The interaction of the GroEL chaperone with early kinetic intermediates of renaturing proteins inhibits the formation of their native structure

The main function of the chaperone GroEL is to prevent nonspecific association of nonnative protein chains and provide their correct folding. In the present work, the renaturation kinetics of three globular proteins (human alpha-lactalbumin, bovine carbonic anhydrase, and yeast phosphoglycerate kinase) in the presence of different molar excess of GroEL (up to 10-fold) was studied. It was shown that the formation of the native structure during the refolding of these proteins is retarded with an increase in GroEL molar excess due to the interaction of kinetic protein intermediates with the chaperone. Mg(2+)-ATP and Mg(2+)-ADP weaken this interaction and decrease the retarding effect of GroEL on the protein refolding kinetics. The theoretical modeling of protein folding in the presence of GroEL showed that the experimentally observed linear increase in the protein refolding half-time with increasing molar excess of GroEL must occur only when the protein adopts its native structure outside of GroEL (i.e. in the free state), while the refolding of the protein in the complex with GroEL is inhibited. The dissociation constants of GroEL complexed with the kinetic intermediates of the proteins studied were evaluated, and a simple mechanism of the functioning of GroEL as a molecular chaperone was proposed.     

Direct refolding of inclusion bodies using reversed micelles

The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.     

Isolation and characterisation of mutants of GroEL that are fully functional as single rings

A key aspect of the reaction mechanism for the molecular chaperone GroEL is the transmission of an allosteric signal between the two rings of the GroEL complex. Thus, the single-ring mutant SR1 is unable to act as a chaperone as it cannot release bound substrate or GroES. We used a simple selection procedure to identify mutants of SR1 that restored chaperone activity in vivo. A large number of single amino acid changes, mapping at diverse positions throughout the protein, enabled SR1 to regain its ability to act as a chaperone while remaining as a single ring. In vivo assays were used to identify the proteins that had regained maximal activity. In some cases, no difference could be detected between strains expressing wild-type GroEL and those expressing the mutated proteins. Three of the most active proteins where the mutations were in distinct parts of the protein were purified to homogeneity and characterised in vitro. All were capable of acting efficiently as chaperones for two different GroES-dependent substrates. All three proteins bound nucleotide as effectively as did GroEL, but the binding of GroES in the presence of ATP or ADP was reduced significantly relative to the wild-type. These active single rings should provide a useful tool for studying the nature of the allosteric changes that occur in the GroEL reaction cycle.     

GroEL and protein disulfide isomerase each binds with folding intermediates of D-glyceraldehyde-3-phosphate dehydrogenase released from complexes formed with the other

Simultaneous presence of two chaperones, GroEL and protein disulfide isomerase (PDI), assists the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an additive way. Delayed addition of chaperones to the refolding solution after dilution of denatured GAPDH indicates an interaction with intermediates formed mainly in the first 5 min for PDI and formed within a longer time period for GroEL-ATP. The above indicate that the two chaperones interact with different folding intermediates of GAPDH. After delayed addition of one chaperone to the refolding mixture containing the other at 4°C, GroEL binds with all GAPDH intermediates dissociated from PDI, and PDI interacts with the intermediates released from GroEL during the first 10–20 min. It is suggested that the GAPDH folding intermediates released from the chaperone-bound complex are still partially folded so as to be rebound by the other chaperone. The above results clearly support the network model of GroEL and PDI.     

Glycosylation inhibits the interaction of invertase with the chaperone GroEL.

During refolding and reassociation of chemically denatured non-glycosylated invertase from Saccharomyces cerevisiae, aggregation competes with correct folding, leading to low yields of reactivation (Kern et al. (1992) Protein Sci. 1, 120-131). In the presence of the chaperone GroEL, refolding is completely arrested. This suggests the formation of a stable complex between GroEL and non-native non-glycosylated invertase. Addition of MgATP results in a slow release of active invertase from the chaperone complex. When GroEL/ES and MgATP are present during refolding, the final reactivation yield increases from 14% to 36%. In contrast, refolding of the core-glycosylated and the high-mannose glycosylated forms of invertase is not arrested by GroEL. Only a short lag phase at the beginning of reactivation and a slightly increased reactivation yield (64% to 86% for core-glycosylated and 62% to 76% for external invertase) indicate a weak interaction of the glycosylated forms with the chaperone.     

