Intermediates in the chaperonin-assisted refolding of rhodanese are trapped at low temperature and show a small stoichiometry.

In vitro refolding of the urea-unfolded, monomeric, mitochondrial enzyme rhodanese (thiosulfate sulfur-transferase; EC 2.8.1.1) is facilitated by the chaperonin proteins cpn60 and cpn10 from Escherichia coli at 37 degrees C, but the refolding is strongly inhibited at 10 degrees C. In contrast, the unassisted refolding of rhodanese is efficient at 10 degrees C, but the refolding efficiency decreases as the temperature is raised. These observations provided two measures of the cpn60-rhodanese complex. Thus, we monitored either 1) the cpn60-dependent inhibition of spontaneous folding at 10 degrees C or 2) the recovery of active rhodanese in the complete chaperonin system at 25 degrees C, after first forming a cpn60-rhodanese complex at 10 degrees C. These procedures minimized the aggregation of interactive folding intermediates that tend to overestimate the apparent number of cpn60 14-mers in determining the stoichiometry of protein-cpn60 14-mer interactions. Both procedures used here gave results that were consistent with there being 1 rhodanese binding site/cpn60 tetradecamer. This stoichiometry is significantly less than might be expected from the fact that cpn60 is composed of 14 identical subunits, and it may indicate that rhodanese interacts with a restricted region that is formed when the cpn60 tetradecamer is assembled. The ability to stabilize chaperonin-protein complexes that can subsequently be reactivated will aid studies of the mode of action of the ubiquitous chaperonin proteins.     

Sulfhydryl modification ofE. coli cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex

Differential chemical modification ofE. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4′-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.     

Characterization of a stable, reactivatable complex between chaperonin 60 and mitochondrial rhodanese.

Efficient formation of the cpn60-rhodanese complex can be achieved by mixing unfolded rhodanese with excess cpn60 at low temperature. By employing these conditions, a stable and highly reactivatable complex is formed. The complex is not formed when native enzyme is used. Concentrations of NaCl, as high as 0.75 M, do not have any effect on the formation or disruption of the binary complex. cpn60-bound rhodanese contains an exposed hydrophobic surface, as detected by the binding of the fluorescent reporter, 1-anilinonaphthalene-8-sulfonic acid. The intrinsic fluorescence of cpn60-bound rhodanese reports that the average tryptophan is in an intermediate environment between that found in unfolded and native states. This form of rhodanese has an accessibility to quenching by acrylamide or iodide that is intermediate between the unfolded and native forms of the enzyme. Protease susceptibility studies show that rhodanese bound to cpn60 exhibits a trypsin digestion pattern similar to the native enzyme, although it is more rapidly proteolyzed. The results suggest that the conformation of cpn60-bound rhodanese resembles a native-like conformation, but with increased flexibility. Further, only intact rhodanese or enzyme lacking its N-terminal sequence can interact with cpn60 and form a stable binary complex. The protein fragment corresponding to the rhodanese N-terminal sequence did not form part of a stable complex with cpn60.     

The chaperonin assisted and unassisted refolding of rhodanese can be modulated by its N-terminal peptide

Thein vitro refolding of the monomeric, mitochondrial enzyme rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), which is assisted by theE. coli chaperonins, is modulated by the 23 amino acid peptide (VHQVLYRALVSTKWLAESVRAGK) corresponding to the amino terminal sequence (1–23) of rhodanese. In the absence of the peptide, a maximum recovery of active enzyme of about 65% is achieved after 90 min of initiation of the chaperonin assisted folding reaction. In contrast, this process is substantially inhibited in the presence of the peptide. The maximum recovery of active enzyme is peptide concentration-dependent. The peptide, however, does not prevent the interaction of rhodanese with the chaperonin 60 (cpn60), which leads to the formation of the cpn60-rhodanese complex. In addition, the peptide does not affect the rate of recovery of active enzyme, although it does affect the extent of recovery. Further, the unassisted refolding of rhodanese is also inhibited by the peptide. Thus, the peptide interferes with the folding of rhodanese in either the chaperonin assisted or the unassisted refolding of the enzyme. A 13 amino acid peptide (STKWLAESVRAGK) corresponding to the amino terminal sequence (11–23) of rhodanese does not show any significant effect on the chaperonin assisted or unassisted refolding of the enzyme. The results suggest that other sequences of rhodanese, in addition to the N-terminus, may be required for the binding of cpn60, in accord with a model in which cpn60 interacts with polypeptides through multiple binding sites.     

