GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.     

Exploring the cooperativity of the fast folding reaction of a small protein using pulsed thiol labeling and mass spectrometry

It has been difficult to obtain directly residue-specific information on side chain packing during a fast (ms) protein folding reaction. Such information is necessary to determine the extent to which structural changes in different parts of the protein molecule are coupled together in defining the cooperativity of the overall folding transition. In this study, structural changes occurring during the major fast folding reaction of the small protein barstar have been characterized at the level of individual residue side chains. A pulsed cysteine labeling methodology has been employed in conjunction with mass spectrometry. This provides, with ms temporal resolution, direct information on structure formation at 10 different locations in barstar during its folding. Cysteine residues located on the surface of native barstar, at four different positions, remain fully solvent-accessible throughout the folding process, indicating the absence of any ephemeral nonnative structure in which these four cysteine residues get transiently buried. For buried cysteine residues, the rates of the change in cysteine-thiol accessibility to rapid chemical labeling by the thiol reagent methyl methanethiosulfonate appear to be dependent upon the location of the cysteine residue in the protein and are different from the rate measured by the change in tryptophan fluorescence. But the rates vary over only a 3-fold range. Nevertheless, a comparison of the kinetics of the change in accessibility of the cysteine 3 thiol with those of the change in the fluorescence of tryptophan 53, as well as of their denaturant dependences, indicates that the major folding reaction comprises more than one step.     

On the nature of conformational openings: native and unfolded-state hydrogen and thiol-disulfide exchange studies of ferric aquomyoglobin.

Native-state amide hydrogen exchange (HX) of proteins in the presence of denaturant has provided valuable details on the structures of equilibrium folding intermediates. Here, we extend HX theory to model thiol group exchange (SX) in single cysteine-containing variants of sperm whale ferric aquomyoglobin. SX is complementary to HX in that it monitors conformational opening events that expose side-chains, rather than the main chain, to solvent. A simple two-process model, consisting of EX2-limited local structural fluctuations and EX1-limited global unfolding, adequately accounts for all HX data. SX is described by the same model except at very low denaturant concentrations and when the bulky labeling reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) is used. Under these conditions SX can occur by a novel denaturant-dependent process. This anomalous behavior is not observed when the smaller labeling reagent methyl methanethiosulfonate is employed, suggesting that it reflects a denaturant-induced increase in the amplitudes of local structural fluctuations. It also is not seen in heme-free apomyoglobin, which may indicate that local openings are sufficiently large in the absence of denaturant to allow DTNB unhindered access. Differences in SX kinetics obtained using the two labeling reagents provide estimates of the sizes of local opening reactions at different sites in the protein. At all sequence positions examined except for position 73, the same opening event appears to facilitate exchange of both backbone amide and side-chain thiol groups. The C73 thiol group is exposed by a low-energy fluctuation that does not expose its amide group to exchange.     

Origin of apparent fast and non-exponential kinetics of lysozyme folding measured in pulsed hydrogen exchange experiments.

Folding of lysozyme at pH 5.2 is a complex processes. After rapid collapse (<1 ms) kinetic partitioning into a slow and fast folding pathway occurs. The fast pathway leads directly to the native structure (N), whereas the slow pathway goes through a partially folded intermediate (I(1)) with native-like secondary structure in the alpha-domain. This mechanism is in agreement with data from a large number of spectroscopic probes, from changes in the radius of gyration and from measurements on the time-course of the populations of the different species. Results from pulsed hydrogen exchange experiments, in contrast, revealed that the secondary structure of I(1) and of N is formed significantly faster than changes in spectroscopic properties occur and showed large variations in the protection kinetics of individual amide sites. We investigated the molecular origin of the rapid amide protection by quantitatively simulating all kinetic processes during the pulse-labeling experiments. Absorbance and fluorescence-detected folding kinetics showed that the early events in lysozyme folding are accelerated under exchange conditions (pH 9.2) and that a change in folding mechanism occurs due to the transient population of an additional intermediate (I(2)). This leads to kinetic competition between exchange and folding during the exchange pulse and to incomplete labeling of amide sites with slow intrinsic exchange rates. As a result, apparently faster and non-exponential kinetics of amide protection are measured in the labeling experiments. Our results further suggest that collapsed lysozyme (C) and I(1) have five and ten-times reduced free exchange rates, respectively, due to limited solvent accessibility.     

