Stable Folding Intermediates Research Articles

TMPfold, a Computational Method for Assessment of Stability of Transmembrane α-Helical Assemblies

A new physics-based computational method TMPfold has been developed and implemented in a public web server for assessment of stability of TM α-helical complexes (accessible at https://opm.phar.umich.edu/tmpfold_server). Using the available 3D structure of an arbitrary TM α-bundle formed by a single or multiple subunits as input, the method calculates the free energies of subunit-subunit and helix-helix association (ΔGasc), identifies stable two-helical units that can initiate folding, and predict the plausible folding pathways for multi-pass transmembrane (TM) proteins. The free energy components include van der Waals, hydrogen bonding and dipole interactions, side-chain conformational entropy, and solvation energy in the anisotropic lipid environment. The method was verified using the experimental ΔGasc values for 36 TM complexes, including dimers of 10 glycophorin A mutants. TMPfold was applied for evaluation of TM helix association energies in 554 PDB structures of 85 seven-helical TM proteins and identification of their stable two-helical folding intermediates. The proposed tentative paths of co-translational helix assembly of several polytopic proteins were qualitatively consistent with experimental studies of their folding. The assessment of free energy changes caused by mutations in TM helices demonstrated the good correlation between experimental ΔΔGfolding and calculated ΔΔGasc values for 42 mutants of bacteriorhodopsin (R2 of 0.41, RMSE of 0.81 kcal/mol) and 25 mutants of rhomboid protease (R2 of 0.43, RMSE of 0.40 kcal/mol).

TMPfold: A Web Tool for Predicting Stability of Transmembrane α-Helix Association

Estimating energies of transmembrane (TM) α-helix association is essential for understanding folding of membrane proteins and formation of their functional assemblies. A new physics-based method was developed and implemented in the TMPfold web server for the calculation of the free energy of TM helix association (ΔGasc) in TM α-bundles of known structure. The method was verified using the experimental ΔGasc values for 36 TM complexes, including dimers of 10 glycophorin A mutants. The calculated free energy changes (ΔΔGasc) caused by mutations in TM helices correlated with experimental changes in the stability of 42 mutants of bacteriorhodopsin and 25 mutants of rhomboid protease. TMPfold was applied for evaluation of ΔGasc in 554 PDB structures of 85 seven-helical TM proteins and identification of stable two-helical folding intermediates. The proposed tentative paths of cotranslational helix assembly of several polytopic proteins were consistent with experimental studies of their folding. TMPfold is accessible at (https://opm.phar.umich.edu/tmpfold_server).

Phosphorylation Induced Global Structural Destabilization of a Small Protein Domain

Protein phosphorylation at a single or multiple sites is utilized to regulate protein functional outcomes and overall cellular activities through signaling pathways. At a molecular level, the addition of a phosphoryl group may alter the function of a protein through distinct and versatile mechanisms. These include allosteric structural and dynamic changes, direct positive or negative modulation of affinities, masking or unmasking of cofactor binding sites, autoinhibition and local disorder-to-order or order-to-disorder transitions. Here we present evidence from NMR spectroscopy and other biophysical techniques that protein phosphorylation may lead to a global domain destabilization. Upon phosphorylation a small protein domain acquires a molten globule state, where at least two stable folding intermediates exist in equilibrium with a heterogeneous conformational ensemble. This conformational transition modulates in turn the affinity for protein partners but also permits rapid domain refolding upon removal of the phosphoryl group.

Stable folding intermediates prevent fast interconversion between the closed and open states of Mad2 through its denatured state.

Different states of metamorphic proteins can interconvert under physiological conditions to realize corresponding functions. The mechanism behind the conversion is critical for understanding how these proteins work. We report a combined thermodynamic and kinetic study on the folding/unfolding process of the open and closed conformers of mitotic arrest deficient protein 2 (Mad2), a metamorphic protein. It has been observed that open Mad2 (O-Mad2) can convert to closed Mad2 (C-Mad2). Our results show that O-Mad2 and C-Mad2 have similar thermodynamic stability, which explains the presence of metamorphosis. The folding/unfolding kinetics suggest that the conversion between O-Mad2 and C-Mad2 would be much faster than that reported previously if this conversion goes through the denatured state (U) directly, i.e. through an O-Mad2-denatured state (U)-C-Mad2 (O-U-C) pathway. This inconsistency implies that there exist stable intermediates in between the native and denatured states of Mad2, which would either slow down the O-U-C interconversion or prevent it going through the denatured state.

Pseudoknots in RNA folding landscapes.