Sulfhydryl modification ofE. coli cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex

Differential chemical modification ofE. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.     

Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese.

In vitro refolding of the monomeric mitochondrial enzyme, rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is facilitated by molecular chaperonins. The four components: two proteins from Escherichia coli, chaperonin 60 (groEL) and chaperonin 10 (groES), MgATP, and K+, are necessary for the in vitro folding of rhodanese. These were previously shown to be necessary for the in vitro folding of ribulose-1,5-bisphosphate carboxylase at temperatures in excess of 25 degrees C (Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O'Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5671). The labile folding intermediate, rhodanese-I, which rapidly aggregates at 37 degrees C in the absence of the chaperonins, can be stabilized by forming a binary complex with chaperonin 60. The discharge of the binary chaperonin 60-rhodanese-I complex, results in the formation of active rhodanese, and requires the presence of chaperonin 10. Optimal refolding is associated with a K(+)-dependent hydrolysis of ATP. At lower protein concentrations and 25 degrees C, where aggregation is reduced, a fraction of the rhodanese refolds to an active form in the absence of the chaperonins. This spontaneous refolding can be arrested by chaperonin 60. There is some refolding (approximately equal to 20%) when ATP is replaced by nonhydrolyzable analogs, but there is no refolding in the presence of ADP or AMP. ATP analogs may interfere with the interaction of rhodanese-I with the chaperonins. Nondenaturing detergents facilitate rhodanese refolding by interacting with exposed hydrophobic surfaces of folding intermediates and thereby prevent aggregation (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). The chaperonin proteins appear to play a similar role in as much as they can replace the detergents. Consistent with this view, chaperonin 60, but not chaperonin 10, binds 2-3 molecules of the hydrophobic fluorescent reporter, 1,1'-bi(4-anilino)naphthalene-S,5'-disulfonic acid, indicating the presence of hydrophobic surfaces on chaperonin 60. The number of bound probe molecules is reduced to 1-2 molecules when chaperonin 10 and MgATP are added. The results support a model in which chaperonins facilitate folding, at least in part, by interacting with partly folded intermediates, thus preventing the interactions of hydrophobic surfaces that lead to aggregation.     

The lower hydrolysis of ATP by the stress protein GroEL is a major factor responsible for the diminished chaperonin activity at low temperature

The chaperonins GroEL and GroES were shown to facilitate the refolding of urea-unfolded rhodanese in an ATP-dependent process at 25 or 37 degrees C. A diminished chaperonin activity was observed at 10 degrees C, however. At low temperature, GroEL retains its ability to form a complex with urea-unfolded rhodanese or with GroES. GroEL is also able to bind ATP at 10 degrees C. Interestingly, the ATPase activity of GroEL was highly decreased at low temperatures. Hydrolysis of ATP by GroEL was 60% less at 10 degrees C than at 25 degrees C. We conclude that the reduced hydrolysis of ATP by GroEL is a major but perhaps not the only factor responsible for the diminished chaperonin activity at 10 degrees C. GroEL may function primarily at higher temperatures in which the ability of GroEL to hydrolyze ATP is not compromised.     

Unassisted refolding of urea unfolded rhodanese

In vitro refolding after urea unfolding of the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) normally requires the assistance of detergents or chaperonin proteins. No efficient, unassisted, reversible unfolding/folding transition has been demonstrated to date. The detergents or the chaperonin proteins have been proposed to stabilize folding intermediates that kinetically limit folding by aggregating. Based on this hypothesis, we have investigated a number of experimental conditions and have developed a protocol for refolding, without assistants, that gives evidence of a reversible unfolding transition and leads to greater than 80% recovery of native enzyme. In addition to low protein concentration (10 micrograms/ml), low temperatures are required to maximize refolding. Otherwise optimal conditions give less than 10% refolding at 37 degrees C, whereas at 10 degrees C the recovery approaches 80%. The unfolding/refolding phases of the transition curves are most similar in the region of the transition, and refolding yields are significantly reduced when unfolded rhodanese is diluted to low urea concentrations, rather than to concentrations near the transition region. This is consistent with the formation of "sticky" intermediates that can remain soluble close to the transition region. Apparently, nonnative structures, e.g. aggregates, can form rapidly at low denaturant concentrations, and their subsequent conversion to the native structure is slow.     

GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle

GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.     

On the chaperonin activity of GroEL at heat-shock temperature

The studies of GroEL, almost exclusively, have been concerned with the function of the chaperonin under non-stress conditions, and little is known about the role of GroEL during heat shock. Being a heat shock protein, GroEL deserves to be studied under heat shock temperature. As a model for heat shock in vitro, we have investigated the interaction of GroEL with the enzyme rhodanese undergoing thermal unfolding at 43 degrees C. GroEL interacted strongly with the unfolding enzyme forming a binary complex. Active rhodanese (82%) could be recovered by releasing the enzyme from GroEL after the addition of several components, e.g. ATP and the co-chaperonin GroES. After evaluating the stability of the GroEL-rhodanese complex, as a function of the percentage of active rhodanese that could be released from GroEL with time, we found that the complex had a half-life of only one and half-hours at 43 degrees C; while, it remained stable at 25 degrees C for more than 2 weeks. Interestingly, the GroEL-rhodanese complex remained intact and only 13% of its ATPase activity was lost during its incubation at 43 degrees C. Further, rhodanese underwent a conformational change over time while it was bound to GroEL at 43 degrees C. Overall, our results indicated that the inability to recover active enzyme at 43 degrees C from the GroEL-rhodanese complex was not due to the disruption of the complex or aggregation of rhodanese, but rather to the partial loss of its ATPase activity and/or to the inability of rhodanese to be released from GroEL due to a conformational change.     

Functional consequences of single:double ring transitions in chaperonins: life in the cold

The cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5 degrees C to -13.7 degrees C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266-1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)(7) was the active oligomer at 4-10 degrees C, whereas at > 10 degrees C, this complex was converted to (O.Cpn60)(14). The dissociation reduces the ATP consumption (energy-saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double-ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild-type chaperonin was 24-28 degrees C and 4-18 degrees C, whereas that for the mutants was 45-55 degrees C and 14-36 degrees C respectively. The temperature inducing unfolding (T(M)) increased from 45 degrees C to more than 65 degrees C. In contrast, a single ring mutant, O.Cpn60(SR), with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10 degrees C. Above 10 degrees C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4 degrees C as the single ring variant. We demonstrated that expression of O.Cpn60(WT) and O.Cpn60(SR) leads to a higher growth of E. coli at 4 degrees C ( micro (max), 0.22 and 0.36 h(-1) respectively), whereas at 10-15 degrees C, only E. coli cells expressing O.Cpn60 or O.Cpn60(DR) grew better than parental cells (-cpn). These results clearly indicate that the single-to-double ring transition in Oleispira chaperonin is a wild-type mechanism for its thermal acclimation. Although previous studies have also reported single-to-double ring transitions under many circumstances, this is the first clear indication that single-ring chaperonins are necessary to support growth when the temperature falls from 37 degrees C to 4 degrees C.     

Affinity purification of fusion chaperonin Cpn60-(His)(6) from thermophilic bacterium Bacillus strain MS and its use in facilitating protein refolding and preventing heat denaturation

The cpn60 gene from Bacillus strain MS, which is highly homologous to Bacillus stearothermophilus, was cloned. Cpn60 with a hexahistidine affinity tag (His)(6) fused to its C-terminus (cpn60-(His)(6)) was overproduced in Escherichia coli. Cpn60-(His)(6) was expressed in a soluble form in E. coli. and purified to homogeneity in a single step by nickel chelate affinity chromatography. Cpn60-(His)(6) formed a tetradecamer and had ATPase activity. Cpn60-(His)(6) mediated refolding of guanidine hydrochloride unfolded pig heart malic dehydrogenase (MDH) and Thermus flavus MDH at 25 and 70 degrees C, respectively, in an ATP-dependent manner. In addition, cpn60-(His)(6) prevented heat denaturation of pig heart MDH and T. flavus MDH at 30 and 80 degrees C, respectively, in an ATP-dependent manner. Therefore, cpn60-(His)(6) facilitates protein refolding and prevents heat denaturation of proteins across a wide temperature range.     

Purification and characterization of chaperonins 60 and 10 from Methylobacillus glycogenes

Two proteins belonging to the group I chaperonin family were isolated from an obligate methanotroph, Methylobacillus glycogenes. The two proteins, one a GroEL homologue (cpn60: M. glycogenes 60 kDa chaperonin) and the other a GroES homologue (cpn10: M. glycogenes 10 kDa chaperonin), composed a heteropolymeric complex in the presence of ATP. Both proteins were purified from crude extracts of M. glycogenes by anion-exchange (DEAE-Toyopearl) and gel-filtration (Sephacryl S-400) chromatography. The native molecular weights of each chaperonin protein as determined by high-performance liquid chromatography (HPLC) gel-filtration were 820 000 for cpn60 and 65 000 for cpnl0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the subunit molecular weights of cpn60 and cpnl0 were 58 000 and 10 000, respectively. Both cpn60 and cpnl0 possessed amino acid sequences which were highly homologous to other group I chaperonins. M. glycogenes cpn60 displayed an ATPase activity which was inhibited in the presence of cpn10. The chaperonins also displayed an ability to interact with and facilitate the refolding of Thermus malate dehydrogenase and yeast enolase in a manner similar to that of GroEL/ES. The similarities between the Escherichia coli GroE proteins are discussed.     

Functional characterization of the recombinant group II chaperonin alpha from Thermoplasma acidophilum

The functional characteristics of group II chaperonins, especially those from archaea, have not been elucidated extensively. Here, we performed a detailed functional characterization of recombinant chaperonin alpha subunits (16-mer) (Ta-cpn alpha) from the thermophilic archaea Thermoplasma acidophilum as a model protein of archaeal group II chaperonins. Recombinant Ta-cpn alpha formed an oligomeric ring structure similar to that of native protein, and displayed an ATP hydrolysis activity (optimal temperature: 60 degrees C) in the presence of either magnesium, manganese or cobalt ions. Ta-cpn alpha was able to bind refolding intermediates of Thermus MDH and GFP in the absence of ATP, and to promote the refolding of Thermus MDH at 50 degrees C in the presence of Mg2+-, Mn2+-, or Co2+-ATP. Ta-cpn alpha also prevented thermal aggregation of rhodanese and luciferase at 50 degrees C. Interestingly, Ta-cpn alpha in the presence of Mn2+ ion showed an increased hydrophobicity, which correlated with an increased efficiency in substrate protein binding. Our finding that Ta-cpn alpha chaperonin system displays folding assistance ability with ATP-dependent substrate release may provide a detailed look at the potential functional capabilities of archaeal chaperonins.     

The aggregation state of rhodanese during folding influences the ability of GroEL to assist reactivation

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).     

Isolation and characterization of rhodanese intermediates during thermal inactivation and their implications for the mechanism of protein aggregation

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.     

Differential effects of co-chaperonin homologs on cpn60 oligomers

In this study, we have investigated the relationship between chaperonin/co-chaperonin binding, ATP hydrolysis, and protein refolding in heterologous chaperonin systems from bacteria, chloroplast, and mitochondria. We characterized two types of chloroplast cpn60 oligomers, ch-cpn60 composed of α and β subunits (α₇β₇ ch-cpn60) and one composed of all β subunits (β₁₄ ch-cpn60). In terms of ATPase activity, the rate of ATP hydrolysis increased with protein concentration up to 60 μM, reflecting a concentration at which the oligomers are stable. At high concentrations of cpn60, all cpn10 homologs inhibited ATPase activity of α₇β₇ ch-cpn60. In contrast, ATPase of β₁₄ ch-cpn60 was inhibited only by mitochondrial cpn10, supporting previous reports showing that β₁₄ is functional only with mitochondrial cpn10 and not with other cpn10 homologs. Surprisingly, direct binding assays showed that both ch-cpn60 oligomer types bind to bacterial, mitochondrial, and chloroplast cpn10 homologs with an equal apparent affinity. Moreover, mitochondrial cpn60 binds chloroplast cpn20 with which it is not able to refold denatured proteins. Protein refolding experiments showed that in such instances, the bound protein is released in a conformation that is not able to refold. The presence of glycerol, or subsequent addition of mitochondrial cpn10, allows us to recover enzymatic activity of the substrate protein. Thus, in our systems, the formation of co-chaperonin/chaperonin complexes does not necessarily lead to protein folding. By using heterologous oligomer systems, we are able to separate the functions of binding and refolding in order to better understand the chaperonin mechanism.     

A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes.

A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.     

Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies

Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.