Denaturation and reassembly of chaperonin GroEL studied by solution X-ray scattering

We measured the denaturation and reassembly of Escherichia coli chaperonin GroEL using small-angle solution X-ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea-induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil-like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X-ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same structure and the molecular mass as the native GroEL. These results demonstrate that the fully unfolded GroEL monomer can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis and preexisting chaperones. Therefore, GroEL can, in principle, fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under in vivo conditions, in order to avoid misfolding and aggregation.     

Comparison of refolding activities between nanogel artificial chaperone and GroEL systems

The chaperone-like activity of a nanogel of cholesteryl group-bearing pullulan (CHP) was compared with that of GroEL for refolding acid-denatured green fluorescent protein (GFP). The refolding of denatured GFP was carried out by dilution of acid-denatured GFP in the presence of the nanogel or GroEL. GFP fluorescence was increasingly repressed with increases in the concentration of the CHP nanogel or GroEL added to the dilution buffer. The concentrations of 50% inhibition of recovery of GFP fluorescence were 0.03 microM (GroEL) and 0.08 microM (CHP nanogel), respectively. The refolding was resumed by the addition of ATP into the GroEL (0.20 microM) system or by the addition of methyl-beta-cyclodextrin into the nanogel (0.20 microM) system. In the nanogel-GFP system, about 90% of the intensity was recovered within 10 min. The half time (t(1/2)) for refolding in the CHP nanogel system (36 s) is almost equal to that of the natural chaperone GroEL-GroES system.     

The kinetic pathway of folding of barnase

To search for folding intermediates, we have examined the folding and unfolding kinetics of wild-type barnase and four representative mutants under a wide range of conditions that span two-state and multi-state kinetics. The choice of mutants and conditions provided in-built controls for artifacts that might distort the interpretation of kinetics, such as the non-linearity of kinetic and equilibrium data with concentration of denaturant. We measured unfolding rate constants over a complete range of denaturant concentration by using by 1H/2H-exchange kinetics under conditions that favour folding, conventional stopped-flow methods at higher denaturant concentrations and continuous flow. Under conditions that favour multi-state kinetics, plots of the rate constants for unfolding against denaturant concentration fitted quantitatively to the equation for three-state kinetics, with a sigmoid component for a change of rate determining step, as did the refolding kinetics. The position of the transition state on the reaction pathway, as measured by solvent exposure (the Tanford beta value) also moved with denaturant concentration, fitting quantitatively to the same equations with a change of rate determining step. The sigmoid behaviour disappeared under conditions that favoured two-state kinetics. Those data combined with direct structural observations and simulation support a minimal reaction pathway for the folding of barnase that involves two detectable folding intermediates. The first intermediate, I(1), is the denatured state under physiological conditions, D(Phys), which has native-like topology, is lower in energy than the random-flight denatured state U and is suggested by molecular dynamics simulation of unfolding to be on-pathway. The second intermediate, I(2), is high energy, and is proven by the change in rate determining step in the unfolding kinetics to be on-pathway. The change in rate determining step in unfolding with structure or environment reflects the change in partitioning of this intermediate to products or starting materials.     

Interaction of GroE with an all-beta-protein.

Molecular chaperones are involved in protein folding both in vivo and in vitro. The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins. A common structural motif of nonnative proteins, which is recognized by GroEL, has not yet been identified. In order to study the role of beta-sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the F(ab) fragment of a monoclonal antibody as a model substrate protein. Here we show that GroEL interacts functionally with this all-beta-protein during reactivation. Antibody fragments refold spontaneously in good yield from the guanidine-denatured state. Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES. The yield of these unspecifically released GroEL-bound F(ab) fragments corresponds to that of the spontaneous reactivation in the absence of chaperones. However, the refolding kinetics in the presence of GroEL are considerably slower. The addition of Mg-ATP to the GroEL.F(ab) complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted antibody fragments compared to spontaneous reactivation. GroES is not essential for functional GroEL-mediated refolding of the F(ab) fragment but affects the reactivation yield to a small extent. Interestingly, stimulation of the GroEL-mediated F(ab) refolding depends primarily on the binding and not on hydrolysis of adenosine triphosphates. Previous results indicate the binding of alpha-helices to GroEL. The results presented in this paper suggest that beta-sheet secondary structural elements are recognized by GroEL. We therefore conclude that the interaction of a nonnative protein with GroEL depends mainly on the nature of the early folding intermediate but not on a specific element of secondary structure.     

