linearly with the number of atoms. SIMS is superior to Connolly's MS (molecular surface) program: it is faster, more accurate, and more stable, and it smooths singularities of the molecular surface. Solvation free energies calculated with these two programs do not depend on molecular position or orientation and are stable along a molecular dynamics trajectory. We have applied this method to calculate the conformational free energy of native and intentionally misfolded globular conformations of proteins (the EMBL set of deliberately misfolded proteins) and have obtained good discrimination in favor of the native conformations in all instances.     

UNIFAC Modeling of Cosolvent Phase Partitioning in Nonaqueous Phase Liquid-Water Systems

In this study, an existing thermodynamic model was used to predict equilibrium phase partitioning behavior of a cosolvent in a two-phase nonaqueous phase liquid (NAPL)–water system. The activity coefficients are calculated using the universal quasichemical functional group activity coefficient (UNIFAC) method. We examined an assortment of cosolvent–NAPL pairs of environmental interest and compared the UNIFAC-predicted ternary phase diagrams against published experimentally derived ternary phase diagrams. Results show that the UNIFAC model is a promising method for predicting equilibrium cosolvent partitioning behavior in NAPL–water systems, and thus can be useful in estimating the potential for NAPL solubilization and mobilization in remediation processes. The cosolvent partitioning behavior is interpreted with regard to changes in the physical properties of the NAPL-water system. Changes in interfacial tension between the two phases were estimated using an existing correlation. A viscosity experiment was conducted for selected mixtures of ethanol, toluene, and water; and the viscosity was found to increase with increasing amounts of the cosolvent.     

Osmotic interaction of plasma proteins with interstitial macromolecules

The osmotic interaction of plasma proteins with collagen and hyaluronate has been evaluated by measuring the oncotic pressure of mixed solutions of varying composition. Collagen, despite its insolubility, exhibits a pronounced volume exclusion effect on plasma proteins, and the oncotic pressure of mixed solutions is considerably higher than that of the plasma protein stock solution. The volume exclusion of collagen on small molecules such as sucrose is negligible. A solution composed of 1.6% plasma proteins, 20% collagen, and .4% hyaluronate in Ringer solution, approximating the composition of the interstitium, was found to yield higher oncotic pressures than those previously reported from the interstitium. The probable role of impurities and degradation in the isolation process is discussed. Results reported earlier from in vitro and in vivo studies indicated that tissue oncotic pressures are considerably higher than generally recognized and that tissue fluid is in probable osmotic equilibrium with lymph in skin and muscle.     

Influence of Osmotic Suction on the Soil-Water Characteristic Curves of Compacted Expansive Clay

Unsaturated clays are subject to osmotic suction gradients in geoenvironmental engineering applications and it therefore becomes important to understand the effect of these chemical concentration gradients on soil-water characteristic curves (SWCCs). This paper brings out the influence of induced osmotic suction gradient on the wetting SWCCs of compacted clay specimens inundated with sodium chloride solutions/distilled water at vertical stress of 6.25 kPa in oedometer cells. The experimental results illustrate that variations in initial osmotic suction difference induce different magnitudes of osmotic induced consolidation and osmotic consolidation strains thereby impacting the wetting SWCCs and equilibrium water contents of identically compacted clay specimens. Osmotic suction induced by chemical concentration gradients between reservoir salt solution and soil-water can be treated as an equivalent net stress component, (pπ) that decreases the swelling strains of unsaturated specimens from reduction in microstructural and macrostructural swelling components. The direction of osmotic flow affects the matric SWCCs. Unsaturated specimens experiencing osmotic induced consolidation and osmotic consolidation develop lower equilibrium water content than specimens experiencing osmotic swelling during the wetting path. The findings of the study illustrate the need to incorporate the influence of osmotic suction in determination of the matric SWCCs.     

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.     

