Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Hydration of denatured and molten globule proteins

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of alpha-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.     

Protein precipitation and denaturation by dimethyl sulfoxide

Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements. The proteins used, i.e., ribonuclease, lysozyme, beta-lactoglobulin and chymotrypsinogen, all showed negative preferential DMSO binding, or preferential hydration, at low DMSO concentrations, where they are in the native state. As the DMSO concentration was increased, the preferential interaction changed from preferential hydration to preferential DMSO binding, except for ribonuclease. The preferential DMSO binding correlated with structural changes and unfolding of these proteins observed at higher DMSO concentrations. Amino acid solubility measurements showed that the interactions between glycine and DMSO are highly unfavorable, while the interactions of DMSO with aromatic and hydrophobic side chains are favorable. The observed preferential hydration of the native protein may be explained from a combination of the excluded volume effects of DMSO and the unfavorable interaction of DMSO with a polar surface, as manifested by the unfavorable interactions of DMSO with the polar uncharged glycine molecule. Such an unfavorable interaction of DMSO with the native protein correlates with the enhanced self-association and precipitation of proteins by DMSO. Conversely, the observed conformational changes at higher DMSO concentration are due to increased binding of DMSO to hydrophobic and aromatic side chains, which had been newly exposed on protein unfolding.     

Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding

The preferential interactions of proteins with solvent components were studied in concentrated aqueous solutions of the sulfate, acetate, and chloride salts of Mg2+, Ba2+, Ca2+, Mn2+ and Ni2+ [except for CaSO4, BaSO4, Mn-(OAc)2, and Ni(OAc)2], and results were compared with those of the Na+ salts. It was found that, for all the salts, the preferential hydration increased in the order of Cl- less than CH3-COO- less than SO42- regardless of the cationic species used, in agreement with the anionic lyotropic series, and that the same parameter exhibited a tendency to increase in the order of Mn2+, Ni2+ less than Ca2+, Ba2+ less than Mg2+ less than Na+. The salting-out and stabilizing or salting-in and destabilizing effectiveness of the salts were interpreted in terms of the observed preferential interactions. The surface tension increment of salts, which is a major factor responsible for the preferential interactions of the Na+ salts, had no correlation with those of the divalent cation salts. It was shown that the binding of divalent cations to the proteins overcomes the salt exclusion due to the surface tension increase, leading to a decrease in the preferential hydration. In conformity with this mechanism, the preferential interaction of MgCl2 was strongly pH dependent, because of the protein charge-dependent affinity of Mg2+ for proteins, while NaCl showed no pH dependence of the preferential interaction. The proposed mechanism was supported by a strong correlation between the preferential interaction results and the interaction of these salts with the model peptide compound acetyltetraglycine ethyl ester, described by Robinson and Jencks.     

Abnormal solubility behavior of beta-lactoglobulin: salting-in by glycine and NaCl

The causes of the salting-in of beta-lactoglobulin by glycine and NaCl, a solubility behavior contrary to expectations, were probed by a detailed study of the interactions between these solvent components and the protein. The preferential interactions of beta-lactoglobulin with solvent components in aqueous glycine and NaCl systems have been compared with those of bovine serum albumin and lysozyme. At neutral pH, beta-lactoglobulin exhibited insignificant preferential interactions in glycine and NaCl at low cosolvent concentrations and an increasing preferential hydration at higher concentrations, the levels approaching the values expected from the other two proteins. These results indicate considerable binding of the electrolytes to beta-lactoglobulin, sufficient to compensate for the exclusion due to perturbation of the solvent surface tension. The difference between the preferential interactions of beta-lactoglobulin and the other proteins with these two solvent additives was shown to be the cause of the increase of beta-lactoglobulin solubility even at high concentrations of the additives, at which they have salting-out effects on the other proteins. The preferential interactions of NaCl with the three proteins were examined as a function of pH. The results showed no pH dependence of the preferential hydration for bovine serum albumin and lysozyme, while this parameter increased significantly for beta-lactoglobulin at lower pH. This suggests that the binding of electrolytes to beta-lactoglobulin is due to a unique charge distribution on the surface of the protein around neutral pH, which imparts to this protein a large dipole moment.     

