Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.     

Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes.

The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.     

Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.     

Exploring the inhibitory activity of short-chain phospholipids against amyloid fibrillogenesis of hen egg-white lysozyme

Amyloid fibrillogenesis is an important pathological feature of a group of degenerative human diseases. The 129-residue enzyme hen egg-white lysozyme has been shown to form fibrils in vitro at pH 2.0 and 55°C. In this research, using various spectroscopic techniques, light scattering, and transmission electron microscopy, we first examined the influence of short-chain phospholipids on the amyloid fibrillogenesis and the structural changes derived from hen lysozyme in vitro. Both model short-chain phospholipids were observed to mitigate the fibrillogenesis of hen lysozyme. Also, urea-induced unfolding results suggested that the susceptibility of hen lysozyme to conformational changes elicited by the denaturant was observed to decrease upon addition of short-chain phospholipids. Moreover, our molecular dynamics simulations results demonstrated that the observed inhibitory action of short-chain phosoholipids against hen lysozyme fibrillogenesis might be attributable to the interference of β-strand extension by the binding of phospholipids to lysozyme's β-sheet-rich region. We believe that the outcome from this study may contribute to a better understanding the molecular factors affecting amyloid fibrillogenesis and the molecular mechanism(s) of the interactions between phospholipids/lipids and amyloid-forming proteins.     

The quantitative comparison of the effects of denaturants on the stability of proteins

A novel quantitative comparison of denaturants involving the complete reversible unfolding of proteins is presented. Ribonuclease A was denatured with guanidinium chloride in the presence of low fixed concentrations of various partial denaturants, with the unfolding process being monitored by circular dichroism and difference spectroscopy. The major advantage of this method is that it allows a direct quantitative comparison of the effects of denaturants on the stability of proteins. The effect on the stability of ribonuclease A was shown to be linearly dependent upon the concentration of denaturant. An investigation of the constitutive ions of salts revealed that their effects were additive only in the case of salts that have no specific binding capability. This method can also be useful in detecting the specific binding of salts.     

Process evaluations and economic analyses of recombinant human lysozyme and hen egg-white lysozyme purifications

Human lysozyme and hen egg-white lysozyme have antibacterial, antiviral, and antifungal properties with numerous potential commercial applications. Currently, hen egg-white lysozyme dominates low cost applications but the recent high-level expression of human lysozyme in rice could provide an economical source of lysozyme. This work compares human lysozyme and hen egg-white lysozyme adsorption to the cation exchange resin, SP-Sepharose FF, and the effect of rice extract components on lysozyme purification. With one exception, the dynamic binding capacities of human lysozyme were lower than those of hen egg-white at pH 4.5, 6, and 7.5 with ionic strengths ranging from 0 to 100 mM (5-20 mS). Ionic strength and pH had a similar effect on the adsorption capacities, but human lysozyme was more sensitive to these two factors than hen egg-white lysozyme. In the presence of rice extract, the dynamic binding capacities of human and hen egg-white lysozymes were reduced by 20-30% and by 32-39% at pH 6. Hen egg-white lysozyme was used as a benchmark to compare the effectiveness of human lysozyme purification from transgenic rice extract. Process simulation and cost analyses for human lysozyme purification from rice and hen egg-white lysozyme purification from egg-white resulted in similar unit production costs at 1 ton per year scale.     

Participation of the catalytic carboxyls, Asp 52 and Glu 35, and Asp 101 in the binding of substrate analogues to hen lysozyme.

The interactions of the substrate analogues, GlcNAc, beta-methyl GlcNAc, (GlcNAc)2, and (GlcNAc)3, with turkey egg-white lysozyme [ED 3.2.1.17], in which the Asp 101 of hen lysozyme is replaced by Gly, were studied at various pH values by measuring changes in the circular dichroic (CD) band at 295 nm. Results were compared with those for hen egg-white lysozyme. The modes of binding of these substrate analogues to turkey lysozyme were very similar to those hen lysozyme except for the participation of Asp 101 in hen lysozyme. The ionization constants of the catalytic carboxyls, Glu 35 and Asp 52, in the turkey lysozyme-(GlcNAc)3 complex were determined by measuring the pH dependence of the CD band at 304 nm, which originates from Trp 108 near the catalytic carboxyls. The ionization behavior of the catalytic carboxyls of turkey lysozyme in the presence and absence of (GlcNAc)3 was essentially the same as that for hen lysozyme. The pH dependence of the binding constant of (GlcNAc)3 to hen lysozyme was compared with that to turkey lysozyme between pH 2 and 8. The pH dependence of the binding constant for (GlcNAc)3 to turkey lysozyme could be interpreted entirely in terms of perturbation of catalytic carboxyls. In the case of hen lysozyme, it was interpreted in terms of perturbation of the catalytic carboxyls and Asp 101 in the substrate-binding site. The pK values of Asp 101 in hen lysozyme and the hen lysozyme-(GLcNAc)3 complex were 4.5 and 3.4, respectively. The binding constants of (GlcNAc)3 to lysozyme molecules with different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated. The binding constant of lysozyme, in which Asp 52 and Glu 35 are deprotonated, to (GlcNAc)3 was the smallest. The other three species had similar binding constant to (GlcNAc)3.     

