Unfolding/refolding studies of smooth muscle tropomyosin. Evidence for a chain exchange mechanism in the preferential assembly of the native heterodimer

The thermal and the urea-induced unfolding profiles of the coiled-coil alpha-helix of native and refolded tropomyosin from chicken gizzard were studied by circular dichroism. Refolding of tropomyosin at low temperature from alpha + beta subunits, dissociated by guanidinium chloride, urea, or high temperature, predominantly produced alpha alpha + beta beta homodimers in agreement with earlier studies of refolding from guanidinium chloride (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The presence of two unfolding transitions in low salt solutions with about equal helix loss verified the composition with the first unfolding transition of the homodimer mixture originating from alpha alpha. In contrast, refolding by equilibrating at temperatures close to physiological, however, produced the native alpha beta heterodimer, which unfolded in a single transition. The refolding kinetics of dissociated alpha + beta subunits indicated that beta beta homodimers form first, leading to alpha alpha homodimers both of which are relatively stable against chain exchange below approximately 25 degrees C. Equilibrating the homodimer mixture at 37-40 degrees C for long times, however, produced the native alpha beta molecule via chain exchange. The equilibria involved indicate that the free energy of formation from subunits of alpha beta is much less than that of (alpha alpha + beta beta)/2. In vivo folding of alpha beta from the two separate alpha and beta gene products is, therefore, thermodynamically favored over the formation of homodimers and biological factors need not be considered to explain the native preferred alpha beta composition.     

The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation.

1. The thermodynamically reversible unfolding and refolding of penicillinase between the native and fully unfolded states were followed by using guanidinium chloride as denaturant. 2. The equilibria, studied by optical rotation, u.v. absorption, viscosity and enzyme activity, show the presence of a state of intermediate conformation, termed state H, which is stable at 20 degrees C in 0.8 M-guanidinium chloride. 3. The physical properties of this state show that it is slightly expanded with an intrinsic viscosity of 8 ml-g-1, that the 13 tyrosine residues, which are distributed through the primary sequence, are maximally exposed to the solvent and that the helix content is the same as that of the native state. 4. The kinetics of the transition between the native state, state H and the fully unfolded state were followed by u.v. absorption and by optical rotation. They are interpreted as showing that state H lies on the folding pathway between the native and fully unfolded states. 5. The transition between the native state and state H exhibits monophasic unfolding kinetics and biphasic refolding kinetics. This indicates that there must be at least two intermediate states in this process, at least one of which lies on the folding pathway which may also involve cul-de-sac paths. 6. The results are discussed in terms of a mechanism involving rapid stabilization of nucleation regions in a moderately compact but internally solvated structure, with 'native format' [Anfinsen (1973) Science 181, 233-230] secondary structure stabilized by tertiary interaction. The final and rate-limiting step in refolding involves shuffling of these structural elements into the native state. 7. This model is discussed in relation to folding in vivo.     

Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase

The reversible denaturation by urea of beta-lactamase from Staphylococcus aureus was followed in the presence and absence of ammonium sulphate by circular dichroism studies, difference absorption spectroscopy and measurement of enzyme activity. The multiple unfolding and refolding transitions demonstrate the existence of a thermodynamically stable state of intermediate conformation in equilibrium with the native (N) and fully unfolded (U) states. Its physical properties show that it is identical to the state H found on denaturation by guanidinium chloride. State H is 10.1 (+/-1.5) kJ mol-1 less stable than the native state and 10.1 (+/-1.6) kJ mol-1 more stable than the unfolded state. Ammonium sulphate shifts both the N in equilibrium H and H in equilibrium U transitions to concentrations of urea higher by 5.3 M per mole of sulphate. It has markedly different effects on the thermodynamic stabilities of states N and H, making delta G'N-H, O and delta G'H-U, O more negative by 41 kJ mol and 20 kJ mole, respectively, per mole of ammonium sulphate. The change in equilibrium constant for the N-H transition is reflected almost exclusively in a dramatic change of the unfolding rate constant, which is decreased by a factor of 10(11) on addition of 1.4 M-sulphate. The presence of the substrate benzyl penicillin has little effect on the equilibria or kinetics of the N-H transition. The results are discussed in terms of the nature of the N-H transition and of the ordering of intermediate states on the folding pathway.     