Relationship between the native-state hydrogen exchange and the folding pathways of barnase

The previous native-state hydrogen exchange experiment with barnase failed to detect any partially unfolded intermediate state which was contrary to the experimental results from kinetic deuterium hydrogen exchange pulse labeling and protein engineering studies. This has been taken to suggest that the native-state hydrogen exchange method cannot be used alone as an analytical tool to study the folding pathways of proteins. Here, we revisited the pulse labeling experiment with barnase and detected no stable folding intermediate. This finding allows a reconciliation of the native-state HX data and the folding pathway of barnase. Along with alternative theoretical interpretations for a curved chevron plot of protein folding, these data suggest that further investigation of the nature of the intermediate of barnase is needed.     

Compact dimension of denatured states of staphylococcal nuclease

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.     

Protein folding mechanisms studied by pulsed oxidative labeling and mass spectrometry

Deciphering the mechanisms of protein folding remains a considerable challenge. In this review we discuss the application of pulsed oxidative labeling for tracking protein structural changes in a time-resolved fashion. Exposure to a microsecond OH pulse at selected time points during folding induces the oxidation of solvent-accessible side chains, whereas buried residues are protected. Oxidative modifications can be detected by mass spectrometry. Folding is associated with dramatic accessibility changes, and therefore this method can provide detailed mechanistic insights. Solvent accessibility patterns are complementary to H/D exchange investigations, which report on the extent of hydrogen bonding. This review highlights the application of pulsed OH labeling to soluble proteins as well as membrane proteins.     

Kinetic folding mechanism of an integral membrane protein examined by pulsed oxidative labeling and mass spectrometry

We report the application of pulsed oxidative labeling for deciphering the folding mechanism of a membrane protein. SDS-denatured bacteriorhodopsin (BR) was refolded by mixing with bicelles in the presence of free retinal. At various time points (20 ms to 1 day), the protein was exposed to a microsecond ·OH pulse that induces oxidative modifications at solvent-accessible methionine side chains. The extent of labeling was determined by mass spectrometry. These measurements were complemented by stopped-flow spectroscopy. Major time-dependent changes in solvent accessibility were detected for M20 (helix A) and M118 (helix D). Our kinetic data indicate a sequential folding mechanism, consistent with models previously suggested by others on the basis of optical data. Yet, ·OH labeling provides additional structural insights. An initial folding intermediate I(1) gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I(2). Noncovalent retinal binding to I(2) induces folding of helix D, thereby generating an intermediate I(R). In the absence of retinal, the latter transition does not take place. Hence, formation of helix D depends on retinal binding, whereas this is not the case for helix A. As the cofactor settles deeper into its binding pocket, a final transient species I(R) is generated. This intermediate converts into native BR within minutes by formation of the retinal-K216 Schiff base linkage. The combination of pulsed covalent labeling and optical spectroscopy employed here should also be suitable for exploring the folding mechanisms of other membrane proteins.     

Disulfide-linked bovine beta-lactoglobulin dimers fold slowly, navigating a glassy folding landscape

To gain insight into the folding of large proteins, we constructed a bovine beta-lactoglobulin (beta-lg) dimeric mutant, A34C/C121A beta-lg. In the mutant, a free thiol group of wild-type beta-lg at Cys121 was removed and two beta-lg molecules were linked by a disulfide bridge through Cys34 created at the dimer's interface. Under strongly native conditions at low concentrations of urea, the refolding yield of A34C/C121A beta-lg was low when monitored by heteronuclear NMR spectroscopy. However, under marginally native conditions, the yield improved notably, although the refolding was still slow. H-D exchange pulse labeling monitored using heteronuclear NMR spectroscopy indicated that A34C/C121A beta-lg forms a folding intermediate similar to monomeric C121A beta-lg in spite of its slow folding. These results indicate that the rapid formation of folding intermediates driven by local interactions occurs in a manner independent of the molecular size and that, if the non-native interactions are too strong, the kinetic trap is set, leading to a glasslike misfolded state. The results suggest the important roles of marginal stability and pathways in making the folding of large proteins possible.     

