Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.     

Thermal denaturation of cytochromes c of horse cow, and Candida krusei in aqueous guanidine hydrochloride.

Thermal denaturation of cytochromes c of horse, cow, and Candida krusei in aqueous guanidine hydrochloride in the neutral pH region was studied by means of absorption and optical rotation measurements. The values of standard free energy change upon denaturation were estimated over the temperature range from 3 to 51 degrees C. Large differences in the heat capacity of the native and denatured states amounting to several kcal/mol-deg were obtained for all three kinds of cytochromes c. These lead to a change in the sign of both the enthalpy and entropy change of denaturation, with maximum stability of the native state at 12 degrees C for horse and bovine cytochromes c and at 9 degrees C for Candida krusei.     

Two-domain structure of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase.

NADH-cytochrome b₅ reductase is an amphiphilic protein consisting of a hydrophilic (FAD-containing) moiety and a hydrophobic (membrane-binding) segment and exists in aqueous media as an oligomeric aggregate. Circular dichroism studies have shown that denaturation of the reductase by guanidine hydrochloride in the presence of Emulgen 109P, a nonionic detergent, is a two-stage process as a function of the denaturant concentration. The first transition occurs at about 1 m guanidine hydrochloride and the second one at much higher concentrations. The guanidine hydrochloride concentration causing the second-stage unfolding depends on the concentration of Emulgen 109P. A hydrophilic fragment of the reductase lacking the hydrophobic segment undergoes one-stage denaturation at about 1 m guandine hydrochloride regardless of the presence and absence of Emulgen 109P. Both the reductase as well as the hydrophilic fragment lose their NADH-ferricyanide reductase activity and FAD also at about 1 m guanidine hydrochloride in the presence of the detergent. These findings suggest that the first-stage denaturation of the reductase represents the unfolding of the hydrophilic moiety and the second one that of the hydrophobic segment. Gel chromatography experiments have suggested that in the presence of Emulgen 109P the reductase exists as a mixed micelle with the detergent and this aggregation state persists even after the first-stage denaturation (unfolding of the hydrophilic moiety). The dissociation of the mixed micelle seems to take place concomitant with the second-stage denaturation. It is concluded that the two moieties of the reductase molecule, though linked to each other covalently, exist as independent domains undergoing unfolding separately at least in the presence of Emulgen 109P. This structural feature of the reductase is similar to that of cytochrome b₅ reported by us. The reductase is, therefore, another example of amphiphilic membrane proteins having two structurally independent domains in the molecule.     

A guanidine hydrochloride induced change in ribonuclease without gross unfolding

Although denaturation of ribonuclease by guanidine hydrochloride to a random coil has been considered to be a simple two-state mechanism, the time dependence of our calorimetric data indicate that a cooperative endothermic pretransition may occur near 1.25 M. guanidine hydrochloride (pH 6 and 25°C) without gross unfolding of the protein. Reexamination of other observables as a function of guanidine hydrochloride concentrations as well as activity measurements suggests the possibility of some process other than simple binding occurring in the concentration range below the onset of gross denaturation.     

Protein stability: functional dependence of denaturational Gibbs energy on urea concentration

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.     

Stability of proteins in the presence of polyols estimated from their guanidinium chloride-induced transition curves at different pH values and 25 degrees C

We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.     

Increased thermal stability of proteins in the presence of sugars and polyols.

Sugars and polyols stablize proteins against heat denaturation. Scanning calorimetry was used to obtain a quantitative estimate of the degree of stabilization. Solutions of ovalbumin, lysozyme, conalbumin, and alpha-chymotrypsinogen were heated at a constant rate, and the temperature of the maximum rate of denaturation was estimated (Tm). Addition of a sugar or polyol raised Tm. The magnitude of the stabilizing effect (delta Tm) depended on both the nature of the protein and the nature of the sugar or polyol, ranging from 18.5 degrees C for lysozyme at pH 3 in the presence of 50% (w/w) sorbitol to 0 degrees C for conalbumin at pH 7 in 50% glycerol solution. It is argued that this stablization is due to the effects of sugars and polyols on hydrophobic interactions. The strength of the hydrophobic interaction was measured in model systems in sucrose and glycerol solutions. Sucrose and glycerol strengthened the pairwise hydrophobic interaction between hydrophobic groups; however, they reduced the tendency for complete transfer of hydrophobic groups from an aqueous to a nonpolar environment. The extent of stabliziation by different sugars and polyols is explained by their different influences on the structure of water. The difference between the partial molar volume of the sugar or polyol and its van der Waals volume was used as a rough quantitative measure of the structure-making or structure-breaking effect. There was a linear relationship between this quantity and delta Tm.     

Evidence for an intermediate in the denaturation and assembly of phosphoglucose isomerase

The denaturation of dimeric rabbit muscle phosphoglucose isomerase in guanidine hydrochloride occurs in two discrete steps consisting of partial unfolding followed by subunit dissociation. In 3.5 to 4.5 m guanidine hydrochloride the enzyme forms a stable denaturation intermediate. Formation of this intermediate abolishes catalytic activity, shifts the protein fluorescence emission maximum from 332 to 345 nm, exposes all of the unavailable sulfhydryl groups, and decreases the s_20,w from 6.8 to 4.6 S. The intermediate dissociates into fully unfolded polypeptide chains with further increases in the concentration of the denaturant. The fluorescence maximum shifts to 352 nm and the s_20,w of the denatured monomer is 1.6 S. From the equilibrium constant for subunit association, 3 × 10⁴M^?1, in 4.7 m guanidine hydrochloride, the apparent free energy of association is estimated to be ?6 kcal mol^?1. Reconstitution of the enzyme protein takes place by the reversal of the steps observed upon denaturation. The denatured monomers refold and associate to reform the dimeric intermediate which then anneals to yield the intact enzyme molecule.     

Thermodynamic stabilities of yeast iso-1-cytochromes c having amino acid substitutions for lysine 32

Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.     

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.     

An acid induced conformational transition of denatured cytochrome c in urea and guanidine hydrochloride solutions.

Previous work has shown that at neutral pH ferricytochrome c (horse heart) retains certain residual structures in concentrated solutions of urea or guanidine hydrochloride (Tsong, T. Y. (1974), J. Biol. Chem. 249, 1988). Present studies reveal that cooperative unfolding of these residual structures can be achieved by acidification of the protein to pH 4 in 9 M urea but can only be partially achieved in a 6 M guanidine hydrochloride solution. The evidence that the residual structures unfold in 9 M urea upon acidification is twofold. (1) Further uncoupling of the Trp-59-heme interaction occurs; this is reflected in the intensification of the tryptophan fluorescence from 55 to 90 percent relative to that of free tryptophan in the same solvent. (2) The intrinsic viscosity of the protein solution increases from 15.0 to 21 ml/g. The acidification also induces a spin-state transformation of the heme group at pH 5 both in urea and in guanidine hydrochloride. Acidic titration of the protein in urea and guanidine hydrochloride indicates that the unfolding involves the absorption of a single proton. However, the kinetics of the spin-state transformation are triphasic. These results suggest that the displacement of the ligand His-18 by a solvent molecule and the subsequent disintegration of the residual structures are complex processes and involve at least three kinetic steps. The ineffectiveness of guanidine hydrochloride as a denaturant for ferricytochrome c is shown to be due to the presence of the high concentration of Cl minus which can stabilize certain elements of the protein structure.     

Comparison of denaturation of tryptophan synthase alpha-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

Artocarpus hirsuta lectin. Differential modes of chemical and thermal denaturation.

Unfolding, inactivation and dissociation of the lectin from Artocarpus hirsuta seeds were studied by chemical (guanidine hydrochloride, GdnHCl) and thermal denaturation. Conformational transitions were monitored by intrinsic fluorescence and circular dichroism. The gradual red shift in the emission maxima of the native protein from 335 to 356 nm, change in the ellipticity at 218 nm and simultaneous decrease in the sugar binding activity were observed with increasing concentration of GdnHCl in the pH range between 4.0 and 9.0. The unfolding and inactivation by GdnHCl were partially reversible. Gel filtration of the lectin in presence of 1-6 m GdnHCl showed that the protein dissociates reversibly into partially unfolded dimer and then irreversibly into unfolded inactive monomer. Thermal denaturation was irreversible. The lectin loses activity rapidly above 45 degrees C. The exposure of hydrophobic patches, distorted secondary structure and formation of insoluble aggregates of the thermally inactivated protein probably leads to the irreversible denaturation.     

Inhibition by Polyphenolic Phytochemicals and Sulfurous Compounds of the Formation of 8-Chloroguanosine Mediated by Hypochlorous Acid,Human Myeloperoxidase,and Activated Human Neutrophils

The thermal denaturation temperature of a soy protein isolate was increased, but its gel-melting temperature was decreased by the addition of polyols with increasing concentration and number of hydroxyl groups of the polyols. This inverse stabilizing effect of polyols on the protein structure and gel is discussed in terms of the competing solvent effects on intra- and intermolecular hydrophobic interactions and on the peptide-peptide hydrogen bonds of the protein.     

Domain interaction in rabbit muscle pyruvate kinase. I. Effects of ligands on protein denaturation induced by guanidine hydrochloride

The structural stability of rabbit muscle pyruvate kinase was examined. The unfolding of pyruvate kinase was induced by guanidine hydrochloride, and the process was monitored by spectroscopic techniques (fluorescence and UV absorption) and hydrodynamic measurements (sedimentation velocity, sedimentation equilibrium, densimetry, and viscometry). The spectroscopic techniques revealed that the unfolding of pyruvate kinase induced by guanidine hydrochloride is not a simple cooperative process. This suggests that different regions of pyruvate kinase are unfolding with different efficiencies in response to the denaturant. These regions are most likely related to the domain structures observed by x-ray crystallography. In the presence of L-phenylalanine, the allosteric inhibitor, the denaturation process became more cooperative, and the enzyme dissociated and unfolded at a higher denaturant concentration. The binding of phenylalanine also induced a structural change in the enzyme, rendering it more susceptible to tryptic digestion. One of the peptides, the production rate of which was increased, was isolated and sequenced. Its N terminus is located at the interface between two domains, one of which contains the active site. This evidence indicates structural changes, probably involving domain-domain interaction, for pyruvate kinase in response to phenylalanine binding.     