Programmed diffusional release rate from encapsulated cosolvent system

The programmed diffusional release rate of an active agent through a rate-controlling membrane from a cosolvent system is discussed. At initial conditions, the drug is present below saturation in solution in a solvent mixture, enclosed by the rate-controlling membrane; the solvent is composed of the main solvent and a consolvent, which increases the drug solubility in the main solvent. During operation, the active agent and cosolvent diffuse from the capsule at a rate controlled by the membrane. Equations were derived describing the release rate of the active agent as a function of the permeability of the cosolvent and agent, the capsule dimensions, and the system's initial conditions. A great variety of release rate profiles can be programmed from declining to increasing delivery rate patterns as a function of time. Experimental data are presented for the drug progesterone in solution in cyclohexane with methyl, heptyl, or cetyl alcohol as the cosolvent in a polyethylene capsule. The theory qualitatively predicts the theory qualitatively predicts the experimental results.     

Progress in the design of DNA sequence-specific lexitropsins

Polymeric microparticles containing two ceftiofur salts as antimicrobial agents for intramammary application in dry cows were prepared by modified o/w-solvent evaporation methods (dispersion or cosolvent method) or by a w/o/w-multiple emulsion solvent evaporation method. The microspheres were characterized with respect to drug loading, drug release, and morphological properties. The three methods resulted in high encapsulation efficiencies. The choice of organic solvent/solvent mixture strongly affected the structure of the microparticles; both matrix and reservoir-type structures with different porosities were obtained. Scaling up to larger batch sizes resulted in microspheres with a faster drug release. The addition of water-miscible cosolvents to the water-immiscible polymer solution allowed the preparation of microparticles from a drug solution rather than a drug dispersion. Microparticles prepared by the cosolvent method could be separated after shorter time intervals from the aqueous phase; the microspheres had a denser matrix with finely dispersed drug crystals and a slower drug release when compared with microspheres prepared by the dispersion method, which had a more porous structure with larger embedded drug crystals. The cosolvent and dispersion methods present a simple alternative to the w/o/w-solvent evaporation method for the encapsulation of water-soluble drugs with an external water phase.     

Stability of hydrophobic nuclei of protein molecules depending of the degree of packing

The values of free energies for the formation of hydrophobic cores with different degree of packing of nonpolar side chains have been calculated. It has been shown that the difference between the values of free energy for tightly packed cores inaccessible to water solvent molecules and the values of free energies for less tightly packed hydrophobic cores whose surface is in contact with the water solvent, is 50-60 kJ/mol and coincides with the value of the enzyme free energy change during its interaction with the substrate.     

The function and activity of certain membrane enzymes when localized on- and off- the membrane

A group of enzymes known to be involved in group translocation-type transport mechanisms for the uptake of a variety of nucleotide precursors are enzymatically active both in their natural membrane milieu and in aqueous solution. The activity in aqueous solution markedly differ, however, from the enzymatic activity when the enzyme is membrane localized. The adenine phosphoribosyltransferase (PRT) of E. coli (Hochstadt-Ozer and Stadtman, 71a) is capable of carrying out an exchange reaction between the base moieties of adenine and AMP without requiring P-ribose-PP as an intermediate; the enzyme in aqueous solution requires P-ribose-PP, indicating a different reaction mechanism in the two environments. Like the adenine PRT of E. coli, the hypoxanthine PRT of Salmonella typhimurium (Jackman and Hochstadt, '76) also carried out an exchange reaction on the membrane only and also is more sensitive to a number of inhibitors in aqueous solution relative to the sensitivity when embedded in the membrane. In addition, however, the hypoxanthine PRT, while restricted to hypoxanthine as a substrate in the membrane, also accepts guanine as substrate in its soluble form. The membrane capacities reas determined in a guanine PRT deletion strain (Jackman and Hochstadt, '76). Finally, in mammalian cell lines purine nucleoside phosphorylase, which translocates the ribose moiety of inosine across the plasma membrane of mouse fibroblasts undergoes a 30-fold increase in substrate turnover number upon liberation from the membrane. These data raise two important caveats with respect to study of membrane enzymes and transport. Firstly, an enzyme once solubilized and found to differ kinetically from substrate transport in situ cannot be excluded from participating in translocations in the membrane on the basis of its activity in aqueous solution. Secondly, an enzyme which "appears" largely soluble upon cell rupture cannot be assumed to be a cycloplasmic enzyme because of majority of the solubilized activity may represent only a small fraction of the enzyme molecules highly activated concomitant to their solubilization. In this latter case the ability to activate enzyme still residing on the membrane (e.g., with detergents) would be necessary in order to estimate total membrane associated activity after cell rupture.     