The volume and compressibility changes of lysozyme associated with guanidinium chloride and pressure-assisted unfolding.

The apparent specific volumes and isentropic compressibilities of hen egg white lysozyme were measured in aqueous guanidinium chloride solutions at 25 degrees C by means of a vibrational densimeter and a sing-around ultrasonic velocimeter. Little transition attributable to a protein unfolding was detected in the partial specific volume, while the partial specific isentropic compressibility decreased slightly around the transition region. The pressure-assisted unfolding was also investigated in aqueous guanidinium chloride solutions by means of ultraviolet spectroscopy. Assuming a two-state transition model, it was found that the free energy change of unfolding depends almost linearly on pressure and the unfolding reaction is accompanied by a small decrease in volume. The compressibility behavior is in conflict with the notion that a protein structure is almost completely unfolded by guanidinium chloride and most of the amino acid residues in the protein interior are exposed to solvent. These results support the current view that globular proteins have some residual structures even in the unfolded state induced by a strong denaturant.     

Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model

The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).     

Effect of ethanol on folding of hen egg-white lysozyme under acidic condition

The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.     

Conversion of Trp 62 of hen egg-white lysozyme to Tyr by site-directed mutagenesis

An expression plasmid for hen egg-white lysozyme in Saccharomyces cerevisiae was constructed by inserting almost full-length cDNA (about 600 base pairs) encoding hen egg-white pre-lysozyme into a yeast expression vector, pAM 82. The hen lysozyme was expressed under the control of the repressible acid phosphatase promoter of pAM 82 in S. cerevisiae. About half of the expressed lysozyme was secreted in the yeast growth medium as a precise mature protein which exhibited specific activity consistent with that of authentic hen egg-white lysozyme. The replacement of Trp 62 of hen egg-white lysozyme with a tyrosine residue was performed by site-directed mutagenesis using a 19-mer oligodeoxyribonucleotide. The mutant lysozyme with Tyr 62 was found to exhibit enhanced bacteriolytic activity.     

The simultaneous binding of lanthanide and N-acetylglucosamine inhibitors to hen egg-white lysozyme in solution by 1H and 13C nuclear magnetic resonance.

Lanthanide ions and the N-acetylglucosamine (GlcNAc) sugars are able to bind simultaneously to hen egg-white lysozyme (EC 3.2.1.17). The present study characterizes the properties of the ternary complexes with lysozyme, which involve up to seven paramagnetic lanthanides and two diamagnetic lanthanides, together with alpha GlcNAc, beta GlcNAc, alpha MeGlcNAc and beta MeGlcNAc. pH titrations and binding titrations of the GlcNAc sugars with lysozyme-La(III) complexes show that the GlcNAc sugars bind to at least two independent sites and that one of them competes with La(III) for binding to lysozyme. Given the known binding site of lanthanides at Asp-52 and Glu-35, the competitive binding site of GlcNAc is identified as subsite E. A simple analysis of the paramagnetic-lanthanide-induced shifts shows that the GlcNAc sugar binds in subsite C, in accordance with crystallographic results [Perkins, Johnson, Machin & Phillips (1979) Biochem. J. 181, 21-36]. This finding was refined by several computer analyses of the lanthanide-induced shifts of 17 proton and carbon resonances of beta MeGlcNAc. Good fits were obtained for all the signals, except for two that were affected by exchange broadening phenomena. No distinction could be made between a fit for a two-position model of Ln(III) binding with axial symmetry to lysozyme, according to the crystallographic result, or a one-position model with axial symmetry where the Ln(III) is positioned mid-way between Asp-52 and Glu-35. Although this work establishes the feasibility of lanthanide shift reagents for study of protein-ligand complexes, further work is required to establish the manner in which lanthanides bind to lysozyme in solution.     

Crystal structure of a phage library-derived single-chain Fv fragment complexed with turkey egg-white lysozyme at 2.0 A resolution

The three-dimensional structure of the single-chain Fv fragment 1F9 in complex with turkey egg-white lysozyme (TEL) has been determined to a nominal resolution of 2.0 A by X-ray diffraction. The scFv fragment 1F9 was derived from phage-display libraries in two steps and binds both hen and turkey egg-white lysozyme, although the level of binding affinity is two orders of magnitude greater for the turkey lysozyme. The comparison of the crystal structure with a model of the single-chain Fv fragment 1F9 in complex with hen egg-white lysozyme (HEL) reveals that in the latter a clash between Asp101 in lysozyme and Trp98 of the complementarity determining region H3 of the heavy chain variable domain occurs. This is the only explanation apparent from the crystal structure for the better binding of TEL compared to HEL.The binding site topology on the paratope is not simply a planar surface as is usually found in antibody-protein interfaces, but includes a cleft between the light chain variable domain and heavy chain variable domain large enough to accommodate a loop from the lysozyme. The scFv fragment 1F9 recognizes an epitope on TEL that differs from the three antigenic determinants recognized in other known crystal structures of monoclonal antibodies in complex with lysozyme.     

