Effect of temperature, pressure, and cosolvents on structural and dynamic properties of the hydration shell of SNase: a molecular dynamics computer simulation study

It is now generally agreed that the hydration water and solvational properties play a crucial role in determining the dynamics and hence the functionality of proteins. We present molecular dynamics computer simulation studies on staphylococcal nuclease (SNase) at various temperatures and pressures as well as in different cosolvent solutions containing various concentrations of urea and glycerol. The aim is to provide a molecular level understanding of how different types of cosolvents (chaotropic and kosmotropic) as well as temperature and high hydrostatic pressure modify the structure and dynamics of the hydration water. Taken together, these three intrinsic thermodynamic variables, temperature, pressure, and chemical potential (or activity) of the solvent, are able to influence the stability and function of the protein by protein-solvent dynamic coupling in different ways. A detailed analysis of the structural and dynamical properties of the water and cosolvents at the protein surface (density profile, coordination numbers, hydrogen-bond distribution, average H-bond lifetimes (water-protein and water-water), and average residence time of water in the hydration shell) was carried out, and differences in the structural and dynamical properties of the hydration water in the presence of the different cosolvents and at temperatures between 300 and 400 K and pressures up to 5000 bar are discussed. Furthermore, the results obtained help understand various thermodynamic properties measured for the protein.     

The Kirkwood-Buff theory and the effect of cosolvents on biochemical reactions

Cosolvents added to aqueous solutions of biomolecules profoundly affect protein stability, as well as biochemical equilibria. Some cosolvents, such as urea and guanidine hydrochloride, denature proteins, whereas others, such as osmolytes and crowders, stabilize the native structures of proteins. The way cosolvents interact with biomolecules is crucial information required to understand the cosolvent effect at a molecular level. We present a statistical mechanical framework based upon Kirkwood-Buff theory, which enables one to extract this picture from experimental data. The combination of two experimental results, namely, the cosolvent-induced equilibrium shift and the partial molar volume change upon the reaction, supplimented by the structural change, is shown to yield the number of water and cosolvent molecules bound or released during a reaction. Previously, denaturation experiments (e.g., m-value analysis) were analyzed by empirical and stoichiometric solvent-binding models, while the effects of osmolytes and crowders were analyzed by the approximate molecular crowding approach for low cosolvent concentration. Here we synthesize these previous approaches in a rigorous statistical mechanical treatment, which is applicable at any cosolvent concentration. The usefulness and accuracy of previous approaches was also evaluated.     

Estimation of excess solvation numbers of water and cosolvents from preferential interaction and volumetric experiments

To elucidate, at a molecular level, how cosolvents influence protein stability, it is indispensable to understand the distribution of water and cosolvent molecules around proteins. Calculation of excess solvation numbers of water and cosolvents serves this purpose, and I show that they can be extracted from preferential interaction parameter and volumeric data via the Kirkwood-Buff theory. This scheme was applied to trehalose and glycerol (stabilizers) and urea (denaturant). Important insights from the application include stabilizer-induced enhancement of protein hydration, which, together with the stabilizer's exclusion from protein surfaces, may contribute to protein stabilization at high osmolyte concentrations.     

Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution--experiments and theoretical interpretation

We used pressure perturbation calorimetry (PPC), a relatively new and efficient technique, to study the solvation and volumetric properties of amino acids and peptides as well as of proteins in their native and unfolded state. In PPC, the coefficient of thermal expansion of the partial volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. Furthermore, the effects of various chaotropic and kosmotropic cosolvents on the volume and expansivity changes of proteins were measured over a wide concentration range with high precision. Depending on the type of cosolvent and its concentration, specific differences were found for the solvation properties and unfolding behaviour of the proteins, and the volume change upon unfolding may even change sign. To yield a molecular interpretation of the different terms contributing to the partial protein volume and its temperature dependence, and hence a better understanding of the PPC data, molecular dynamics computer simulations on SNase were also carried out and compared with the experimental data. The PPC studies introduced aim to obtain more insight into the basic thermodynamic properties of protein solvation and volume effects accompanying structural transformations of proteins in various cosolvents on one hand, as these form the basis for understanding their physiological functions and their use in drug designing and formulations, but also to initiate further valuable applications in studies of other biomolecular and chemical systems.     

