Spectral studies of horse heart porphyrin cytochrome c

Removal of the heme iron from cytochrome c to generate porphyrin cytochrome c relieves the quenching of porphyrin fluorescence and enhances the fluorescence of the single tryptophan residue and the 4 tyrosine residues. The intensity of the porphyrin fluorescence is not perturbed by denaturation of the protein at neutral pH using either urea or guanidine hydrochloride. However, the amplitude of tryptophan fluorescence is increased by these denaturants from 5 to about 85% of a model tryptophan residue using solutions of 2 microM tryptophan. In contrast to cytochrome c, the tryptophan fluorescence amplitude of denatured porphyrin cytochrome c is independent of pH over the range pH 3.0 to 7.4. Acidification of solutions of either native or denatured porphyrin cytochrome c markedly alters both the visible absorbance and fluorescence of the protein consistent with protonation of two pyrrole nitrogens on the porphyrin. Preliminary analysis of the spectral changes occurring in the acid transition suggests the presence of an intermediate form having only one of these two pyrrole nitrogens protonated.     

Domain unfolding and the stability of thermolysin in guanidine hydrochloride

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.     

Refolding a disulfide dimer of cytochrome c

A covalent dimer of Saccharomyces cerevisiae iso-1 cytochrome c is stabilized by an interchain disulfide bond involving the cysteine residue penultimate to the C-terminus. The individual chains in the dimer appear to retain the tertiary structural features characteristic for monomeric cytochrome c albeit with some perturbation. The dimer is reversibly denatured by heat, urea, or guanidine hydrochloride in a single cooperative transition whose midpoint is less than that of the monomeric protein. The kinetic profile observed for the refolding of the denatured dimer is characteristic for monomeric cytochromes except for a markedly enhanced slow-phase amplitude.     

Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.     

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.     

A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography.

The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.     

Kinetic studies on the effect of the heme iron(III) on the protein folding of ferricytochrome c.

The three-dimensional conformation of ferricytochrome c results from specific folding of the polypeptide chain around the covalently bound heme so that His-18 and Met-80 are axially coordinated to the Fe(III). The Fe(III)-free, porphyrin protein has an intrinsic viscosity, sedimentation coefficient, and circular dichroism indicative of a compact, globular protein conformation comparable to the holoprotein. Both the porphyrin protein and ferricytochrome c are reversibly denatured by guanidinium chloride. Refolding of the porphyrin protein occurs in essentially a single, exceptionally rapid kinetic phase (tau = 14 ms, 0.75 M guanidinium chloride, pH 6.5, 25 degrees C); whereas refolding of ferricytochrome c occurs in two slower kinetic phases (TAU 1 = 0.10 S, TAU 2 = 20 S) UNDER COMPARABLE CONDITIONS. The presence of Fe(III) in the metalloporphyrin of ferricytochrome c thus has a major effect on the protein folding kinetics. The slow kinetic phase is evidently due to this effect of Fe(III) and not to the slow cis-trans isomerism of the peptide bond of proline residues as has been suggested.     

Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding

The native serpin fold is metastable and possesses the inherent ability to convert into more stable, but inactive, conformations. In order to understand why serpins attain the native fold instead of other more thermodynamically favourable folds we have investigated the presence of residual structure within denatured antichymotrypsin (ACT). Through mutagenesis we created a single tryptophan variant of ACT in which a Trp residue (276) is situated on the H-helix, located within a region known as the B/C barrel. The presence of residual structure around Trp 276 in 5 M guanidine hydrochloride (GdnHCl) was shown by fluorescence and circular dichroism spectroscopy and fluorescence lifetime experiments. The residual structure was disrupted in the presence of 5 M guanidine thiocyanate (GdnSCN). Protein refolding studies showed that significant refolding could be achieved from the GdnHCl denatured state but not the GdnSCN denatured form. The implications of these data on the folding and misfolding of the serpin superfamily are discussed.     

Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c

Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Folding of yeast iso-1-AM cytochrome c

We describe a specific modification of iso-1 cytochrome c which results in blocking a single free sulfhydryl group. The derivative differs from the unmodified protein by the introduction of a small, uncharged group, thus maintaining the same charge balance as the native protein. The modified protein, obtained by treatment of iso-1 cytochrome c with iodoacetamide, has an activity indistinguishable from that of the unmodified protein in the lactate dehydrogenase-cytochrome c reductase system from yeast and has the same stability toward denaturation by guanidine hydrochloride. The kinetics of fluorescence changes associated with the guanidine hydrochloride induced folding-unfolding transition for modified iso-1 cytochrome c (iso-1-AM) have been investigated throughout the transition zone by using stopped-flow mixing. The results are compared to those for the yeast isozyme, iso-2 cytochrome c. The main features of the fluorescence-detected folding kinetics are similar, as might be expected for homologous proteins; however, the limiting value of the fraction of fast refolding protein (alpha 2) below the transition zone is smaller for iso-1-AM (approximately 0.7) than for iso-2 (approximately 0.9).     

Use of fluorescence energy transfer to characterize the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding

The CL fragment of a type-kappa immunoglobulin light chain in which the C-terminal cysteine residue was modified with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (CL-AEDANS fragment) was prepared. This fragment has only one tryptophan residue at position 148. The compactness of the fragment whose intrachain disulfide bond was reduced in order for the tryptophan residue to fluoresce (reduced CL-AEDANS fragment) was studied in the early stages of refolding from 4 M guanidine hydrochloride by fluorescence energy transfer from Trp 148 to the AEDANS group. The AEDANS group attached to the SH group of a cysteine scarcely fluoresced when excited at 295 nm. For the reduced CL-AEDANS fragment, the fluorescence emission band of the Trp residue overlapped with the absorption band of the AEDANS group, and the fluorescence energy transfer was observed between Trp 148 and the AEDANS group in the absence of guanidine hydrochloride. In 4 M guanidine hydrochloride, the distance between the donor and the acceptor was larger, and the efficiency of the energy transfer became lower. The distance between Trp 148 and the AEDANS group for the intact protein estimated by using the energy-transfer data was in good agreement with that obtained by X-ray crystallographic analysis. By the use of fluorescence energy transfer, tryptophyl fluorescence, and circular dichroism at 218 nm, the kinetics of unfolding and refolding of the reduced fragment were studied. These three methods gave the same unfolding kinetic pattern. However, the refolding kinetics measured by fluorescence energy transfer were different from those measured by tryptophyl fluorescence and circular dichroism, the latter two giving the same kinetic pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

Folding pathways of immunoglobulin domains. The folding kinetics of the Cgamma3 domain of human IgG1.

The in vitro folding kinetics of a fragment corresponding to an intact dimer of the Cgamma3 domain of human IgG1 (pFc') were monitored via the large changes in tryptophan fluorescence which accompany these processes. In going from the guanidine hydrochloride (Gdn.HCl) induced unfolded state (4.0 M Gdn.HCl) to the native state (0.5 M Gdn.HCl), three well-separated first-order processes were observed having time constants of 5, 50, and 350 s and roughly equal amplitudes. These values were concentration independent, a fact consistent with there being no fluorescence change accompanying dimerization. These time constants are one to two orders of magnitude slower than those observed for proteins of similar size such as ribonuclease or cytochrome c, most probably reflecting the complex processes involved in forming the correct beta-sheet arrangement of immunoglobulin domains. The corresponding unfolding transition is biphasic having time constant values of 50 and 500 s, the latter comprising 80% of the fluorescence change. These data indicate the presence of at least one species with intermediate fluorescence along the unfolding pathway. Gdn.HCl concentration jumps were also performed over various intervals within the transition zone. The results are not consistent with a fully reversible mechanism. In the absence of the intrachain disulfide bond, pFc' exists in an unfolded state even at 0.5 M Gdn.HCl. In a concomitant refolding and reoxidation experiment (at 0.5 M Gdn.HCl and using an optimal disulfide interchange catalytic system), the time constant for disulfide formation was in the range of 80--200 s and the fluorescence change revealed a lag phase analyzable in terms of rate-limiting reoxidation and refolding times consistent with those observed for the initially disulfide bonded species. Under similar conditions but a 4 M Gdn.HCl, reoxidation was more than two orders of magnitude slower, suggesting that reoxidation is directed by a refolding nucleation event.     

Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force.

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

Highly nonexponential kinetics in the early-phase refolding of proteins at low temperatures

Theory and simulations predict that the folding kinetics of protein-like heteropolymers become nonexponential and glassy (i.e., controlled by escape from different low-energy misfolded states) at low temperatures, but there was little experimental evidence for such behavior of proteins. We have developed a stopped-flow instrument working reliably down to -40 degrees C with high mixing capability and applied it to study the refolding kinetics of horse cytochrome c (cyt c) and hen egg white lysozyme at temperatures below 0 degrees C in the presence of antifreeze NaCl, LiCl, or ethylene glycol and above 0 degrees C in the presence and absence of antifreeze. The refolding was initiated by rapid dilution of the guanidine hydrochloride unfolded proteins, and the kinetics were monitored by intrinsic tryptophan fluorescence. Highly nonexponential kinetics extended over 3 decades in time (0.01-10 s) were observed in the early phases of the refolding of cyt c and lysozyme in the temperature range of -35 to 5 degrees C. These results are in agreement with the theoretical prediction, suggesting that the folding energy landscapes of these proteins are rugged in the upper portions.     

Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.     

Factors affecting protein refolding yields in a fed-batch and batch-refolding system

The refolding of recombinant protein from inclusion bodies expressed in Escherichia coli can present a process bottleneck. Yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. This article studies the effect of five factors upon the dilution refolding of protein in a twin impeller fed-batch system using refold buffer containing only the oxidized form of the redox reagent. Such a buffer is easier to prepare and more stable than a buffer containing both reduced and oxidized forms. The five factors chosen were: bulk impeller Reynolds number, mini-impeller Reynolds number, injection rate of denatured protein, redox ratio, and guanidine hydrochloride (GdHCl) concentration. A 2(5) factorial experiment was conducted at an industrially relevant protein concentration using lysozyme as the test system. The study identified that in the system used, the guanidine hydrochloride concentration, redox ratio, and injection rate were the most important factors in determining refolding yields. Two interactions were found to be important: redox ratio/guanidine hydrochloride concentration and guanidine hydrochloride concentration/injection rate. Conditions were also found at which high refolding yields could be achieved even with rapid injection and poor mixing efficiency. Therefore, a comparative assessment was carried out with minimal mixing in a simple batch-refolding mode of operation, which revealed different behavior to that of fed-batch. A graphical (windows of operation) analysis of the batch data suggested that optimal yields and productivity are obtained at high guanidine hydrochloride concentrations (1.2 M) and redox ratios of unity or greater.     

Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.