Molten globule intermediates of human serum albumin in low concentration of urea

Interaction of non-electrolytes such as urea with proteins especially at lower concentrations is opening-up newer concepts in the understanding of protein stability and folding in proteomics. In this study, the secondary and tertiary structural characteristics and thermal stability of human serum albumin at lower concentrations of urea have been monitored. The protein attains a molten globule like structure at concentration urea below 2 M. This structural state also shows an increase in the alpha-helical content as compared to the native state. At concentrations of urea above 2 M, human serum albumin starts unfolding, resulting in a three-state transition with two mid points of transitions at around 4 M and 7 M urea concentrations. The characteristics of the partially folded intermediates are discussed with respect to the three component system analyses. Preferential hydration dominates over preferential interaction at lower concentration of urea (up to 2.5 M) and at higher concentration, the preferential interaction overtakes preferential hydration in a competitive manner. Formation of structural intermediates at lower concentration of urea is hypothesized as a general phenomenon in proteins and fits in with the observation with preferential interaction parameters by Timasheff and co-workers in the case of lysozyme and ribonuclease at different pH values.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Thermodynamic and kinetic examination of protein stabilization by glycerol

The effect of concentrated glycerol on the thermal transitions of chymotrypsinogen and ribonuclease has been examined by differential spectrophotometry at 293 and 287 mm, respectively. It was found that for both proteins addition of glycerol raises the transition temperature, the increase in Tm being greater for ribonuclease than for chymotrypsinogen. This increase in the free energy of denaturation appears to reflect primarily a decrease in the entropy change. Analysis in terms of the Wyman linkage equation shows that, for both proteins, the exclusion of glycerol from the protein domain increases on denaturation i.e., the chemical potential of glycerol becomes even more positive when the protein unfolds relative to the native structure. This provides the thermodynamic stabilization free energy. Results of the kinetic examination of the slow unfolding reaction are consistent with the concept that the preferential exclusion of glycerol is related, at least in part, to enhanced solvent ordering.     

Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility

The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.     

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

Spectrofluorimetric detection of two states during melting of ribonuclease T1

The melting temperature of ribonuclease T1 was studied by the fluorescent method. It was shown that in the melting region the tryptophanyl fluorescence spectrum of the protein containing a single tryptophanyl is the sum of two simple spectra typical for tryptophanyl located in the hydrophobic environment and for tryptophanyl completely accessible to aqueous solvent, correspondingly. This implies the evidence of two forms of the protein, i.e. native (folded) and denatured (unfolded), in the transition region. No intermediate states were found in measured quantities. Therefore, ribonuclease T1 melting process corresponds to the two states model. The free energy of native structure stabilization of the protein at room temperature is delta G approximately equal to 37 kJ/mol.     

Interaction of ribonuclease A with aqueous 2-methyl-2,4-pentanediol at pH 5.8.

The interactions between ribonuclease A and solvent components in aqueous 2-methyl-2,4-pentanediol (MPD) have been investigated by differential refractometry and light scattering at pH 5.8, i.e., conditions similar to those used to crystallize the protein from this solvent system. Application of multicomponent thermodynamic theory shows that, at all solvent compositions up to 50% (v/v) MPD, the protein is preferentially hydrated; i.e., addition of ribonuclease to the mixed solvent leads to an increase in the chemical potential of MPD. This unfavorable thermodynamic interaction leads to phase separation, probably caused by local salting out of the MPD by the charges on the surface of the protein molecule. A parallel examination by circular dichroism (CD) has shown that the CD spectrum of ribonuclease in 50% MPD is indistinguishable from that in dilute buffer.     

Differential salt-induced stabilization of structure in the initial folding intermediate ensemble of barstar

The effects of two salts, KCl and MgCl(2), on the stability and folding kinetics of barstar have been studied at pH 8. Equilibrium urea unfolding curves were used to show that the free energy of unfolding, deltaG(UN), of barstar increased from a value of 4.7 kcalmol(-1) in the absence of salt to a value of 6.9 kcalmol(-1) in the presence of 1M KCl or 1M MgCl(2). For both salts, deltaG(UN) increases linearly with an increase in concentration of salt from 0M to 1M, suggesting that stabilization of the native state occurs primarily through a Hofmeister effect. Refolding kinetics were studied in detail in the presence of 1M KCl as well as in the presence of 1M MgCl(2), and it is shown that the basic folding mechanism is not altered upon addition of salt. The major effects on the refolding kinetics can be attributed to the stabilization of the initial burst phase ensemble, I(E), by salt. Stabilization of structure in I(E) by KCl causes the fluorescence properties of I(E) to change, so that there is an initial burst phase change in fluorescence at 320 nm, during refolding. The structure in I(E) is stabilized by MgCl(2), but no burst phase change in fluorescence at 320 nm is observed during refolding. The fluorescence emission spectra of I(E) show that when refolding is initiated in 1M KCl, the three tryptophan residues in I(E) are less solvent exposed than when folding is initiated in 1M MgCl(2). Stabilization of I(E) leads to an acceleration in the rate of the fast observable phase of folding by both salts, suggesting that structure of the transition state resembles that of I(E). The stabilization of I(E) by salts can be accounted for largely by the same mechanism that accounts for the stabilization of the native state of the protein, namely through the Hofmeister effect. The salts do not affect the rates of the slower phases of folding, indicating that the late intermediate ensemble, I(L), is not stabilized by salts. Stabilization of the native state results in deceleration of the fast unfolding rate, which has virtually no dependence on the concentration of KCl or MgCl(2) at high concentrations. The observation that the salt-induced stabilization of structure in I(E) is accompanied by an acceleration in the fast folding rate, suggests that I(E) is likely to be a productive on-pathway intermediate.     

Acceleration of oxidative folding of bovine pancreatic ribonuclease A by anion-induced stabilization and formation of structured native-like intermediates

Phosphate anions accelerate the oxidative folding of reduced bovine pancreatic ribonuclease A with dithiothreitol at several temperatures and ionic strengths. The addition of 400 mM phosphate at pH 8.1 increased the regeneration rate of native protein 2.5-fold at 15 degrees C, 3.5-fold at 25 degrees C, and 20-fold at 37 degrees C, compared to the rate in the absence of phosphate. In addition, the effects of other ions on the oxidative folding of RNase A were examined. Fluoride was found to accelerate the formation of native protein under the same oxidizing conditions. In contrast, cations of high charge density or ions with low charge density appear to have an opposite effect on the folding of RNase A. The catalysis of oxidative folding results largely from an anion-dependent stabilization and formation of tertiary structure in productive disulfide intermediates (des-species). Phosphate and fluoride also accelerate the initial equilibration of unstructured disulfide ensembles, presumably due to non-specific electrostatic and hydrogen bonding effects on the protein and solvent.     

Hydration state change of proteins upon unfolding in sugar solutions

Change in hydration number of proteins upon unfolding, Deltan, was obtained from the analysis of thermal unfolding behavior of proteins in various sugar solutions with water activity, a(W), varied. By applying the reciprocal form of Wyman-Tanford equation, Deltan was determined to be 133.9, 124.1, and 139.2 per protein molecule for ribonuclease A at pH=5.5, 4.2, and 2.8, respectively, 201.4 for lysozyme at pH=5.5, and 100.1 for alpha-chymotripnogen A at pH=2.0. Among the sugars tested, reducing sugars gave the lower apparent Deltan as compared with nonreducing sugars probably because of the direct interaction of reducing terminal with amino group of proteins at a high temperature. From the knowledge of Deltan, a new thermodynamic model for protein stability was proposed with explicit consideration for hydration state change of protein upon unfolding. From this model, the contribution of a(W) was proven to be always positive for stabilization of proteins and its effect is not negligible depending on Deltan and a(W).     

The effects of pH and ionic strength on the partitioning of four proteins in reverse micelle systems

Four proteins with different physicochemical properties have been partitioned in reversed micelle systems: thaumatin, ribonuclease A, soybean trypsin inhibitor, and alpha-lactalbumin. The organic phase was formed by sodium salt (AOT) in isooctane, and the aqueous phase contained KCl, KBr, MgCl(2), or NaCl. Aqueous phase pH was varied between 2 and 13 and ionic strength from 0.1 to 1.0 M. Small changes in pH [around the isoelecric point (pl)] were found to influence the solubilization of ribonuclease A and trypsin inhibitor, but for thaumatin the pH change necessary to affect partition was much greater as a consequence of the difference in net charge (titration curves) of these protein molecules as pH changes. The type of ions present in the system was also a determining factor for partition; the larger ions (K(+)) produced more electrostatic screening and hence less protein solubilization than the smaller ions (Na(+)). With changes in ionic strength surface hydrophobicity was a dominant factor affecting solubilization of thaumatin in NaCl-containing systems at high pH. Charge distribution and hydrophobicity are thought to be important parameters when partitioning the protein alpha-lactalbumin. (c) 1994 John Wiley & Sons, Inc.