Theoretical study of volume changes accompanying xenon-lysozyme binding: implications for the molecular mechanism of pressure reversal of anesthesia

The change in partial molar volume (PMV) accompanying the xenon-lysozyme binding was investigated for elucidating the molecular mechanism of the pressure reversal of general anesthesia, using the three-dimensional reference interaction site model theory of molecular solvation. An increase of the PMV from xenon binding to the substrate binding site of lysozyme was found, and the binding is suppressed by pressure, while the internal site binding did not change the PMV. The PMV change was analyzed by decomposing it into several contributions from geometry and hydration. We also analyzed the hydration change due to the binding. From the results, we draw a molecular picture of the PMV change accompanying xenon-lysozyme binding, which gives a possible mechanism of pressure reversal of anesthesia.     

Control of protein interfacial affinity by nonionic cosolvents

In a biological cell, proteins perform their functions in a highly complex environment comprising crowding and confinement effects as well as interactions with interfaces, cosolvents, and other biomolecules. Cosolvents can stabilize or destabilize the native folded structure of proteins in solution. In this study, we show that nonionic cosolvents also affect the interfacial affinity of proteins. We use bovine ribonuclease A and a planar silica-water interface as model system and apply neutron and optical reflectometry to analyze this system. The degree of protein adsorption and the density profile of adsorbed protein molecules were determined in the absence and the presence of cosolvents. It has been found that both the protein stabilizing glycerol and the protein destabilizing urea cause a distinct reduction in protein interfacial affinity, which may represent a rather unexpected result. However, it is suggested that different mechanisms are underlying the similar effects of glycerol and urea.     

Partial molar volume of proteins studied by the three-dimensional reference interaction site model theory

The three-dimensional reference interaction site model (3D-RISM) theory is applied to the analysis of hydration effects on the partial molar volume of proteins. For the native structure of some proteins, the partial molar volume is decomposed into geometric and hydration contributions using the 3D-RISM theory combined with the geometric volume calculation. The hydration contributions are correlated with the surface properties of the protein. The thermal volume, which is the volume of voids around the protein induced by the thermal fluctuation of water molecules, is directly proportional to the accessible surface area of the protein. The interaction volume, which is the contribution of electrostatic interactions between the protein and water molecules, is apparently governed by the charged atomic groups on the protein surface. The polar atomic groups do not make any contribution to the interaction volume. The volume differences between low- and high-pressure structures of lysozyme are also analyzed by the present method.     

Hydrophobic effects on partial molar volume

The hydrophobic effects on partial molar volume (PMV) are investigated as a PMV change in the transfer of a benzenelike nonpolar solute from the nonpolar solvent to water, using an integral equation theory of liquids. The volume change is divided into two effects. One is the "packing" effect in the transfer from the nonpolar solvent to hypothetical "nonpolar water" without hydrogen bonding networks. The other is the "iceberg" effect in the transfer from nonpolar water to water. The results indicate that the packing effect is negative and a half compensated by the positive iceberg effect. The packing effect is explained by the difference in the solvent compressibility. Further investigation shows that the sign and magnitude of the volume change depend on the solute size and the solvent compressibility. The finding gives a significant implication that the exposure of a hydrophobic residue caused by protein denaturation can either increase or decrease the PMV of protein depending on the size of the residue and the fluctuation of its surroundings.     

Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy

We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially.     

Crucial importance of translational entropy of water in pressure denaturation of proteins

We present statistical thermodynamics of pressure denaturation of proteins, in which the three-dimensional integral equation theory is employed. It is applied to a simple model system focusing on the translational entropy of the solvent. The partial molar volume governing the pressure dependence of the structural stability of a protein is expressed for each structure in terms of the excluded volume for the solvent molecules, the solvent-accessible surface area (ASA), and a parameter related to the solvent-density profile formed near the protein surface. It is argued that the entropic effect originating from the translational movement of water molecules plays critical roles in the pressure-induced denaturation. We also show that the exceptionally small size of water molecules among dense liquids in nature is crucial for pressure denaturation. An unfolded structure, which is only moderately less compact than the native structure but has much larger ASA, is shown to turn more stable than the native one at an elevated pressure. The water entropy for the native structure is higher than that for the unfolded structure in the low-pressure region, whereas the opposite is true in the high-pressure region. Such a structure is characterized by the cleft and/or swelling and the water penetration into the interior. In another solvent whose molecular size is 1.5 times larger than that of water, however, the inversion of the stability does not occur any longer. The random coil becomes relatively more destabilized with rising pressure, irrespective of the molecular size of the solvent. These theoretical predictions are in qualitatively good agreement with the experimental observations.     

Effect of Osmolytes on Pressure‐Induced Unfolding of Proteins: A High‐Pressure SAXS Study

Herein, we explore the effect of different types of osmolytes on the high‐pressure stability and tertiary structure of a well‐characterized monomeric protein, staphylococcal nuclease (SNase). Changes in the denaturation pressure and the radius of gyration are obtained in the presence of different concentrations of trimethylamine N‐oxide (TMAO), glycerol and urea. To reveal structural changes in the protein upon compression at various osmolyte conditions, small‐angle X‐ray scattering (SAXS) experiments were carried out. To this end, a new high‐pressure cell suitable for high‐precision SAXS studies at synchrotron sources was built, which allows one to carry out scattering experiments up to maximum pressures of about 7 kbar. Our data clearly indicate that the osmolytes that stabilize proteins against temperature‐induced unfolding drastically increase their pressure stability and that the elliptically shaped curve of the pressure–temperature–stability diagram of proteins is shifted to higher temperatures and pressures with increasing osmolyte concentration. A drastic stabilization is observed for the osmolyte TMAO, which exhibits not only a significant stabilization against temperature‐induced unfolding, but also a particularly strong stabilization of the protein against pressure. In fact, such findings are in accordance with in vivo studies (for example P. J. Yancey, J. Exp. Biol. 2005 , 208, 2819–2830), where unusually high TMAO concentrations in some deep‐sea animals were found. Conversely, chaotropic agents such as urea have a strong destabilizing effect on both the temperature and pressure stability of the protein. Our data also indicate that sufficiently high TMAO concentrations might be able to largely offset the destabilizing effect of urea. The different scenarios observed are discussed in the context of recent experimental and theoretical studies.     

Effects of sugars and polyols on the stability of azurin in ice

This study represents the first attempt to gain a quantitative estimate of the protective influence of sugars (sucrose and trehalose) and polyols (sorbitol and glycerol) on the thermodynamic stability (DeltaG degrees ) of a protein in low-temperature part-frozen aqueous solutions. The method, based on guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, distinguishes between the effects of cooling to subfreezing temperatures from those induced specifically by the formation of a solid ice phase. The results point out that in the liquid state the generally stabilizing effect (at molar concentrations) of these polyhydric compounds is markedly attenuated on cooling to subfreezing temperatures such that at -15 degrees C, only sucrose still exerts a significant increase in DeltaG degrees . At this temperature, and in the absence of additives, the formation of ice caused a progressive destabilization of the native fold, DeltaG degrees decreasing up to 3-4 kcal/mol as the fraction of liquid water in equilibrium with ice (V(L) was reduced to less than 1%. Unexpectedly, denaturation profiles in ice at selected V(L) demonstrate that none of the above sugars and polyols counters effectively the decrease in protein stability at small V(L). Only trehalose was able to partly attenuate the ice perturbation, raising DeltaG degrees by a modest 0.6-0.8 kcal/mol relative to the salt reference. In all cases the reduction in DeltaG degrees caused by the solidification of water correlates with the decrease in m-value. The implication is that DeltaASA of unfolding is smaller in ice because protein-ice interactions either increase the solvent-accessible surface area (ASA) of the native fold (partial unfolding) or reduce the ASA of the denatured state (compaction), or both. Information on the protein tertiary structure in ice, in the absence and in the presence of sucrose or glycerol, suggests that these osmolytes play an important role in maintaining a compact native state that in their absence is expanded and partly unfolded. Thus, it appears that the prevailing mechanism by which these osmolytes act as cryoprotectants is through preservation of the native conformation in the liquidus rather than by increasing the thermodynamic stability of the native fold.     

FLUORESCENCE LIFETIME STUDY OF THE DENATURATION OF RIBONUCLEASE-A

Abstract. Fluorescence quantum yield and lifetime measurements of the tyrosine residues in ribonuclease-A (RNase) were used to study the conformational changes involved in the denaturation of the enzyme. Measurements were done on RNase and on selectively acetylated RNase in the native, the partly denatured (reductive cleavage of S-S bridges or treatment with 8 M urea) and in the fully denatured state. The data were interpreted to mean that the opening of the S-S bridges causes large parts of the enzyme chain to unfold while leaving a hydrophobic region; including one of the tyrosine residues, intact. The biological activity of RNase is destroyed by this unfolding. Urea apparently does penetrate the protein coil but does not greatly affect the RNase structure since some of its biological activity is still retained. The opening of the S-S bridges in the presence of urea destroys the native conformation (and biological activity) completely leaving the protein in the form of an uncoiled polypeptide chain. It is suggested which parts of the protein structure might be affected by partial denaturation.     

Hydration of thermally denatured lysozyme studied by sorption calorimetry and differential scanning calorimetry

We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90 degrees C, in the other procedure a sample that contained 20% of water was denatured at 150 degrees C. The protein denatured at 90 degrees C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150 degrees C were studied at several temperatures in the range of 25-60 degrees C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.     

Isoelectric focusing in low-denaturing media: visualization in renal disease of variation of the isoelectric point of albumin not related to a remarkable conformational variation

The isoelectric properties of serum and urinary albumin from normal subjects and patients with nephrotic syndrome have been investigated in various conditions of denaturation, obtained by using urea (0-8 M) as a support in isoelectric focusing. In normal human serum, albumin is rather acidic (pI = 4.7) when focused in glycerol while the denatured form obtained by exposing the protein to 8 M urea has a much higher pI (6.1). Albumin from nephrotic patients is acidic in glycerol but at very low levels of urea (2M) it shifts from pI 4.7 to pI 6.1; the same effect has been induced by treating albumin with activated charcoal at low pH. In order to obtain more information on urea-induced changes, we have recorded the circular dichroic spectra of albumin when exposed to the concentration of urea used in gels, and we found that no conformational transition occurs for urea concentrations less than 5 M. Taken together, these observations reveal that variation of the pI of albumin in nephrotic syndrome occurs mainly due to a dissociating effect of urea on charged substances bound to this protein.     

Effect of denaturation by preheating on the foam fractionation behavior of ovalbumin

Ovalbumin is a globular protein. When it is denatured, it can produce molecular species with different conformational states, each of which has different adsorption properties at a gas-liquid interface. Such changes in adsorption can then affect the foaming behaviors of ovalbumin. Results of semi-batch foam fractionation of both native and denatured ovalbumin aqueous solutions are reported in this paper, along with possible relationships between denaturation and foam fractionation outcomes, such as the enrichment ratio and mass recovery. Bubble size and foam stability are determined in the experiments to show the effect of denaturation on these measured parameters in this system. The relationships between the bubble size, void fraction, and ovalbumin enrichment are also reported to reflect the effect of the presence of denatured species.     

Peptide conformational preferences in osmolyte solutions: transfer free energies of decaalanine

The nature in which the protecting osmolyte trimethylamine N-oxide (TMAO) and the denaturing osmolyte urea affect protein stability is investigated, simulating a decaalanine peptide model in multiple conformations of the denatured ensemble. Binary solutions of both osmolytes and mixed osmolyte solutions at physiologically relevant concentrations of 2:1 (urea:TMAO) are studied using standard molecular dynamics simulations and solvation free energy calculations. Component analysis reveals the differences in the importance of the van der Waals (vdW) and electrostatic interactions for protecting and denaturing osmolytes. We find that urea denaturation governed by transfer free energy differences is dominated by vdW attractions, whereas TMAO exerts its effect by causing unfavorable electrostatic interactions both in the binary solution and mixed osmolyte solution. Analysis of the results showed no evidence in the ternary solution of disruption of the correlations among the peptide and osmolytes, nor of significant changes in the strength of the water hydrogen bond network.     

On the molecular mechanism of stabilization of proteins by cosolvents: role of Lifshitz electrodynamic forces

Several ionic and nonionic additives are known to affect structural stability of proteins in aqueous solutions. At a fundamental level, the mechanism of stabilization or destabilization of proteins by cosolvents must be related to three-body interactions between the protein, additive, and the water medium. In this study, the role of the Lifshitz-van der Waals electrodynamic interaction between various additives (sucrose, glycerol, urea, poly(ethylene glycol)-200, betaine, taurine, proline, and valine) and bovine serum albumin (BSA) in water medium was examined. The electrodynamic interaction energy was attractive for all of the additives studied here when both far ultraviolet and infrared relaxations of the additives were included in their dielectric susceptibility representations. However, when only the infrared contribution was included for structure stabilizers and both far ultraviolet and infrared contributions for the structure destabilizers, the resulting electrodynamic interaction energy (E/kT) followed the structure stabilizing and/or destabilizing behavior of the additives; that is, the interaction was attractive for urea and PEG200 (structure destabilizers), whereas it was repulsive for sucrose, glycerol, betaine, taurine, alanine, valine, and proline (structure stabilizers). The electrodynamic interaction energy E/kT at any given surface-to-surface separation distance between the additives and BSA was positively correlated (r(2) = 0.92) with the experimental thermal denaturation temperature (T(d)) of BSA in 1 M solutions of the additives. These analyses provided a mechanistic basis for the experimental observations of exclusion of the structure-stabilizing additives from the protein-water interface and binding of the structure-destabilizing additives to the protein surface. The role of water structure in the three-body electrodynamic interaction is discussed. It is hypothesized that in the case of additives that enhance water structure the hydration shells formed around the additives effectively dampen the contribution of ultraviolet frequencies to the dielectric susceptibility of the additives and thus impart repulsive electrodyanamic interaction between the additive and the protein, whereas the opposite occurs in the case of additives that breakdown the hydrogen-bonded structure of water.     

Culation of partial molar volume and its components for molecular dynamics models of dilute solutions

This paper is a review of our recent computational studies of volumetric characteristics using computer models of dilute solutions. Partial molar volume (PMV) and its components are calculated for simple and complex molecules in water (methane, noble gases, surfactants, polypeptides). Advantages and disadvantages of various computational methods are discussed. It is proposed to use the Voronoi-Delaunay technique to determine the reasonable boundary between a solute molecule and solvent molecules and to identify the PMV components related to the molecule, the boundary layer, and the solvent. It is noted that the observed increase in PMV with temperature for large molecules is due to an increase in the volume of voids in the boundary layer, i.e., due to the “thermal volume.” In this case, the solvent gives a negative contribution to the PMV. In contrast, for simple molecules (methane), the contribution from the solvent is positive and is the main factor in the increase in the PMV, which is associated with a specific change in water structure around a spherical hydrophobic particle outside the boundary layer. For surfactant molecules, the contribution from the solvent changes sign (from negative to positive) with increasing temperature.     

Microcalorimetric study of thermal unfolding of lysozyme in water/glycerol mixtures: an analysis by solvent exchange model

Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition.