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.     

Conformational transitions of thioredoxin in guanidine hydrochloride

Spectral and hydrodynamic measurements of thioredoxin from Escherichia coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25 degrees C. This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative. Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 +/- 0.2 s. Refolding measurements in 2 M denaturant reveal three kinetic phases having relaxation times of 0.54 +/- 0.23, 14 +/- 6, and 500 +/- 130 s, accounting for 12 +/- 2%, 10 +/- 1%, and 78 +/- 3% of the observed change in tryptophan fluorescence. The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant. Both the slowest phase observed in refolding and the generation of the slowest phase in the denatured state have an activation enthalpy of 22 +/- 1 kcal/mol. These features of the slowest phase are compatible with an obligatory peptide isomerization of proline-76 to its cis isomer prior to refolding.     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

Denaturation of thymidylate synthetase from amethopterin-resistant Lactobacillus casei.

The effects of various concentrations of urea and guanidine hydrochloride on enzyme activity and on subunit association were determined. Incubation of thymidylate synthetase with buffered solutions of 3M to 3.5M guanidine hydrochloride or 5 M to 6 M urea resulted in the loss of about 90% of the enzyme activity. Under these denaturing conditions a red shift of the fluorescence emission maximum from 340 nm to 351 nm was observed together with a significant decrease in the relative fluorescence intensity of the protein. Studies at both 4 degrees C and 25 degrees C indicated that the enzyme was in the dimer form in 2 M guanidine hydrochloride but was dissociated into monomers in concentrations of this denaturant of 3 M and above. Although only monomeric species were evident at 4 degrees C in 6 M urea, at 25 25 degrees C this denaturant caused protein aggregation which increased with decreasing phosphate buffer concentration. Enzyme (5 mg/ml) in 0.5 M potassium phosphate buffer, pH 6.8, containing 4 M guanidine hydrochloride gave a minimum S20, w value of 1.22S at 25 degrees C. Sedimentation behavior of the native enzyme in the range of 5 to 20 mg/ml was only slightly concentration-dependent (4.28 S to 4.86 S) but extensive aggregation occurred above 20 mg/ml.     

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2. 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23. 5 A). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.     

Viscometric characterization of pennisetin from pearl millet

Pennisetin, the alcohol soluble storage protein of pearl millet (Pennisetum americanum), was isolated in a homogeneous state. The intrinsic viscosity [n] of this protein was found to be in the range of 16.5-17.7 ml/g in 70% (v/v) aqueous ethanol. The [eta] changed marginally when temperature was increased from 20 to 70 degrees C and also in the presence of 10 mM NaCl. The data indicated that pennisetin was a rigid, rod shaped asymmetric hydrodynamic particle with molecular dimensions in the range of 301 x 14.4 A - 317.7 x 14.2 A. During denaturation with guanidine hydrochloride (Gdn.HCl), the intrinsic viscosity of pennisetin increased from 16 to 25ml/g with a mid point at 3.6 M of the denaturant. The native protein structure was unfolded in 6 M Gdn.HCl as shown by the exposure of aromatic amino acid residues buried in the native state and this transition was found to be reversible. The intrinsic viscosity of pennisetin in 5.9 M Gdn.HCl corresponded to Mr 25,000 which was comparable to that determined by SDS-PAGE.     

Protein stabilization by urea and guanidine hydrochloride

The urea, guanidine hydrochloride, salt, and temperature dependence of the rate of dissociation of CO from a nonequilibrium state of CO-bound native ferrocytochrome c has been studied at pH 7. The heme iron of ferrocytochrome c in the presence of denaturing concentrations of guanidine hydrochloride (GdnHCl) and urea prepared in 0.1 M phosphate, pH 7, binds CO. When the unfolded protein solution is diluted 101-fold into CO-free folding buffer, the protein chain refolds completely, leaving the CO molecule bonded to the heme iron. Subsequently, slow thermal dissociation of the CO molecule yields to the heme coordination of the native M80 ligand. Thus, the reaction monitors the rate of thermal conversion of the CO-liganded native ferrocytochrome c to the M80-liganded native protein. The rate of this reaction, k(diss), shows a characteristic dependence on the presence of nondenaturing concentrations of the denaturants in the reaction medium. The rate decreases by approximately 1.9-3-fold as the concentration of GdnHCl in the refolding medium increases from nearly 0 to approximately 2.1 M. Similarly, the rate decreases by 1.8-fold as the urea concentration is raised from 0.l to approximately 5 M. At still higher concentrations of the denaturants the denaturing effect sets in, the protein is destabilized, and hence the CO dissociation rate increases sharply. The activation energy of the reaction, E(a), increases when the denaturant concentration in the reaction medium is raised: from 24.1 to 28.3 kcal mol(-1) for a 0.05-2.1 M rise in GdnHCl and from 25.2 to 26.9 kcal mol(-1) for a 0.1-26.9 M increase in urea. Corresponding to these increases in denaturant concentrations are also increases in the activation entropy, S(diss)/R, where R is the gas constant of the reaction. The denaturant dependence of these kinetic and thermodynamic parameters of the CO dissociation reaction suggests that binding interactions with GdnHCl and urea can increase the structural and energetic stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na(2)SO(4), which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.     