Folding kinetics of the all-beta-sheet protein human basic fibroblast growth factor, a structural homolog of interleukin-1beta

The refolding and unfolding kinetics of the all-beta-sheet protein human basic fibroblast growth factor (hFGF-2) were studied by fluorescence spectroscopy. The kinetics of the unfolding transition are monophasic. The refolding reaction at high and low guanidinium chloride (GdmCl) concentrations is best described by mono- and biphasic folding, respectively. Refolding and unfolding of hFGF-2 (155 amino acids) is very slow compared with other non-disulfide-bonded monomeric proteins of similar size. For example, the rate constant for unfolding at 4.5 mol.liter(-1) GdmCl is 0.006 s(-1), and the refolding rate constants at 0.4 mol.liter(-1) GdmCl are 0.01 s(-1) and 0.0009 s(-1) (15 degrees C, pH 7.0). A characterization of the thermodynamic nature of the folding process using transition state theory revealed that the slow refolding is almost exclusively controlled by entropic factors, namely the strong loss of conformational freedom during refolding. The rate of the slow unfolding kinetics is mainly (and at low denaturant concentrations exclusively) controlled by the large positive change in enthalpy. hFGF-2 shows similar slow folding kinetics to that of its structural homolog interleukin-1beta. Since both proteins show very little sequence identity, it is suggested that their slow folding kinetics are determined by the complex beta-sheet arrangement of the native molecules.     

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.     

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.     

Aggregation as the basis for complex behaviour of cutinase in different denaturants

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.     

A comparative study of the unfolding of the endoglucanase Cel45 from Humicola insolens in denaturant and surfactant.

Cellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Cel45 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Cel45 unfolds in GdmCl according to a simple two-state model under equilibrium conditions, it accumulates a transient intermediate during refolding. The four disulfide bonds do not contribute detectably to the stability of the native state. Cel45 is unfolded by very low concentrations of C12-LAS (1-4 mM). An analysis of 16 mutants of Cel45 shows a very weak correlation between unfolding rates in denaturant and detergent; mutants that have the same unfolding rate in GdmCl (within a factor of 1.5) vary 1,000-fold in their unfolding rates in C12-LAS. The data support a simple model for unfolding by detergent, in which the introduction of positive charges or removal of negative charges greatly increases detergent sensitivity, while interactions with the hydrophobic detergent tail contribute to a smaller extent. This implies that different detergent-mediated unfolding pathways exist, whose accessibilities depend on individual residues. Double-mutant cycles reveal that mutations in two proximal residues lead to repulsion and a destabilization greater than the sum of the individual mutations as measured by GdmCl denaturation, but they also reduce the affinity for LAS and therefore actually stabilize the protein relative to wild-type. Ligands that interact strongly with the denatured state may therefore alter the unfolding process.     

Unfolding free energy changes determined by the linear extrapolation method. 2. Incorporation of delta G degrees N-U values in a thermodynamic cycle

The linear extrapolation method was used to evaluate the unfolding free energy changes (delta G degrees N-U) for phenylmethanesulfonyl chymotrypsin (PMS-Ct) at pH 6.0. The nonlinear least-squares fits of difference spectral data using urea and guanidinium chloride as denaturants gave identical values for delta G degrees N-U and delta epsilon degrees U, the latter being extinction coefficient differences between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of these parameters from the nature of solvent suggests strongly that they are characteristic properties of the protein alone. The delta G degrees N-U data at pH 6.0 and 4.0, which differ by more than 100-fold in stability of the protein, were incorporated into a thermodynamic cycle involving free energy changes for titration of native and unfolded PMS-Ct from pH 4.0 to 6.0. The purpose of the cycle was to test whether delta G degrees N-U obtained by use of the linear extrapolation method exhibits the characteristics required of a thermodynamic function of state. Within error, the thermodynamic cycle was found to accommodate the delta G degrees N-U quantities obtained at pH 4.0 and 6.0 for PMS-Ct.     

Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model.

The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl. This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP. These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible. From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits.     

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.     

Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.     

Folding of horse cytochrome c in the reduced state

Equilibrium and kinetic folding studies of horse cytochrome c in the reduced state have been carried out under strictly anaerobic conditions at neutral pH, 10 degrees C, in the entire range of aqueous solubility of guanidinium hydrochloride (GdnHCl). Equilibrium unfolding transitions observed by Soret heme absorbance, excitation energy transfer from the lone tryptophan residue to the ferrous heme, and far-UV circular dichroism (CD) are all biphasic and superimposable, implying no accumulation of structural intermediates. The thermodynamic parameters obtained by two-state analysis of these transitions yielded DeltaG(H2O)=18.8(+/-1.45) kcal mol(-1), and C(m)=5.1(+/-0.15) M GdnHCl, indicating unusual stability of reduced cytochrome c. These results have been used in conjunction with the redox potential of native cytochrome c and the known stability of oxidized cytochrome c to estimate a value of -164 mV as the redox potential of the unfolded protein. Stopped-flow kinetics of folding and unfolding have been recorded by Soret heme absorbance, and tryptophan fluorescence as observables. The refolding kinetics are monophasic in the transition region, but become biphasic as moderate to strongly native-like conditions are approached. There also is a burst folding reaction unobservable in the stopped-flow time window. Analyses of the two observable rates and their amplitudes indicate that the faster of the two rates corresponds to apparent two-state folding (U<-->N) of 80-90 % of unfolded molecules with a time constant in the range 190-550 micros estimated by linear extrapolation and model calculations. The remaining 10-20 % of the population folds to an off-pathway intermediate, I, which is required to unfold first to the initial unfolded state, U, in order to refold correctly to the native state, N (I<-->U<-->N). The slower of the two observable rates, which has a positive slope in the linear functional dependence on the denaturant concentration indicating that an unfolding process under native-like conditions indeed exists, originates from the unfolding of I to U, which rate-limits the overall folding of these 10-20 % of molecules. Both fast and slow rates are independent of protein concentration and pH of the refolding milieu, suggesting that the off-pathway intermediate is not a protein aggregate or trapped by heme misligation. The nature or type of unfolded-state heme ligation does not interfere with refolding. Equilibrium pH titration of the unfolded state yielded coupled ionization of the two non-native histidine ligands, H26 and H33, with a pK(a) value of 5.85. A substantial fraction of the unfolded population persists as the six-coordinate form even at low pH, suggesting ligation of the two methionine residues, M65 and M80. These results have been used along with the known ligand-binding properties of unfolded cytochrome c to propose a model for heme ligation dynamics. In contrast to refolding kinetics, the unfolding kinetics of reduced cytochrome c recorded by observation of Soret absorbance and tryptophan fluorescence are all slow, simple, and single-exponential. In the presence of 6.8 M GdnHCl, the unfolding time constant is approximately 300(+/-125) ms. There is no burst unfolding reaction. Simulations of the observed folding-unfolding kinetics by numerical solutions of the rate equations corresponding to the three-state I<-->U<-->N scheme have yielded the microscopic rate constants.     

Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding

The refolding of chymotrypsin inhibitor 2 (CI2) is, at least, a triphasic process. The rate constants are 53 s-1 for the major phase (77% of the total amplitude) and 0.43 and 0.024 s-1 for the slower phases (23% of the total amplitude) at 25 degrees C and pH 6.3. The multiphase nature of the refolding reaction results from heterogeneity in the denatured state because of proline isomerization. The fast phase corresponds to the refolding of the fraction of protein that has all its prolines in a native trans conformation in the denatured state. It is not catalyzed by peptidyl-prolyl isomerase. The rate-limiting step of folding for the slower phases, however, is proline isomerization, and they are both catalyzed by peptidyl-prolyl isomerase. The slowest phase has properties consistent with a process involving proline isomerization in a denatured state. In particular, the activation enthalpy is large, 16 kcal mol-1 K-1, and the rate is independent of guanidinium chloride concentration ([GdnHCl]). In comparison, the intermediate phase shows properties consistent with a process involving proline isomerization in a partially structured state. The activation enthalpy is small, 8 kcal mol-1 K-1, and the rate has a strong dependence on [GdnHCl]. Temperature dependences of the rate constants for unfolding and for the fast refolding phase, both in the absence and in the presence of GdnHCl, were used to characterize the thermodynamic nature of the transition state and its relative exposure to solvent. The Eyring plot for unfolding is linear, indicating that there is relatively little change in heat capacity between native state and transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic stability of bacteriorhodopsin mutants measured relative to the bacterioopsin unfolded state

The stability of bacteriorhodopsin (bR) has often been assessed using SDS unfolding assays that monitor the transition of folded bR (bR(f)) to unfolded (bR(u)). While many criteria suggest that the unfolding curves reflect thermodynamic stability, slow retinal (RET) hydrolysis during refolding makes it impossible to perform the most rigorous test for equilibrium, i.e., superimposable unfolding and refolding curves. Here we made a new equilibrium test by asking whether the refolding rate in the transition zone is faster than RET hydrolysis. We find that under conditions we have used previously, refolding is in fact slower than hydrolysis, strongly suggesting that equilibrium is not achieved. Instead, the apparent free energy values reported previously are dominated by unfolding rates. To assess how different the true equilibrium values are, we employed an alternative method by measuring the transition of bR(f) to unfolded bacterioopsin (bO(u)), the RET-free form of unfolded protein. The bR(f)-to-bO(u) transition is fully reversible, particular when we add excess RET. We compared the difference in unfolding free energies for 13 bR mutants measured by both assays. For 12 of the 13 mutants with a wide range of stabilities, the results are essentially the same within experimental error. The congruence of the results is fortuitous and suggests the energetic effects of most mutations may be focused on the folded state. The bR(f)-to-bO(u) reaction is inconvenient because many days are required to reach equilibrium, but it is the preferable measure of thermodynamic stability. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates

The thermodynamics and kinetics of unfolding of 28 bacteriophage T4 lysozyme variants were compared by using urea gradient gel electrophoresis. The mutations studied cause a variety of sequence changes at different residues throughout the polypeptide chain and result in a wide range of thermodynamic stabilities. A striking relationship was observed between the thermodynamic and kinetic effects of the amino acid replacements: All the substitutions that destabilized the native protein by 2 kcal/mol or more also increased the rate of unfolding. The observed increases in unfolding rate corresponded to a decrease in the activation energy of unfolding (delta Gu) at least 35% as large as the decrease in thermodynamic stability (delta Gu). Thus, the destabilizing lesions bring the free energy of the native state closer to that of both the unfolded state and the transition state for folding and unfolding. Since a large fraction of the mutational destabilization is expressed between the transition state and the native conformation, the changes in folding energetics cannot be accounted for by effects on the unfolded state alone. The results also suggest that interactions throughout much of the folded structure are altered in the formation of the transition state during unfolding.     

Compaction of ribosomal protein S6 by sucrose occurs only under native conditions

The effect of osmolyte sucrose on the stability and compaction of the folded and unfolded states of ribosomal protein S6 from Thermus thermophilus was analyzed. Confirming previous results obtained with sodium sulfate and trehalose, refolding stopped-flow measurements of S6 show that sucrose favors the conversion of the unfolded state ensemble to a highly compact structure (75% as compact as the folded state). This conversion occurs when the unfolded state is suddenly placed under native conditions and the compact state accumulates in a transient off-folding pathway. This effect of sucrose on the compaction of the unfolded state ensemble is counteracted by guanidinium hydrochloride. The compact state does not accumulate at higher guanidinium concentrations and the unfolded state ensemble does not display increased compaction in the presence of 6 M guanidinium as evaluated by collisional quenching of tryptophan fluorescence. In contrast, accessibility of the tryptophan residue of folded S6 above 1 M sucrose concentration decreased as a result of an increased compaction of the folded state. Unfolding stopped-flow measurements of S6 reflect this increased compaction of the folded state, but the unfolding pathway is not affected by sucrose. Compaction of folded and unfolded S6 induced by sucrose occurs under native conditions indicating that decreased protein conformational entropy significantly contributes to the mechanism of protein stabilization by osmolytes.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6

The unfolding and refolding of Staphylococcus aureus penicillinase have been followed by urea-gradient electrophoresis. Unfolding of the native state proceeds by an all-or-none transition to fully unfolded protein, with no detectable accumulation of partially unfolded states. In contrast, refolding is complex and proceeds by very rapid, reversible formation of a partially folded state, H, which had been detected and characterized previously, as it is the most stable conformation at intermediate denaturant concentrations. At very low urea concentrations, a more compact conformational state was observed as a transient intermediate in refolding. There was little kinetic heterogeneity of the unfolded protein, as is normally observed with proteins containing proline residues.     

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.