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.     

GroEL-assisted refolding of adrenodoxin during chemical cluster insertion.

Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP. The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possible scaffolding role for two bacterial sulfur transferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates. The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH-cytochrome c reduction assay. These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin-containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times. The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanese, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein.     

The chaperonin GroEL binds a polypeptide in an alpha-helical conformation.

Chaperones facilitate folding and assembly of nascent polypeptides in vivo and prevent aggregation in refolding assays in vitro. A given chaperone acts on a number of different proteins. Thus, chaperones must recognize features present in incompletely folded polypeptide chains and not strictly dependent on primary structural information. We have used transferred nuclear Overhauser effects to demonstrate that the Escherichia coli chaperonin GroEL binds to a peptide corresponding to the N-terminal alpha-helix in rhodanese, a mitochondrial protein whose in vitro refolding is facilitated by addition of GroEL, GroES, and ATP. Furthermore, the peptide, which is unstructured when free in aqueous solution, adopts an alpha-helical conformation upon binding to GroEL. Modification of the peptide to reduce its intrinsic propensity to take up alpha-helical structure lowered its affinity for GroEL, but, nonetheless, it could be bound and took up a helical conformation when bound. We propose that GroEL interacts with sequences in an incompletely folded chain that have the potential to adopt an amphipathic alpha-helix and that the chaperonin binding site promotes formation of a helix.     

In vivo and in vitro function of GroEL mutants with impaired allosteric properties

Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 degrees C. This strain supports the growth of phages T4 and T5 but not phage lambda and produces light at 28 degrees C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 degrees C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is important in vivo in a manner that depends on the physiological conditions and is protein substrate specific.     

Foldability, enzymatic activity, and interacting ability of barnase mutants obtained by permutation of secondary structure units

Barnase, a well-characterized ribonuclease, has been decomposed into six modules (M1-M6) or secondary structure units (S1-S6). We have studied the foldability and activity of the barnase mutants obtained by permutation of the four internal modules (M2-M5) or secondary structure units (S2-S5) to investigate whether permutation of these building blocks is a useful way to create foldable and/or functional proteins. In this study, we found that one of the secondary structure unit mutants was expressed in Escherichia coli only when His102 was substituted by alanine, which is a catalytic residue of wild-type barnase. This mutant (S2354H102A) had ordered conformations, which unfolded cooperatively during urea-induced unfolding experiments. S2354H102A interacted with other barnase mutants to show a distinct RNase activity, although its own activity was quite weak. This interaction was specific, because S2354H102A interacted with only barnase mutants having His 102 and certain orders of the secondary structure units giving a distinct RNase activity. These results suggest that secondary structure units permuted in barnase mutants maintain their intrinsic "interacting ability" that is used for the folding of wild-type barnase, and the units can form certain conformations that complement those of the appropriate counterparts. Seven of 23 secondary structure unit mutants and only 2 of 23 module mutants had RNase activity. On the basis of the results of analyses of foldability and RNase activity of the mutants performed in this and previous studies, we conclude that secondary structure units are more suitable than modules as building blocks to create novel foldable and/or functional proteins in the case of barnase.     

Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR

Phosphate is a competitive inhibitor of transesterification of GpC by the ribonuclease barnase. Barnase is significantly stabilized in the presence of phosphate against urea denaturation. The data are consistent with the existence of a single phosphate binding site in barnase with a dissociation constant, Kd, of 1.3 mM. The 2D 1H NMR spectrum of wild-type barnase with bound phosphate is assigned. Changes in chemical shifts and NOEs for wild type with bound phosphate compared with free wild type indicate that phosphate binds in the active site and that only small conformational changes occur on binding. Site-directed mutagenesis of the active site residues His-102, Lys-27, and Arg-87 to Ala increases the magnitude of Kd for phosphate by more than 20-fold. The 2D 1H NMR spectra of the mutants His-102----Ala, Lys-27----Ala, and Arg-87----Ala are assigned. Comparison with the spectra of wild-type barnase reveals that His-102----Ala and Lys-27----Ala have essentially the same structure as weild type, while some structural changes occur in Arg-87----Ala. It appears that phosphate binding by barnase is effected mainly by positively charge residues including His-102, Lys-27, and Arg-87. This may have applications for the design of phosphate binding sites in other proteins.     

Effects of macromolecular crowding on the refolding of glucose- 6-phosphate dehydrogenase and protein disulfide isomerase.

The effects of polysaccharide, polyethylene glycol, and protein-crowding agents on the refolding of glucose-6-phosphate dehydrogenase (G6PDH) and protein disulfide isomerase have been examined. By increasing concentration during refolding, the reactivation yields of the two proteins decrease with the formation of soluble aggregates. In the presence of high concentrations of crowding agents the reactivation yields remain constant but with decreased refolding rates. The refolding of G6PDH changes from monophasic to biphasic first-order reactions in the presence of crowding agents, and the amplitude of the new slow phase increases with increasing concentrations of crowding agents. The molecular chaperone GroEL reverses the refolding kinetics of G6PDH from biphase back to monophase and accelerates the refolding process. Our results display the complexity and diversity of the effects of macromolecular crowding on both the thermodynamics and kinetics of protein folding.     

Cooperative effects of potassium, magnesium, and magnesium-ADP on the release of Escherichia coli dihydrofolate reductase from the chaperonin GroEL.

Previous investigation has shown that at 22 degrees C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512-525). In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL. The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four- to fivefold. In the absence of potassium, magnesium, and ADP, approximately 80-90% of GroEL resides in the form with the faster rate of release. Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-induced transition is observed only in the presence of potassium. MgADP at low concentrations (0-50 microM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent. Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL. In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild-type GroEL at 22 degrees C reaches a limiting value of 0.014 s(-1). For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s(-1), suggesting that inter-ring negative cooperativity exists for MgADP-induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein.     

From minichaperone to GroEL 3: properties of an active single-ring mutant of GroEL

The next step in our reductional analysis of GroEL was to study the activity of an isolated single seven-membered ring of the 14-mer. A known single-ring mutant, GroEL(SR1), contains four point mutations that prevent the formation of double-rings. That heptameric complex is functionally inactive because it is unable to release GroES. We found that the mutation E191G, which is responsible for the temperature sensitive (ts) Escherichia coli allele groEL44 and is located in the hinge region between the intermediate and apical domains of GroEL, appears to function by weakening the binding of GroES, without destabilizing the overall structure of GroEL44 mutant. We introduced, therefore, the mutation E191G into GroEL(SR1) in order to generate a single-ring mutant that may have weaker binding of GroES and hence be active. The new single-ring mutant, GroEL(SR44), was indeed effective in refolding both heat and dithiothreitol-denatured mitochondrial malate dehydrogenase with great efficiency. Further, unlike all smaller constructs of GroEL, the expression of GroEL(SR44) in E. coli that contained no endogenous GroEL restored biological viability, but not as efficiently as does wild-type GroEL. We envisage the notional evolution of the structure and properties of GroEL. The minichaperone core acts as a primitive chaperone by providing a binding surface for denatured states that prevents their self-aggregation. The assembly of seven minichaperones into a ring then enhances substrate binding by introducing avidity. The acquisition of binding sites for ATP then allows the modulation of substrate binding by introducing the allosteric mechanism that causes cycling between strong and weak binding sites. This is accompanied by the acquisition by the heptamer of the binding of GroES, which functions as a lid to the central cavity and competes for peptide binding sites. Finally, dimerization of the heptamer enhances its biological activity.     

Design of a molecular chaperone-assisted protein folding bioreactor

Escherichia coli molecular chaperone GroEL and co-chaperone GroES are well known to assist the folding/refolding of a diverse range of substrate proteins. Despite this, there have been relatively few reports of the GroEL/GroES molecular chaperone system being used as a biotechnology tool for protein folding/refolding. In this paper, a solution-phase protein folding bioreactor is described that involves the complete GroEL/GroES system. The main features of this bioreactor are the use of a stirred-cell concentrator fitted with a 100 kDa molecular weight cutoff membrane and an attached buffer reservoir. This bioreactor system was used successfully for assisted-batch refolding of guanidinium chloride (Gu-HCl) unfolded mitochondrial malate dehydrogenase (mMDH). We believe that protein folding bioreactor systems of this type could have wide potential utility for the folding/refolding of unfolded protein substrates.     

Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization

The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.