Equilibrium denaturation studies of mouse beta-nerve growth factor.

Equilibrium denaturation of dimeric mouse beta-nerve growth factor (beta-NGF) has been studied by monitoring changes in the protein's spectroscopic characteristics. Denaturation of beta-NGF in guanidine hydrochloride and urea resulted in an altered intrinsic fluorescence emission spectrum, fluorescence depolarization, and diminished negative circular dichroism. Native-like spectroscopic properties and specific biological activity are restored when denaturant is diluted from unfolded samples, demonstrating that this process is fully reversible. However, refolding of denatured beta-NGF is dependent on the three disulfide bonds present in the native protein and does not readily occur when the disulfide bonds are reduced. Graphical analysis and nonlinear least-squares fitting of beta-NGF denaturation data demonstrate that denaturation is dependent on the concentration of beta-NGF and is consistent with a two-state model involving native dimer and denatured monomer (N2 = 2D). The conformational stability of mouse beta-NGF calculated according to this model is 19.3 +/- 1.1 kcal/mol in 100 mM sodium phosphate at pH 7. Increasing the hydrogen ion concentration resulted in a 25% decrease in beta-NGF stability at pH 4 relative to pH 7.     

The unfolding pathway and conformational stability of potato carboxypeptidase inhibitor

The unfolding and denaturation curves of potato carboxypeptidase inhibitor (PCI) were investigated using the technique of disulfide scrambling. In the presence of denaturant and thiol initiator, the native PCI denatures by shuffling its native disulfide bonds and converts to form a mixture of scrambled PCI that consists of 9 out of a possible 14 isomers. The denaturation curve is determined by the fraction of native PCI converted to scrambled isomers under increasing concentrations of denaturant. The concentration of guanidine thiocyanate, guanidine hydrochloride, and urea required to denature 50% of the native PCI was found to be 0.7, 1.45, and 8 m, respectively. The PCI unfolding curve was constructed through the analysis of structures of scrambled isomers that were denatured under increasing concentrations of denaturant. These results reveal the existence of structurally defined unfolding intermediates and a progressive expansion of the polypeptide chain. The yield of the beads-form isomer (Cys(8)-Cys(12), Cys(18)-Cys(24), and Cys(27)-Cys(34)) as a fraction of total denatured PCI was shown to be directly proportional to the strength of the denaturing condition. Furthermore, the PCI sequence was unable to fold quantitatively into a single native structure. Under physiological conditions, the scrambled isomers of PCI that constitute about 4% of the protein were in equilibrium with native PCI.     

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.     

Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).     

Subunit dissociation and unfolding of rabbit muscle phosphofructokinase by guanidine by hydrochloride.

The denaturation of rabbit skeletal muscle phosphofructokinase by guanidine hydrochloride has been studied using fluorescence, light scattering, and enzyme activity measurements. The transition from fully active tetramer (0.1 M potassium phosphate (pH 8.0) at 10 and 23 degrees) to unfolded polypeptide chains occurs in two phases as measured by changes in the fluorescence spectrum and light scattering of the protein: dissociation to monomers at low guanidine hydrochloride concentrations (similar to 0.8 M) followed by an unfolding of the polypeptide chains, which presumably results in a random coil state, at high concentrations of denaturant (greater than 3.5 M). The initial transition can be further divided into two distinct stages. The native enzyme is rapidly dissociated to inactive monomers which then undergo a much slower conformational change that alters the fluorescence spectrum of the protein. The dissociation is complete within 2 min and is reversible, but the conformational change requires about 2 hr for completion and is not reversible under a variety of conditions, including the presence of substrates and allosteric effectors. The conformationally altered protomer reaggregates to form a precipitate at 23 degrees, but is stable below 10 degrees. The second major phase of the denaturation is fully reversible. A simple mechanism is proposed to account for the results, and its implications for the corresponding renaturation process are discussed.     

Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics

The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly pure ovalbumin, the decrease in nondenatured native protein showed first-order dependence. The activation energy obtained with different techniques varied between 430 and 490 kJ*mole(-1). First-order behavior was studied in detail using differential scanning calorimetry. The calorimetric traces were irreversible and highly scan rate-dependent. The shape of the thermograms as well as the scan rate dependence can be explained by assuming that the thermal denaturation takes place according to a simplified kinetic process where N is the native state, D is denatured (or another final state) and k a first-order kinetic constant that changes with temperature, according to the Arrhenius equation. A kinetic model for the temperature-induced denaturation and aggregation of ovalbumin is presented. Commercially obtained ovalbumin was found to contain an intermediate-stable fraction (IS) of about 20% that was unable to form aggregates. The denaturation of this fraction did not satisfy first-order kinetics.     

Kinetics of unfolding and refolding of proteins. 3. Results for lysozyme

The kinetics of the reversible denaturation of lysozyme by guanidine hydrochloride have been studied over a wide range of pH and denaturant concentration. The results contrast sharply with those observed for cytochrome c. The interconversion of native (N) and denatured (D) states is strictly first-order in both directions under most conditions, showing that no intermediate forms of any kind accumulate to a significant extent during the reaction. At one pH (pH 2.6) experiments were extended to extreme denaturant concentrations, and under these conditions kinetic intermediates were observed. Analysis by the procedures of the first paper of this series Ikai &; Tanford 1973 showed that the principal intermediate observed at low denaturant concentration must be different from that observed at high denaturant concentration, and that both must be on the direct pathway from N to D. This suggests that the over-all reaction mechanism is of the form

and this mechanism is able to account for all of the observed results. The intermediate X₁ which accumulates transiently in the renaturation reaction at low concentrations of guanidine hydrochloride is spectrally very similar to the native state, i.e. it is probably a highly ordered state, but most of the interactions between surface acidic and basic groups, which are responsible for the anomalous titration behavior of native lysozyme, do not yet exist in this state. It is probable that the difference between the kinetics of refolding of lysozyme and cytochrome c may be ascribed to the existence of disulfide cross-links in lysozyme, which were intact in these experiments. These cross-links greatly limit the possible pathways for folding and thus make it much less likely that incorrectly folded states on dead-end pathways are readily accessible.     

Refolding of denatured thioredoxin observed by size-exclusion chromatography

Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible dissociation and unfolding of aspartate aminotransferase from Escherichia coli: characterization of a monomeric intermediate

The unfolding and dissociation of the dimeric enzyme aspartate aminotransferase (D) from Escherichia coli by guanidine hydrochloride have been investigated at equilibrium. The overall process was reversible, as judged from almost complete recovery of enzymic activity after dialysis of 0.7 mg of denatured protein/mL against buffer. Unfolding and dissociation were monitored by circular dichroism and fluorescence spectroscopy and occurred in three separate phases: D in equilibrium 2M in equilibrium 2M* in equilibrium 2U. The first transition at about 0.5 M guanidine hydrochloride coincided with loss of enzyme activity. It was displaced toward higher denaturant concentrations by the presence of either pyridoxal 5'-phosphate or pyridoxamine 5'-phosphate and toward lower denaturant concentrations by decreasing the protein concentration. Therefore, bound coenzyme stabilizes the dimeric state, and the monomer (M) is inactive because the shared active sites are destroyed by dissociation of the dimer. M was converted to M* and then to the fully unfolded monomer (U) in two subsequent transitions. M* was stable between 0.9 and 1.1 M guanidine hydrochloride and had the hydrodynamic radius, circular dichroism, and fluorescence of a monomeric, compact "molten globule" state.     

Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease

Three very unstable mutant forms of staphylococcal nuclease were used to quantitate the change in the apparent equilibrium constant for reversible denaturation (Kapp) as a function of denaturant concentration for a variety of different denaturing solutes. The value of this equilibrium constant in the absence of denaturant (Kapp,0) was determined by renaturation of the mutant proteins with a combination of glycerol and calcium ion, the latter of which binds at the active site in the native conformation. Because Kapp,0 fell in the easily measurable range between 0.1 and 1, the change in Kapp, and thus the change in free energy (delta Gapp), at very low concentrations of denaturants could be accurately measured. With guanidine hydrochloride (GuHCl), the rate of change of the apparent free energy of denaturation with respect to denaturant concentration (d(delta Gapp)/dCGuHCl or mGuHCl) was found to be remarkably constant down to zero denaturant concentration, even though this value was different for each of the three proteins. Unlike GuHCl, urea exhibited a slightly reduced value of d delta Gapp/dCurea at low concentrations. Results with a number of thiocyanate, perchlorate, and iodide salts confirmed that the Hofmeister series holds for concentrations below 0.1 M; that is, with regard to efficacy as a denaturant SCN- greater than ClO4- greater than I- and Li+,NH4+ greater than Na+,K+. However, all of the chaotropic salts analyzed exhibited markedly increased values of d(delta Gapp)/dCsalt at concentrations below 0.2 M. One possible explanation for these large deviations from a linear relationship between delta Gapp and salt concentration is that weak binding or adsorption of chaotropic anions is occurring at a saturable number of sites in hydrophobic regions of the denatured state.     

The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant

The guanidine hydrochloride (GdnHCl) mediated denaturation pathway for the apo form of homodimeric Escherichia coli aspartate aminotransferase (eAATase) (molecular mass = 43.5 kDa/monomer) includes a partially folded monomeric intermediate, M* [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913; Birolo, L., Dal Piaz, F., Pucci, P., and Marino, G. (2002) J. Biol. Chem. 277, 17428-17437]. The present investigation of the urea-mediated denaturation of eAATase finds no evidence for an M* species but uncovers a partially denatured dimeric form, D*, that is unpopulated in GdnHCl. Thus, the unfolding process is a function of the employed denaturant. D* retains less than 50% of the native secondary structure (circular dichroism), conserves significant quaternary and tertiary interactions, and unfolds cooperatively (mD*<==>U = 3.4 +/- 0.3 kcal mol-1 M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D <==> 2M 2M* <==> 2U in GdnHCl and D <==> D* <==> 2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D <==> 2U) is 36 +/- 3 kcal mol-1, while that for the D* 2U transition is 24 +/- 2 kcal mol-1, both at 1 M standard state and pH 7.5.     

Denaturation of subtilisin BPN' and its derivatives in aqueous guanidine hydrochloride solutions.

The denaturation of subtilisin BPN' (EC 3.4.21.14) in guanidine hydrochloride was studied in order to find possible reasons for the exceptional stability of this enzyme against the action of denaturing agents including guanidine hydrochloride. Chemically modified subtilisins, i.e., phenylmethanesulfonylsubtilisin and thio-subtilisin, were completely denatured in 2 M guanidine hydrochloride at pH 7 without autolysis but they were stable in 0.5 M guanidine hydrochloride for at least 60 h. On the other hand, once completely denatured, the subtilisins remained inactive and in highly unfolded conformations for 60 h or longer after transfer into 0.5 M guanidine solution at pH 7 or 9. No enzymatic activity was regained when the guanidine concentration was lowered to almost zero. We concluded from these and other results described in this paper that this enzyme was thermodynamically unstable in 2 M guanidine hydrochloride at 20 degrees C and at pH 7. We wish to point out the possibility that the denaturation of this enzyme could indeed be irreversible.     

Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state.

We introduced a novel disulfide bond, modeled on that of bullfrog cytochrome c, into yeast iso-1-cytochrome c. The disulfide spontaneously forms upon purification. A variety of techniques were used to examine the denaturation of this variant and several non-cross-linked controls. Denaturation is reversible and, with the exception of the protein in which the two cysteines are blocked, consistent with a two-state process. Comparison of the calorimetric and van't Hoff enthalpy changes indicates that denaturation is two-state at pH 4.6. Calorimetric and fluorescence-monitored guanidine hydrochloride (GdnHCl) denaturation data indicate that the free energy of denaturation for the cross-linked protein (delta Gd at 300 K) is decreased relative to non-cross-linked controls. The dependence of delta Gd on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (mGdnHCl, Cm, delta Hd, delta Sd, and delta Cp, respectively), all decrease in magnitude upon introduction of the cross-link. The decrease in delta Hd and delta Sd were confirmed by monitoring absorbance at several wavelengths as a function of temperature. The cross-link also decreases the pH dependence of these observables. Circular dichroism studies indicate the denatured state of the cross-linked protein possesses more structure than non-cross-linked proteins, and this structure is refractory to increases in temperature and chemical denaturant. We conclude that the diminished values of delta Gd, delta Hd, delta Sd, delta Cp, and mGdnHCl result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.