Mutation of all nonessential cysteine residues to serines in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). bis-ANS binding studies have shown that C3S has more hydrophobic exposure than WT, although both have similar secondary structures suggesting the flexibility of its structure. Activity of C3S falls once it binds bis-ANS, and covalent binding of bis-ANS to C3S is induced by light. bis-ANS binds to C3S in its C-terminal domain as is shown by gel electophoresis and proteolysis. bis-ANS binding makes the C-terminal domain more susceptible to trypsin cleavage.

The non-covalent fluorescent probe 6-propionyl-2-(dimethylamino) naphthalene sulfonate (prodan) binds to hydrophobic surfaces exposed on the surface of GroEL. Under identical experimental conditions free prodan exhibits a green emission peak of intensity 390,000 cps at 520 nm. However prodan bound to GroEL, GroEL-ATP, and GroEL-ATP-GroES shows emission peaks of intensities 500,000, 540,000, and 480,000 cps at 515, 512 and 515 nm, respectively, thus mimicing the way hydrophobic surfaces on GroEL become exposed during the folding cycle. Other hydrophobic probes like bis-ANS and dansyl lysine were unable to detect the minor changes in hydrophobic exposure of GroEL after it binds to ATP, although they were able to detect hydrophobic exposure in GroEL itself.

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range 0.5–3.0 kbar. The kinetics were studied as a function of temperature in the range 15–35 °C. The dissociation processes at each pressure and temperature showed biphasic behavior. The slower rate (k1,obs) was confirmed to be the self-dissociation of SR1 at any specific temperature at atmospheric pressure. This dissociation was pressure independent and followed concentration-dependent first-order kinetics. The self-dissociation rates followed normal Eyring plots (ln k1,obs/T vs. 1/T) from which the free energy of activation (ΔG≠ = 22 ± 0.3 kcal mol−1), enthalpy of activation (ΔH≠ = 18 ± 0.5 kcal mol−1), and entropy of activation (ΔS≠ = −15 ± 1 kcal mol−1) were evaluated. The effect of pressure on the dissociation rates resulted in nonlinear behavior (ln k2,obs vs. pressure) at all the temperatures studied indicating that the activation volumes were pressure dependent. Activation volumes at zero pressure (V≠o) and compressibility factors (β≠) for the dissociation rates at the specific temperatures were calculated. This is the first systematic study where the self-dissociation of an oligomeric chaperonin as well as its activation parameters are reported.

Recent interest has focused on the influence of protein factors in the folding of other proteins both in vivo and in vitro (). Among these factors are catalysts of folding-related processes, such as peptidyl-prolyl cis-trans isomerase () or protein disulfide isomerase (). Other proteins, called "molecular chaperones," have been described that apparently influence protein folding and interactions by forming noncovalent associations with folding intermediates (). These chaperones include the heat shock proteins of the hsp10, hsp60, and hsp70 classes (, , , , , , , , ). The number indicates the approximate molecular weight of the subunit making up the particular hsp. The molecular chaperones are among the most thoroughly reviewed topics in the field of protein folding and structure. The recent review by Gething and Sambrook may serve as an entre to relevant issues and literature ().

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNAf. All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E. coli. Addition of large amounts of E. coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause-site peptides seen in the expression of wild-type CAT. Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing.

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.

Molecular chaperones GroEL and GroES facilitate reactivation of denatured rhodanese which folds poorly unless the process is assisted. The present work tests the hypothesis that more extensively unfolded forms of rhodanese bind tighter than those forms that appear later in the folding pathway. The study of the interaction of different urea-induced forms of rhodanese with GroEL suggests that species preceding the domain folded form bind directly and productively to GroEL. Rhodanese partially folds while in the GroEL-GroES-ADP complex, but it does not significantly reach an active state. Partially folded rhodanese can be released from the GroEL-GroES-ADP complex by subdenaturing concentrations of urea as a homogeneous species that is committed to fold to the native conformation with little or no partitioning to the aggregated state. Dilution of denatured rhodanese to the same final concentration gives less active enzyme and significant aggregation. Urea denaturation studies show that active rhodanese released from complexes behaves identically to native enzyme, while spontaneously folded rhodanese has a different stability. These results are interpreted using a previously proposed model based on studies of unassisted rhodanese folding [Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120-128. Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63-70].

We investigated the dissociation of tetradecameric GroEL by high hydrostatic pressure in the range of 1-2.5 kbar. Kinetics of the dissociation of GroEL in the absence and presence of Mg(2+) and/or KCl were monitored using light scattering. All of the kinetics were biphasic in nature. At any given pressure, only monomers and 14mers were produced, and below 2.5 kbar, the 14mers only partially dissociated to monomers, which did not significantly reassemble on depressurization. Under identical reaction conditions, the observed dissociation rates decreased by only 2-fold when the concentration of GroEL was increased by 20-fold. At 2.5 kbar the observed rates decreased exponentially with the increase in [KCl] and reached a minimum at approximately 75mM. Similarly, the rates decreased with the increase in [Mg(2+)] and reached a minimum at approximately 3 mM Mg(2+). In the presence of saturating amounts of Mg(2+) (10 mM) and KCl (100 mM), the rates were much faster than with 10 mM Mg(2+) alone. The results could be rationalized in terms of the presence of GroEL heterogeneity, which could not be assessed easily by common techniques such as sedimentation velocity, HPLC, gel electrophoresis, and dissociation by chaotropes. This heterogeneity is evidence of subpopulations of GroEL that dissociate at different pressures. At low pressures, the oligomer without added Mg(2+) only partially dissociates to monomers, leading to an apparent plateau in the kinetics, whereas in the presence of Mg(2+) the species are converted to a tighter Mg(2+)-bound species, leading to a much slower dissociation process. The presence of KCl in the sample also leads to similar heterogeneity.

The molecular chaperone DnaK and trigger factor (TF), a ribosome-associated protein with folding activity, have been implicated in assisting nascent polypeptides to acquire a three-dimensional structure on Escherichia coli ribosomes. We asked whether ribosomes that lack trigger factor would recruit DnaK for synthesis and folding of nascent peptides. For these analyses, translating ribosomes with a homogeneous population of nascent peptides were isolated. Truncated forms of rhodanese and E. coli translation initiation factor 3 (IF3) were generated with tandem rare arginine codons in the coding sequence. These codons cause strong translational pausing during coupled transcription/translation in E. coli extracts, generating nascent polypeptides on ribosomes. Protein synthesis in the TF− extract was initiated with biotin-Met-tRNAf. Ribosomes with nascent polypeptides were isolated by interaction of the N-terminal biotin with streptavidin on magnetobeads. These translating ribosomes that lack TF contain the molecular chaperone DnaK in considerably less than stoichiometric amounts.

We investigated the dissociation of single-ring heptameric GroEL (SRI) by high hydrostatic pressure in the range of 1-2.5 kbar. The kinetics of the dissociation of SRI in the absence and presence of Mg2+, KCl, and nucleotides were monitored using light scattering. The major aim of this investigation was to understand the role of the double-ring structure of GroEL by comparing its dissociation with the dissociation of the single ring. At all the pressures that were studied, SRI dissociates much faster than the GroEL 14mer. As observed with the GroEL 14mer, SRI also showed biphasic kinetics and the dissociated monomers do not reassociate readily back to the oligomer. Unlike the GroEL 14mer, the observed rates for SRI dissociation are independent of the concentrations of Mg2+ and KCl in the studied range. The effects of nucleotides on the observed rates, in the absence or presence of Mg2+ and KCl, are not very significant. The heterogeneity induced in the GroEL molecule with the double-ring structure by ligands such as Mg2+, KCl, and nucleotides is not observed in the case of SRI. This indicates that the inter-ring negative cooperativity in the double-ring GroEL has a major role in this regard. The results presented in this investigation demonstrate that the presence of a second ring in the GroEL 14mer is important for its stability in an environment where the functional ligands of the chaperonin are available.

The initial steps of heat-induced inactivation and aggregation of the enzyme rhodanese have been studied and found to involve the early formation of modified but catalytically active conformations. These intermediates readily form active dimers or small oligomers, as evident from there being only a small increase in light scattering and an increase in fluorescence energy homotransfer from rhodanese labeled with fluorescein. These species are probably not the domain-unfolded form, as they show activity and increased protection of hydrophobic surfaces. Cross-linking with glutaraldehyde and fractionation by gel filtration show the predominant formation of dimer during heat incubation. Comparison between the rates of aggregate formation at 50 degrees C after preincubation at 25 or 40 degrees C gives evidence of product-precursor relationships, and it shows that these dimeric or small oligomeric species are the basis of the irreversible aggregation. The thermally induced species is recognized by and binds to the chaperonin GroEL. The unfoldase activity of GroEL subsequently unfolds rhodanese to produce an inactive conformation and forms a stable, reactivable complex. The release of 80% active rhodanese upon addition of GroES and ATP indicates that the thermal incubation induces an alteration in conformation, rather than any covalent modification, which would lead to formation of irreversibly inactive species. Once oligomeric species are formed from the intermediates, GroEL cannot recognize them. Based on these observations, a model is proposed for rhodanese aggregation that can explain the paradoxical effect in which rhodanese aggregation is reduced at higher protein concentration.

We investigated the effects of high hydrostatic pressure in the range of 1--3 kilobars on tetradecameric GroEL, heptameric GroES, and the GroEL-GroES complex. Unlike GroEL monomers formed by urea dissociation, which can be reassembled back to the tetradecamer, the pressure-dissociated monomers do not reassemble readily. This indicates an alteration of their native structures, an example of conformational drift. Pressure versus time profiles and kinetics of the dissociation of both GroEL and GroES at fixed pressures were monitored by light scattering. Unlike GroEL, GroES monomers do reassociate readily. Reaction conditions were varied by adding ATP, Mg(2+), ADP, AMP-PNP, and KCl. At any individual pressure, the dissociation process is governed by both thermodynamics and kinetics. This leads to the decrease in the yield of monomers at lower pressures. In the presence of Mg(2+) and KCl, GroEL is stable up to 3 kilobars. The presence of either ATP or ADP but not AMP-PNP leads to GroEL dissociation at lower pressures. Interestingly, the GroEL-GroES complex is very stable in the range of 1--2.5 kilobars. However, the addition of ADP destabilizes the complex, which dissociates completely at 1.5 kilobars. The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer. Such allosteric interactions leading to the alteration of quaternary structure of GroEL in the absence of chemical denaturants are important in understanding the mechanism of chaperonin-assisted protein folding by the GroEL-GroES system.

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.

The in vitro folding of rhodanese involves a competition between formation of properly folded enzyme and off-pathway inactive species. Co-solvents like glycerol or low temperature, e.g. refolding at 10 degrees C, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate rhodanese if the enzyme is allowed first to spontaneously fold for longer times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of rhodanese (Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120--128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63--70).

Unfolded (inactive) rhodanese (thiosu1fate:cyanide sulfurtransferase, EC 2.8.1.1) can be reactivated in the presence of detergents, e.g. lauryl maltoside (LM). Here, we report the reactivation of urea-unfolded rhodanese in the presence of mixed micelles containing LM and the anionic mitochondrial phospholipid, cardiolipin (CL). Reactivation times increased as the number of CL molecules/micelle was increased. A maximum of 94% of the activity was recovered at 2.2 CL/micelle. Only 71% of the activity was recovered in the absence of CL. The major zwitterionic mitochondrial phospholipid, phosphatidylcholine (PC), had no effect on the LM-assisted reactivation of rhodanese. Size exclusion chromatography showed that denatured, but not native, rhodanese apparently binds to micellar amounts of LM and CL/LM, but not to PC/LM micelles. The lifetime of the enzyme-micelle complex increased with the number of CL molecules/micelle. Furthermore, chromatographic fractions containing micelle-bound enzyme had no activity, while renatured rhodanesecontaining fractions were active. These results suggest that transient complexes form between enzyme and both LM and CL/LM micelles, and that this complex formation may be necessary for reactivation. For CL/ LM micelles, interactions may occur between the positively charged amino-terminal sequence of rhodanese and the negatively charged CL phosphate. Finally, this work shows that there are similarities between “micelle-assisted” and chaperonin-assisted rhodanese refolding.

The molecular chaperone, GroEL, is completely disassembled into monomers by the addition of 4,4'-dipyridyl disulfide. The dissociation leads to monomers in a kinetically controlled process. The additions of functional ligands of GroEL such as Mg(2+) or adenine nucleotides produced differences in the observed rates, but at the end of the kinetics, the dissociation was complete. In addition to the information obtained from native gels, the fluorescent probe bis-ANS was utilized to follow the monomer formation. The results demonstrate that the formation of monomers was associated with the exposure of hydrophobic surfaces. This assessment was possible without the use of added chaotropes, such as urea, to dissociate GroEL. Dissociation kinetics were also followed by light scattering. The kinetics of dissociation of the 14mer are cooperative with respect to the concentration of 4,4'-DPDS. Thermodynamic parameters for the kinetic process gave a free energy of activation (DeltaG) of 19.3 +/- 1.2 kcal mol(-1), which was decomposed to an enthalpy of activation (DeltaH) of 19.30 +/- 1.2 kcal mol(-1) and an entropy of activation (DeltaS) of -8.2 +/- 3.9 cal mol(-1) K(-1). We conclude that the dissociation of GroEL observed in this investigation is an enthalpy-controlled process.

The enzyme rhodanese contains two globular domains connected by a tether region and associated by strong hydrophobic interactions. The protein has proven to be very difficult to refold without assistance to prevent oxidation and aggregation. For this study, the active site cysteine 247, near the interdomain region, was modified with the environmentally sensitive fluorescent probe, 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS), to yield a derivative that reversibly unfolds. Structural transitions during urea unfolding/refolding were complex and multiphasic. Increasing urea concentrations increased the IAANS fluorescence intensity and polarization. Both values reached maxima at ≈4m urea, where there is a concomitant large exposure of hydrophobic sites as reported by both IAANS and the noncovalent fluorescent probe, bis-ANS. The exposure of the hydrophobic sites arises from the decrease in strong interaction between the domain interfaces, which lead to their partial separation. This correlates with the loss of activity of the unlabeled enzyme. Above 4.5m urea, there is progressive loss of rigid, hydrophobic surfaces, and both fluorescence and polarization of IAANS decrease, with accompanying loss of secondary structure. These results are consistent with a folding model in which there is an initial, rapid hydrophobic collapse of the denatured form to an intermediate with native like secondary structure, with exposed interdomain, hydrophobic surfaces. This step is followed by adjustment of the domain-domain interactions and the proper positioning of reduced cysteine 247 at the active site.

Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM β-ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem.265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (N↔I↔U) followed by fluorescence at 363 nm, (N↔I) by fluorescence at 318 nm, and CD (I↔U) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5–6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced.

The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a decreased refolding yield. With time, a portion of the smaller aggregates can partition back first to intermediates and then to refolded protein, while a fraction of these irreversibly form unproductive higher aggregates. Dynamic light scattering measurements indicate that the average sizes of the aggregates formed during rhodanese folding increase from 225 to 325 nm over 45 min and they become increasingly heterogeneous. Glycerol addition or the application of high hydrostatic pressure improved the final refolding yields by stabilizing smaller particles. Although addition of glycerol into the refolding mixture blocks the formation of unproductive aggregates, it cannot dissociate them back to productive intermediates. The presence of 3.9 M urea keeps the aggregates small, and they can be dissociated to monomers by high hydrostatic pressure even after 1 h of incubation. These studies suggest that early associated intermediates formed during folding can be reversed to give active species.