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

α-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine α-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.     

The mechanism of action of Na glutamate, lysine HCl, and piperazine-N,N'-bis(2-ethanesulfonic acid) in the stabilization of tubulin and microtubule formation

Preferential interaction measurements between proteins and monosodium glutamate were carried out to arrive at an understanding of the mechanism of its strong effect on tubulin stability and self-assembly into microtubules. For all proteins studied, i.e. bovine serum albumin, lysozyme, beta-lactoglobulin, and calf brain tubulin, the protein showed a large preferential hydration in the presence of monosodium glutamate. The enhancement of tubulin self-association by monosodium glutamate can be interpreted in terms of the large unfavorable free energy of interaction between the additive and the protein. Preferential interactions were also examined for lysine hydrochloride, which also gave a preferential hydration of the proteins, except for tubulin. The dependence of the preferential hydration parameter on proteins was different for the two additives, suggesting the importance of net electrostatic charges of proteins in their interaction with glutamate anions and lysinium cations. The zero preferential interaction of lysine hydrochloride with tubulin indicates an affinity of the lysine cation for the protein. Both additives increased the transition temperature of proteins. This can be understood in terms of the unfavorable free energy of interaction between the additive and the protein surface, which should be even more unfavorable when the denaturation causes an increase in the surface area.     

Protein stabilization and destabilization by guanidinium salts

Preferential interactions of bovine serum albumin were measured with guanidine sulfate, guanidine acetate, and guanidine hydrochloride. The results showed an increasing preferential hydration with increasing salt concentration for the sulfate, positive preferential salt binding for the hydrochloride, and an intermediate situation for the acetate. These results correlate well with the known effects of the three salts on protein stability, namely, the stabilizing effect of guanidine sulfate and the denaturing effect of guanidine hydrochloride. Comparison of guanidinium and magnesium salts indicated that the substitution of guanidinium ion for Mg2+ decreases the preferential hydration and increases the preferential salt binding, suggesting that the perturbation by guanidinium ion binding of the surface free energy is greater than that by Mg2+ ion. It was concluded that guanidine salts are not a special class, but their activity toward proteins is modulated by the same fine balance between hydration and salt binding to protein as in the case of other salts, with the second factor being stronger in guanidine salts.     

Influence of Polyols on the Stability and Kinetic Parameters of Invertase from Candida utilis: Correlation with the Conformational Stability and Activity

Invertase (β-d-fructofuranoside fructohydrolase-E.C. 3.2.1.26) is a sucrose hydrolyzing enzyme found in microbial, plant and animal sources. Invertase from Candida utilis is a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. We investigated the mechanism of stabilization of invertase with polyols (glycerol, xylitol, and sorbitol). Activity, thermodynamic and kinetic measurements of invertase were performed as a function of polyol concentration and showed that polyols act as very effective stabilizing agents. The result from the solvent-invertase interaction shows preferential exclusion of the polyols from the protein domain leading to preferential hydration of protein. Apparent thermal denaturation temperature of the protein (T _m ) rose from 75 °C to a maximum of 85 °C in 30% glycerol. The stabilization has been attributed to the preferential hydration of the enzyme.     

Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: Distinctive global and residue level features

We present circular dichroism (CD), steady state fluorescence and multidimensional NMR investigations on the equilibrium unfolding of monomeric dynein light chain protein (DLC8) by urea and guanidine hydrochloride (GdnHCl). Quantitative analysis of the CD and fluorescence denaturation curves reveals that urea unfolding is a two-state process, whereas guanidine unfolding is more complex. NMR investigations in the native state and in the near native states created by low denaturant concentrations enabled residue level characterization of the early structural and dynamic perturbations by the two denaturants. Firstly, (15)N transverse relaxation rates in the native state indicate that the regions around N10, Q27, the loop between beta2 and beta4 strands, and K87 at the C-terminal are potential unfolding initiation sites in the protein. Amide and (15)N chemical shift perturbations indicate different accessibilities of the residues along the chain and help identify locations of the early perturbations by the two denaturants. Guanidine and urea are seen to interact at several sites some of which are different in the two cases. Notable among the common interaction site is that around K87 which is in close proximity to W54 on the protein structure, but the interaction modes of the two denaturants are different. The secondary chemical shifts indicate that the structural perturbation by 1M urea is small, compared to that by guanidine which is more encompassing over the length of the chain. The probable (phi, psi) changes at the individual residues have been calculated using the TALOS algorithm. It appears that the helices in the protein are significantly perturbed by guanidine. Further, comparison of the spectral density functions of the native and the two near native states in the two denaturants implicate greater loosening of the structure by guanidine as compared to that by urea, even though the structures are still in the native state ensemble. These differences in the early perturbations of the native state structure and dynamics by the two denaturants might direct the protein along different pathways, as the unfolding progresses on further increasing the denaturant concentration.