Biological therapy of ovarian cancer: current directions

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.     

Stabilization of optical characteristics of DNA cholesteric liquid-crystalline dispersions

Abnormal optical properties of liquid-crystalline dispersions (phases) formed as a result of phase exclusion of double-stranded DNA and RNA from water-salt poly(ethylene glycol) solutions and X-ray parameters of these phases are compared. It is shown that the cholesteric packing of nucleic acid molecules is realized at the certain osmotic pressure of a solvent only. A comparison of the optical properties of liquid-crystalline phases (dispersions) to their X-ray parameters allows one to put forward a suggestion on various hydratation (fluctuation) regimes of the nucleic acid behaviour under the condensed phases formation and factors, influencing the mode of packing of these molecules in phases formed. It is shown as well, that immobilization of DNA cholesteric liquid-crystalline particles in the content of polymeric matrix is accompanied by the stabilization of these particles and, hence, their specific abnormal optical activity as well as by formation of particles having a structure, which corresponds to the "optically isotropic ordered liquid" without abnormal optical activity. Data on stabilization of the cholesteric structure of liquid-crystalline DNA dispersions by creation of polymeric chelate bridges between the neighbouring DNA molecules, fixed in the structure of liquid-crystalline dispersions, are shown.     

The activity of porcine pancreatic phospholipase A2 in 20% alcohol/aqueous solvent, by experiment and electrostatics calculations

The activity of porcine pancreatic phospholipase A2 (pla2), measured at pH 8, is reduced when methanol or ethanol is added to the aqueous solution. Finite difference electrostatics calculations were used to study the effect of modelling mixed solvents on the pKas of histidine 48 and the amino-terminal group, both of which influence the pH-dependence of catalysis. Calculations and experiment indicate that these pKa values cannot account for the activity reduction. Charge separation in the transition state is destabilized in 20% alcohol solvent relative to 100% aqueous solvent. The calculated values, which are combinations of stabilizing and destabilizing contributions, are in qualitative agreement with experiment. Saturating dielectric theory is used to model solvent water ordering in a high electric field, and water dielectric structure is assumed to dominate at the 20% alcohol level. The observed agreement demonstrates the utility of transition state stabilization theory and continuum solvent modelling. It is further suggested that electrostatic effects on kcat contribute to the pH-dependence of activity around pH 7, and to previously reported activity changes for charge mutants.     

Seasonal regulation of prolactin secretion in hypophyseal stalk transected beef calves

Conformational transitions of holo-alpha-lactalbumin in a hydro-ethanolic cosolvent system was studied by spectrofluorescence, CD in near- and far-uv regions, and high-sensitivity differential scanning calorimetry. Experimental results allow us to propose that in isothermal conditions alpha-lactalbumin undergoes a number of conformational transitions with increasing ethanol concentration: N<=>I<=>D<=>H. The existence of I-state was deduced from spectrofluorometric and near-uv CD data. In this state the aromatic chromophores of the amino acid side chains are more accessible to the solvent displaying higher local mobility. The H-state was detected from far-uv CD spectra as a state corresponding to the content of alpha-helices higher than originally found in native protein. However, calorimetric measurements provide data revealing only the two-state mechanism of alpha-lactalbumin unfolding in both water and in aqueous ethanol solutions. This indicates that the energy levels of N- and I-states as well as of D- and H-states are similar. Thermodynamics of the unfolding of alpha-lactalbumin in hydroethanolic solutions was analyzed with the help of the linear model of solvent denaturation. Unfolding increments of enthalpy, entropy, and Gibbs energy of transfer of the protein from a reference aqueous solution to hydro-ethanolic solutions of different concentrations were determined from the calorimetric data. They are linear functions of molar ethanol fraction. The slope of the unfolding increment of Gibbs energy of transfer was calculated from data on transfer of amino acid residues taking into account the average solvent accessibility of amino acid residues in the native structure of small globular proteins, using the additive group contribution method.