Motivation: The function of an RNA molecule is not only linked to its native structure, which is usually taken to be the ground state of its folding landscape, but also in many cases crucially depends on the details of the folding pathways such as stable folding intermediates or the timing of the folding process itself. To model and understand these processes, it is necessary to go beyond ground state structures. The study of rugged RNA folding landscapes holds the key to answer these questions. Efficient coarse-graining methods are required to reduce the intractably vast energy landscapes into condensed representations such as barrier trees or basin hopping graphs (BHG) that convey an approximate but comprehensive picture of the folding kinetics. So far, exact and heuristic coarse-graining methods have been mostly restricted to the pseudoknot-free secondary structures. Pseudoknots, which are common motifs and have been repeatedly hypothesized to play an important role in guiding folding trajectories, were usually excluded.Results: We generalize the BHG framework to include pseudoknotted RNA structures and systematically study the differences in predicted folding behavior depending on whether pseudoknotted structures are allowed to occur as folding intermediates or not. We observe that RNAs with pseudoknotted ground state structures tend to have more pseudoknotted folding intermediates than RNAs with pseudoknot-free ground state structures. The occurrence and influence of pseudoknotted intermediates on the folding pathway, however, appear to depend very strongly on the individual RNAs so that no general rule can be inferred.Availability and implementation: The algorithms described here are implemented in C++ as standalone programs. Its source code and Supplemental material can be freely downloaded from http://www.tbi.univie.ac.at/bhg.html.Contact: qin@bioinf.uni-leipzig.deSupplementary information: Supplementary data are available at Bioinformatics online.

Formation of Tertiary Interactions during rRNA GTPase Center Folding

The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2+ ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg2+-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2+ addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5ms to 4s and with global folding from 1 to 5s. Site-specific substitution with 15N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2+ and suggest that there are two initial sites of Mg2+ ion association.

Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations

Recently various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. However, the key issue as to the nature of forces driving the folding and recognition processes remains unanswered. In this study, structural changes of 22-mer ht-DNA fragment (Tel22), induced by binding of ions (K+, Na+) and specific bisquinolinium ligands, were monitored by calorimetric and spectroscopic methods and by gel electrophoresis. Using the global model analysis of a wide variety of experimental data, we were able to characterize the thermodynamic forces that govern the formation of stable Tel22 G-quadruplexes, folding intermediates, and ligand-quadruplex complexes, and then predict Tel22 behavior in aqueous solutions as a function of temperature, salt concentration, and ligand concentration. On the basis of the above, we believe that our work sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism.

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca2+ binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca2+ is essential for stability as well as correct folding of the whole protein. In the absence of Ca2+, pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca2+ addition, indicating that these are not dead-end species and that Ca2+ drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6–7M urea, relates to Ca2+ dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca2+ induced folding is not restricted to the RTX domain. Taken together, Ca2+ binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation.

An insight into the biophysical characterization of different states of cefotaxime hydrolyzing β-lactamase 15 (CTX-M-15)

Cefotaxime hydrolyzing β-lactamase-15 (CTX-M-15) is encoded by blaCTX-M-15 gene present on plasmid of various Gram-negative bacteria, such as E. coli, E. cloacae, K. pneumoniae, etc. The widespread dissemination of CTX-M-15 harboring bacteria in hospital as well as community settings is a universal threat as they are resistant to various clinically significant antibiotics. In order to gain an insight into the folding mechanism of CTX-M-15, we carried out pH-induced denaturation study by monitoring Trp fluorescence, far-UV circular dichroism (CD), and ANS fluorescence. We found that the pH-induced denaturation of CTX-M-15 was a three-step process with the accumulation of two stable folding intermediates (XI at pH 2.5 and XII at pH 1.5) in the folding pathway. The intermediates were further characterized by far-UV and near-UV CD analysis, Trp fluorescence, ANS fluorescence, three-dimensional fluorescence, acrylamide quenching, dynamic light scattering, and thermal denaturation studies. We found that XI state lacked tertiary structure but retained most of the secondary structure, its Trp residues were partially exposed to the solvent and its hydrophobic patches were highly accessible to ANS. On the other hand, a complete disruption of tertiary structure along with more than 50% loss in secondary structure was observed in XII state. We conclude that the XI state of CTX-M-15 at pH 2.5 had all the characteristics of a molten globule (MG) state, while its XII state at pH 1.5 was more similar to pre-molten globule (PMG) state. ANS fluorescence also showed that the binding of ANS in XII state was lower than that in the XI state. We propose that the accumulation of MG- and PMG-states was due to separation (at pH 2.5) and then unfolding (at pH 1.5) of the αβα-fold of CTX-M-15, respectively.

A Conserved Interdomain Interaction Is a Determinant of Folding Cooperativity in the GST Fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.

Topology and Sequence in the Folding of a TIM Barrel Protein: Global Analysis Highlights Partitioning between Transient Off-pathway and Stable On-pathway Folding Intermediates in the Complex Folding Mechanism of a (βα) 8 Barrel of Unknown Function from B. subtilis

The relative contributions of chain topology and amino acid sequence in directing the folding of a (βα) 8 TIM barrel protein of unknown function encoded by the Bacillus subtilis iolI gene (IOLI) were assessed by reversible urea denaturation and a combination of circular dichroism, fluorescence and time-resolved fluorescence anisotropy spectroscopy. The equilibrium reaction for IOLI involves, in addition to the native and unfolded species, a stable intermediate with significant secondary structure and stability and self-associated forms of both the native and intermediate states. Global kinetic analysis revealed that the unfolded state partitions between an off-pathway refolding intermediate and the on-pathway equilibrium intermediate early in folding. Comparisons with the folding mechanisms of two other TIM barrel proteins, indole-3-glycerol phosphate synthase from the thermophile Sulfolobus solfataricus (sIGPS) and the alpha subunit of Escherichia coli tryptophan synthase (αTS), reveal striking similarities that argue for a dominant role of the topology in both early and late events in folding. Sequence-specific effects are apparent in the magnitudes of the relaxation times and relative stabilities, in the presence of additional monomeric folding intermediates for αTS and sIGPS and in rate-limiting proline isomerization reactions for αTS.

Mapping the Structure of Folding Cores in TIM Barrel Proteins by Hydrogen Exchange Mass Spectrometry: The Roles of Motif and Sequence for the Indole-3-glycerol Phosphate Synthase from Sulfolobus solfataricus

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at ∼4.5 M urea and contains ∼50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I a and I b. To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I a and I b. Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, α-tryptophan synthase (αTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and αTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.

Modulation of the Folding Energy Landscape of Cytochrome c with Salt

The folding reaction of acid-unfolded cytochrome c in the presence of various amounts of KCl was investigated with Trp fluorescence and resonance Raman spectroscopies. It was found that the too-early-too-much polypeptide chain collapse induced by KCl yields some stable folding intermediates, which need to overcome a higher energy barrier to fold into their native conformation. We propose that the charge distribution on the polypeptide chain is part of the folding codon encoded in the linear amino acid sequence. The charge screening effect introduced by KCl alters the shape of the energy landscape by raising the slope of the upper rim and introduces a rugged energy surface toward the bottom of the folding funnel.

Structure of Stable Protein Folding Intermediates by Equilibrium ϕ-Analysis: The Apoflavodoxin Thermal Intermediate

Protein intermediates in equilibrium with native states may play important roles in protein dynamics but, in cases, can initiate harmful aggregation events. Investigating equilibrium protein intermediates is thus important for understanding protein behaviour (useful or pernicious) but it is hampered by difficulties in gathering structural information. We show here that the phi-analysis techniques developed to investigate transition states of protein folding can be extended to determine low-resolution three-dimensional structures of protein equilibrium intermediates. The analysis proposed is based solely on equilibrium data and is illustrated by determination of the structure of the apoflavodoxin thermal unfolding intermediate. In this conformation, a large part of the protein remains close to natively folded, but a 40 residue region is clearly unfolded. This structure is fully consistent with the NMR data gathered on an apoflavodoxin mutant designed specifically to stabilise the intermediate. The structure shows that the folded region of the intermediate is much larger than the proton slow-exchange core at 25 degrees C. It also reveals that the unfolded region is made of elements whose packing surface is more polar than average. In addition, it constitutes a useful guide to rationally stabilise the native state relative to the intermediate state, a far from trivial task.

Two-state folding of lysozyme versus multiple-state folding of alpha-lactalbumin illustrated by the technique of disulfide scrambling.

The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.

Apolipoprotein E4 Forms a Molten Globule: A POTENTIAL BASIS FOR ITS ASSOCIATION WITH DISEASE

The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration ( approximately 90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75 m ( approximately 80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease.

Structural restoration of inactive recombinant fish growth hormones by chemical chaperonin and solvent restraint approaches.

Recombinant proteins may undergo conformational distortion, leading to aggregation and loss of function, when they are expressed in heterologous systems. The structural and functional restoration of such inactive proteins is highly desirable. We have over-expressed recombinant growth hormones from the fish ayu (Plecoglossus altivelis) and yellow grouper (Epinephelus awoara) by a pET expression system. Both recombinant proteins accumulate as insoluble form in Escherichia coli. We refolded these inactive proteins into the active form using a stepwise refolding process with a dilute denaturing agent as a steric blocker and chemical chaperonin. Optical characterization showed that stable folding intermediates with a helical conformation can be detected in the molten globule state. Moreover, the function of restored recombinant growth hormones was demonstrated by its ability to stimulate proliferation in zebrafish liver cells.

Aggregation events occur prior to stable intermediate formation during refolding of interleukin 1beta.

A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo. The refolding reactions of the WT and K97I proteins are both described by three exponential processes. Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state. The K97I replacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low. Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation.

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.

A stable cold folding intermediate of rabbit muscle D‐glyceraldehyde 3‐phosphate dehydrogenase

With decreasing temperature the reactivation yield of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) upon dilution increases but the reactivation rate decreases. Neither reactivation nor aggregation during refolding can be detected at 4 degrees C in 48 h, and at 3 degrees C even in 6 days. However, the reactivation takes place once the temperature is raised with little decrease of the yield after incubation for 6 days at 3 degrees C. A cold folding intermediate forms in a burst phase of refolding at 4 degrees C as shown by a fast change of the intrinsic fluorescence followed by further conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially folded GAPDH subunit with secondary structure between that of the native and denatured enzymes, a hydrophobic cluster not found in either the native or the denatured state, and an active site similar to but different from that of the native state. Chaperonin 60 (GroEL) binds with all intermediates formed at 4 degrees C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustment when dissociated from GroEL in the presence of ATP and further folded and assembled into the native tetramer.