The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

Folding and stability of sweet protein single-chain monellin. An insight to protein engineering

Engineered single-chain monellin (SCM) proteins were constructed by recombinant technology without disrupting the topology and sweet activity of native protein. Data from 8-anilinonaphthalene-1-sulfonic acid fluorescence, size-exclusion chromatography, and heteronuclear NMR strongly suggest the presence of a folding intermediate at 1.5 m GdnHCl for SCM protein. The structural feature of the folding intermediate from NMR data reveals that the secondary structures became mostly unstable, and protein experiences a dynamic equilibrium between native and unfolded state. All backbone amide protons exchange within 10 min, which imply that no stable hydrogen bonds exist in the secondary structural regions in the folding intermediate. From equilibrium unfolding and mutagenesis studies, the unfolding transition midpoints of mutant proteins gradually shifted toward lower denaturant concentration, indicating stability reductions of mutant proteins. Our results suggest that stability and folding pathways of SCM proteins could be regulated by a combined study of spectroscopy and mutagenesis, and these studies will provide useful information for understanding the folding kinetics of novel engineered proteins.     

Increasing stability reduces conformational heterogeneity in a protein folding intermediate ensemble

A multi-site, time-resolved fluorescence resonance energy transfer methodology has been used to study structural heterogeneity in a late folding intermediate ensemble, IL, of the small protein barstar. Four different intra-molecular distances have been measured within the structural components of IL. The IL ensemble is shown to consist of different sub-populations of molecules, in each of which one or more of the four distances are native-like and the remaining distances are unfolded-like. In very stable conditions that favor formation of IL, all four distances are native-like in most molecules. In less stable conditions, one or more distances are unfolded-like. As stability is decreased, the proportion of molecules with unfolded-like distances increases. Thus, the results show that protein folding intermediates are ensembles of different structural forms, and they demonstrate that conformational entropy increases as structures become less stable. These observations provide direct experimental evidence in support of a basic tenet of energy landscape theory for protein folding, that available conformational space, as represented by structural heterogeneity in IL, becomes restricted as the stability is increased. The results also vindicate an important prediction of energy landscape theory, that different folding pathways may become dominant under different folding conditions. In more stable folding conditions, uniformly native-like compactness is achieved during folding to IL, whereas in less stable conditions, uniformly native-like compactness is achieved only later during the folding of IL to N.     

Hydrogen exchange methods to study protein folding

The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed information so far available on the structure and properties of protein folding intermediates. Direct HX measurements can define the structure of tenuous molten globule forms that are generally inaccessible to the usual crystallographic and NMR methods (C. Redfield review in this issue). HX pulse labeling methods can specify the structure, stability and kinetics of folding intermediates that exist for less than 1 s during kinetic folding. Native state HX methods can detect and characterize folding intermediates that exist as infinitesimally populated high energy excited state forms under native conditions. The results obtained in these ways suggest principles that appear to explain the properties of partially folded intermediates and how they are organized into folding pathways. The application of these methods is detailed here.     

Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased.     

Models for protein folding and nature's choice of protein as catalyst

The study of protein folding and unfolding pathways lends a fascinating dimension to protein biochemistry. Several models for protein folding have been postulated. Two powerful probes used in protein folding study are far UV-CD monitored stopped flow kinetics and pulse hydrogen exchange in conjunction with NMR. The formation of molten globule, which is an intermediate possessing secondary structure but not a well packed tertiary structure, is now emerging as a common feature on the folding pathway of many proteins. The molten globule is recognized by a class of molecules called chaperones which act as accelerators of protein folding. This article ends by elucidating why proteins are Nature's choice as catalysts.     

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.     

Multiple parallel-pathway folding of proline-free Staphylococcal nuclease

When a protein exhibits complex kinetics of refolding, we often ascribe the complexity to slow isomerization events in the denatured protein, such as cis/trans isomerization of peptidyl prolyl bonds. Does the complex folding kinetics arise only from this well-known reason? Here, we have investigated the refolding of a proline-free variant of staphylococcal nuclease by stopped-flow, double-jump techniques, to examine the folding reactions without the slow prolyl isomerizations. As a result, the protein folds into the native state along at least two accessible parallel pathways, starting from a macroscopically single denatured-state ensemble. The presence of intermediates on the individual folding pathways has revealed the existence of multiple parallel pathways, and is characterized by multi-exponential folding kinetics with a lag phase. Therefore, a "single" amino acid sequence can fold along the multiple parallel pathways. This observation in staphylococcal nuclease suggests that the multiple folding may be more general than we have expected, because the multiple parallel-pathway folding cannot be excluded from proteins that show simpler kinetics.     

Intimate view of a kinetic protein folding intermediate: residue-resolved structure, interactions, stability, folding and unfolding rates, homogeneity

A cytochrome c kinetic folding intermediate was studied by hydrogen exchange (HX) pulse labeling. Advances in the technique and analysis made it possible to define the structured and unstructured regions, equilibrium stability, and kinetic opening and closing rates, all at an amino acid-resolved level. The entire N-terminal and C-terminal helices are formed and docked together at their normal native positions. They fray in both directions from the interaction region, due to a progression in both unfolding and refolding rates, leading to the surprising suggestion that helix propagation may proceed very slowly in the condensed milieu. Several native-like beta turns are formed. Some residues in the segment that will form the native 60s helix are protected but others are not, suggesting energy minimization to some locally non-native conformation in the transient intermediate. All other regions are unprotected, presumably dynamically disordered. The intermediate resembles a partially constructed native state. It is early, on-pathway, and all of the refolding molecules pass through it. These and related results consistently point to distinct, homogeneous, native-like intermediates in a stepwise sequential pathway, guided by the same factors that determine the native structure. Previous pulse labeling efforts have always assumed EX2 exchange during the labeling pulse, often leading to the suggestion of heterogeneous intermediates in alternative parallel pathways. The present work reveals a dominant role for EX1 exchange in the high pH labeling pulse, which will mimic heterogeneous behavior when EX2 exchange is assumed. The general problem of homogeneous versus heterogeneous intermediates and pathways is discussed.     

A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK

Protein folding is directed by the sequence of sidechains along the polypeptide backbone, but despite this the developement of sidechain interactions during folding is not well understood. Here, the thiol-active reagent, dithio-nitrobenzoic acid (DTNB), is used to probe the exposure of the cysteine sidechain thiols in the kinetic folding intermediates of the N-terminal domain of phosphoglycerate kinase (N-PGK) and a number of conservative (I-, L-, or V-to-C) single cysteine variants. Rapid dilution of chemically denatured protein into folding conditions in the presence of DTNB allowed the degree of sidechain protection in any rapidly formed intermediate to be determined through the analysis of the kinetics of labelling. The protection factors derived for the intermediate(s) were generally small (<25), indicating only partial burial of the sidechains. The distribution of protection parallels the previously reported backbone amide protection for the folding intermediate of N-PGK. These observations are consistent with the hypothesis that such intermediates resemble molten globule states; i.e. with native-like backbone hydrogen bonding and overall tertiary structure, but with the sidechains that make up the hydrophobic protein core dynamic and intermittently solvent exposed. The success of the competition technique in characterizing this kinetic intermediate invites application to other model systems.     

Measuring “Unmeasurable” Folding Kinetics of Proteins by Single-Molecule Force Spectroscopy

Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to “un-boil” an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the β-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of “un-boiling eggs”, providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies.