Structural stability of covalently linked GroES heptamer: advantages in the formation of oligomeric structure

In order to understand how inter-subunit association stabilizes oligomeric proteins, a single polypeptide chain variant of heptameric co-chaperonin GroES (tandem GroES) was constructed from Escherichia coli heptameric GroES by linking consecutively the C-terminal of one subunit to the N-terminal of the adjacent subunit with a small linker peptide. The tandem GroES (ESC7) showed properties similar to wild-type GroES in structural aspects and co-chaperonin activity. In unfolding and refolding equilibrium experiments using guanidine hydrochloride (Gdn-HCl) as a denaturant at a low protein concentration (50 microg ml(-1)), ESC7 showed a two-state transition with a greater resistance toward Gdn-HCl denaturation (Cm=1.95 M) compared to wild-type GroES (Cm=1.1 M). ESC7 was found to be about 10 kcal mol(-1) more stable than the wild-type GroES heptamer at 50 microg ml(-1). Kinetic unfolding and refolding experiments of ESC7 revealed that the increased stability was mainly attributed to a slower unfolding rate. Also a transient intermediate was detected in the refolding reaction. Interestingly, at the physiological GroES concentration (>1 mg ml(-1)), the free energy of unfolding for GroES heptamer exceeded that for ESC7. These results showed that at low protein concentrations (<1 mg ml(-1)), the covalent linking of subunits contributes to the stability but also complicates the refolding kinetics. At physiological concentrations of GroES, however, the oligomeric state is energetically preferred and the advantages of covalent linkage are lost. This finding highlights a possible advantage in transitioning from multi-domain proteins to oligomeric proteins with small subunits in order to improve structural and kinetic stabilities.     

Unfolding and disassembly of the chaperonin GroEL occurs via a tetradecameric intermediate with a folded equatorial domain

The chaperonin GroEL is a homotetradecamer in which the subunits (M(r) 57 000) are joined through noncovalent forces. This study reports on the unfolding and disassembly of GroEL in guanidine hydrochloride and urea. Kinetic and equilibrium measurements were made using amide hydrogen exchange/mass spectrometry, light scattering, and size-exclusion chromatography. Hydrogen exchange in GroEL destabilized in 1.8 M GdHCl (the unfolding midpoint is 1.2 M GdHCl) shows that the apical and intermediate domains unfold 3.1 times faster than the equatorial domain. Light scattering measurements made under the same conditions show that disassembly of the native GroEL tetradecamer occurs at the same rate as unfolding of the equatorial domain. This study of the kinetics of GroEL unfolding and disassembly demonstrates the existence of an intermediate that was identified as a tetradecamer with the apical and intermediate domains unfolded. Although this intermediate was easily detected in dynamic unfolding measurements, its population in equilibrium measurements at the midpoint for GroEL unfolding was too small to be detected. This study of GroEL unfolding and disassembly points to features that may be important in the folding and assembly of the GroEL macroassembly.     

Structural stability of oligomeric chaperonin 10: the role of two beta-strands at the N and C termini in structural stabilization

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.     

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.     

Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.     

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.     

Equilibrium unfolding of dimeric human prostatic acid phosphatase involves an inactive monomeric intermediate

Guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular weight, was investigated with activity measurements, size exclusion HPLC, tryptophan fluorescence, 1-anilinonaphtalene-8-sulfonate (ANS) binding and reactivity with 2-(4'-maleimidoanilino)naphthalene-6-sulfonate (MIANS). Equilibrium analysis was performed to shed light on the role of dimerization in the folding and stability of the catalytically active oligomeric protein. Unfolding was reversible, as verified by activity measurements and tryptophan fluorescence. The noncoincidence of the unfolding curves obtained by different techniques suggests the occurrence of a multiphasic process.The reaction of hPAP inactivation is accompanied by dissociation of the dimer into two monomers. The midpoint of this transition is at 0.65 M GdnHCl with 4.24+/-0.12 kcalmol(-1) free energy change. Binding of ANS to the inactive phosphatase monomer, especially remarkable in the region from 0.8 to 1.25M GdnHCl, suggests that the hydrophobic probe indicates exposition of the intersubunit hydrophobic surface and a loosening of the monomer's tertiary structure. Strong fluorescence of thiol group derivatives, the products of their reaction with MIANS, appears in a limited range of GdnHCl concentrations (1.2-1.6M). This shows that in the relaxed structure of the intermediate, the reagent is allowed to penetrate into the hydrophobic environment of the partially hidden thiol groups.The equilibrium unfolding reaction of hPAP, as monitored by tryptophan fluorescence, does not depend on the protein concentration and displays a single transition curve with a midpoint at 1.7 M GdnHCl and value of DeltaG(unf)(H(2)O)=3.38+/-0.08 kcalmol(-1) per monomer, a result implying that this transition is related to the conformational change of the earlier dissociated and already inactive subunit of the protein.     

Reversible unfolding of bovine odorant binding protein induced by guanidinium hydrochloride at neutral pH

An analysis of the unfolding and refolding curves at equilibrium of dimeric bovine odorant binding protein (bOBP) has been performed. Unfolding induced by guanidinium chloride (GdnHCl) is completely reversible as far as structure and ligand binding capacity are concerned. The transition curves, as obtained by fluorescence and ellipticity measurements, are very similar and have the same protein concentration-independent midpoint (C1/2 approximately 2.6 M). This result implies a sequential, rather than a concerted, unfolding mechanism, with the involvement of an intermediate. However, since it has not been detected, this intermediate must be present in small amounts or have the same optical properties of either native or denatured protein. The thermodynamic best fit parameters, obtained according to a simple two-state model, are: deltaG degrees un,w = 5.0 +/- 0.6 kcal mol(-1), m = 1.9 +/- 0.2 kcal mol(-1) M(-1) and C1/2 = 2.6 +/- 0.1 M. The presence of the ligand dihydromyrcenol has a stabilising effect against unfolding by GdnHCl, with an extrapolated deltaG degrees un,w of 22.2 +/- 0.9 kcal mol(-1), a cooperative index of 3.2 +/- 0.3 and a midpoint of 4.6 +/- 0.4 M. The refolding curves, recorded after 24 h from dilution of denaturant are not yet at equilibrium: they show an apparently lower midpoint (C1/2 = 2.2 M), but tend to overlap the unfolding curve after several days. In contrast to chromatographic unfolding data, which fail to reveal the presence of folded intermediates, chromatographic refolding data as a function of time clearly show a rapid formation of folded monomers, followed by a slower step leading to folded dimers. Therefore, according to this result, we believe that the preferential unfolding/refolding mechanism is one in which dimer dissociation occurs before unfolding rather than the reverse.     

Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1

Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.     

Refolding of denatured ribonuclease observed by size exclusion chromatography

The unfolding and refolding of pancreatic ribonuclease have been observed by absorbance, fluorescence, and size exclusion chromatographic measurements in solutions of guanidinium chloride continuously maintained at pH 6.0 and 4 degrees C. The spectral measurements were fitted with a minimal number of kinetic phases while the chromatographic measurements were simulated from an explicit mechanism. All of the measurements are consistent with a minimal mechanism involving seven components. The folded components include the native protein and two transiently stable intermediates each having the same hydrodynamic volume. The intermediate having all native peptide isomers has an unfolding midpoint in 3.8 M denaturant while the intermediate having one nonnative peptide isomer has an unfolding midpoint in 1.3 M denaturant. The unfolded protein is distributed among four components having the same hydrodynamic volume but differing peptide isomers. At equilibrium, 10% of the denatured protein has all native isomers, 60% has one nonnative isomer, 5% has a different nonnative isomer, and 25% has both nonnative isomers. In low denaturant concentrations, the dominant component with one nonnative isomer can refold to transiently populate the compact intermediate with the same nonnative isomer.     

Structural stability and solution structure of chaperonin GroES heptamer studied by synchrotron small-angle X-ray scattering

The GroES protein from Escherichia coli is a well-known member of the molecular chaperones. GroES consists of seven identical 10 kDa subunits, and forms a dome-like oligomeric structure. In order to obtain information on the structural stability and unfolding-refolding mechanism of GroES protein, especially at protein concentrations (0.4-1.2 mM GroES monomer) that would mimic heat stress conditions in vivo, we have performed synchrotron small-angle X-ray scattering (SAXS) experiments. Surprisingly, in spite of the high protein concentration, reversibility in the unfolding-refolding reaction was confirmed by SAXS experiments structurally. Although the unfolding-refolding reaction showed an apparent single transition with a Cm of 1.1 M guanidium hydrochloride, a more detailed analysis of this transition demonstrated that the unfolding mechanism could be best explained by a sequential three-state model, which consists of native heptamer, dissociated monomer, and unfolded monomer. Together with our previous result that GroES unfolded completely via a partially folded monomer according to a three-state model at low protein concentration (5 microM monomer), the unfolding-refolding mechanism of GroES protein could be explained uniformly by the three-state model from low to high protein concentrations. Furthermore, to clarify an ambiguity of the native GroES structure in solution, especially mobile loop structures, we have estimated a solution structure of GroES using SAXS profiles obtained from experiments and simulation analysis. The result suggested that the native structure of GroES in solution was very similar to that seen in GroES-GroEL complex determined by crystallography.     

Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates

The isolated apical domain of GroEL consisting of residues 191–345 (known as “minichaperone”) binds and assists the folding of a wide variety of client proteins without GroES and ATP, but the mechanism of its action is still unknown. In order to probe into the matter, we have examined minichaperone-mediated folding of a large aggregation prone protein Maltodextrin-glucosidase (MalZ). The key objective was to identify whether MalZ exists free in solution, or remains bound to, or cycling on and off the minichaperone during the refolding process. When GroES was introduced during refolding process, production of the native MalZ was inhibited. We also observed the same findings with a trap mutant of GroEL, which stably captures a predominantly non-native MalZ released from minichaperone during refolding process, but does not release it. Tryptophan and ANS fluorescence measurements indicated that refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. Surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ± 0.04 μM, which are significantly higher than for most GroEL clients. This showed that minichaperone interacts loosely with MalZ to allow the protein to change its conformation and fold while bound during the refolding process. These observations suggest that the minichaperone works by carrying out repeated cycles of binding aggregation-prone protein MalZ in a relatively compact conformation and in a partially folded but active state, and releasing them to attempt to fold in solution.     

Pressure-jump studies of the folding/unfolding of trp repressor.

The dimeric protein, trp apo-repressor of Escherichia coli has been subjected to high hydrostatic pressure under a variety of conditions, and the effects have been monitored by fluorescence spectroscopic and infra-red absorption techniques. Under conditions of micromolar protein concentration and low, non-denaturing concentrations of guanidinium hydrochloride (GuHCl), tryptophan and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence detected high pressure profiles demonstrate that pressures below 3 kbar result in dissociation of the dimer to a monomeric species that presents no hydrophobic binding sites for ANS. The FTIR-detected high pressure profile obtained under significantly different solution conditions (30 mM trp repressor in absence of denaturant) exhibits a much smaller pressure dependence than the fluorescence detected profiles. The pressure-denatured form obtained under the FTIR conditions retains about 50 % alpha-helical structure. From this we conclude that the secondary structure present in the high pressure state achieved under the conditions of the fluorescence experiments is at least as disrupted as that achieved under FTIR conditions. Fluorescence-detected pressure-jump relaxation studies in the presence of non-denaturing concentrations of GuHCl reveal a positive activation volume for the association/folding reaction and a negative activation volume for dissociation/unfolding reaction, implicating dehydration as the rate-limiting step for association/folding and hydration as the rate-limiting step for unfolding. The GuHCl concentration dependence of the kinetic parameters place the transition state at least half-way along the reaction coordinate between the unfolded and folded states. The temperature dependence of the pressure-jump fluorescence-detected dissociation/unfolding reaction in the presence of non-denaturing GuHCl suggests that the curvature in the temperature dependence of the stability arises from non-Arrhenius behavior of the folding rate constant, consistent with a large decrease in heat capacity upon formation of the transition state from the unfolded state. The decrease in the equilibrium volume change for folding with increasing temperature (due to differences in thermal expansivity of the folded and unfolded states) arises from a decrease in the absolute value for the activation volume for unfolding, thus indicating that the thermal expansivity of the transition state is similar to that of the unfolded state.     

pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C _m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.     

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.     

Folding and stability of trp aporepressor from Escherichia coli

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels.     

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.     

Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.

We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence. The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method. When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites. The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity. A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin. The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone. The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability

The urea and guanidine hydrochloride (GdnHCl)-induced denaturation of tetrameric concanavalin A (ConA) at pH 7.2 has been studied by using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and size-exclusion chromatography. The equilibrium denaturation pathway of ConA, as monitored by steady state fluorescence, exhibits a three-state mechanism involving an intermediate state, which has been characterized as a structured monomer of the protein by ANS binding, far-UV CD and gel filtration size analysis. The three-state equilibrium is analyzed in terms of two distinct and separate dissociation (native tetramer<-->structured monomer) and unfolding (structured monomer<-->unfolded monomer) reaction steps, with the apparent transition midpoints (C(m)), respectively, at 1.4 and 4.5 M in urea, and at 0.8 and 2.4 M in GdnHCl. The results show that the free energy of stabilization of structured monomer relative to the unfolded state (-DeltaG(unf, aq)), is 4.4-5.5 kcal mol(-1), and that of native tetramer relative to structured monomer (-DeltaG(dis, aq)) is 7.2-7.4 kcal mol(-1), giving an overall free energy of stabilization (-DeltaG(dis&unf, aq)) of 11.6-12.9 kcal mol(-1) (monomer mass) for the native protein. However, the free energy preference at the level of quaternary tetrameric structure is found to be far greater than that at the tertiary monomeric level, which reveals that the structural stability of ConA is maintained mostly by subunit association.