Catalytic reaction mechanism of goose egg-white lysozyme by molecular modelling of enzyme-substrate complex

Despite the low similarity between their amino acid sequences, the core structures of the fold between chicken-type and goose-type lysozymes are conserved. However, their enzymatic activities are quite different. Both of them exhibit hydrolytic activities, but the goose-type lysozyme does not exhibit transglycosylation activity. The chicken-type lysozyme has a retaining-type reaction mechanism, while the reaction mechanism of the goose-type lysozyme has not been clarified. To clarify the latter mechanism, goose egg-white lysozyme (GEL)-N-acetyl-D-glucosamine (GlcNAc)6 complexes were modelled and compared with hen egg-white lysozyme (HEL)-(GlcNAc)6 complexes. By systematic conformational search, 48 GEL-(GlcNAc)6 complexes were modelled. The right and left side, and the amino acid residues in subsites E-G were identified in GEL. The GlcNAc residue D could bind towards the right side without distortion and there was enough room for a water molecule to attack the C1 carbon of GlcNAc residue D from alpha-side in the right side and not for acceptor molecule. The result of molecular dynamics simulation suggests that GEL would be an inverting enzyme, and Asp97 would act as a second carboxylate and that the narrow space of the binding cleft at subsites E-G in GEL may prohibit the sugar chain to bind alternative site that might be essential for transglycosylation.     

Unfolding of lysozyme by breaking its disulphide bridges results in exposure of hydrophobic sites.

The interaction of 1-anilino-naphthalene-8-sulphonate (ANS), a probe whose fluorescence is strongly dependent on hydrophobicity of the environment, with native lysozyme and lysozyme partially unfolded by breaking the disulphide bridges and reacting the free -SH groups with iodoacetamide, has been investigated. Monitoring the intensity of ANS fluorescence and the position of the emission maximum in the presence of native and partially unfolded lysozyme indicated that unfolding resulted in the exposure of hydrophobic sites. Hydrophobic sites could not be detected when native and partially unfolded lysozyme were denatured with urea or guanidinium chloride. Protein components of the cells export machinery like 'chaperones' associate only with partially unfolded proteins and not native, folded proteins. Hence, hydrophobic regions of proteins, exposed on partial unfolding, could be the sites of recognition by 'chaperone' proteins.     

Three-dimensional structure of goose-type lysozyme from the egg white of the Australian black swan, Cygnus atratus

The egg white of C. atratus contains two forms of lysozyme, a 'chick-type' which is similar to that found in the egg white of the domestic hen, and a 'goose-type' similar to that found in the egg white of the Embden goose. The molecular structure of the goose-type lysozyme has been determined at a resolution of a 2.8 A by X-ray crystallographic analysis. The structure consists of two domains linked by a long stretch of alpha-helix. In all, there are seven helical segments in the structure. While there is no amino acid sequence homology with either hen egg-white or bacteriophage T4 lysozymes, there are portions of the structure where the folding of the main chain is similar to that found in portions of either hen egg-white lysozyme or T4 lysozyme or both. In particular, there is a consistency of structure in the arrangement of acid groups in the catalytic site. G-o plots calculated for this structure and for the bacteriophage T4 lysozyme structure show that both have similar 'modules' of structure with boundaries occurring at structurally equivalent positions. Three of the common boundaries are equivalent structurally to three of the four module boundaries observed in G-o plots of hen egg-white lysozyme. The variation in the position of the remaining boundary may be related to differences in substrate binding.     

Structures of partridge egg-white lysozyme with and without tri-N-acetylchitotriose inhibitor at 1.9 A resolution.

The three-dimensional structures of native partridge egg-white lysozyme (PEWL) and PEWL complexed with tri-N-acetylchitotriose inhibitor have been determined crystallographically and refined at 1.9 A resolution. Crystals of native and complexed protein are isomorphous and have space group and cell dimensions that are identical to those of hen egg-white lysozyme (HEWL) under similar crystallization conditions. Full occupancy of the trisaccharide in the inhibitor complex has allowed definitive modeling and refinement of all three sugar residues, located at subsites A, B, and C in the PEWL active site. A comparison has been made with HEWL/inhibitor complexes in which coordinates were either not refined (Blake CCF, et al., 1967, Proc R Soc B 167:378-388) or were refined at partial occupancy (Cheetham JC, Artymiuk PJ, Phillips DC, 1992, J Mol Biol 224:613-628). Although the loop comprising residues 70-75 is located on the surface of the protein and not near the active site, it appears to be affected indirectly by trisaccharide binding such that the loop shifts toward the active site and becomes relatively immobilized. The source of this loop movement appears to be the anchoring of Trp62, located in the active site cleft, as it forms a hydrogen bond with O6 of the N-acetylglucosamine at site C. Good electron density for the trisaccharide in the PEWL complex structure shows that Asp 101 is involved in hydrogen bonding interactions with the terminal sugar residue.     

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue.

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 A resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is close to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.     

The transition state in the folding-unfolding reaction of four species of three-disulfide variant of hen lysozyme: the role of each disulfide bridge

The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.     

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.