Rhodanese can partially refold in its GroEL-GroES-ADP complex and can be released to give a homogeneous product

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].     

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.     

A thermodynamic coupling mechanism can explain the GroEL-mediated acceleration of the folding of barstar

Despite extensive structural and kinetic studies, the mechanism by which the Escherichia coli chaperonin GroEL assists protein folding has remained somewhat elusive. It appears that GroEL might play an active role in facilitating folding, in addition to its role in restricting protein aggregation by secluding folding intermediates. We have investigated the kinetic mechanism of GroEL-mediated refolding of the small protein barstar. GroEL accelerates the observed fast (millisecond) refolding rate, but it does not affect the slow refolding kinetics. A thermodynamic coupling mechanism, in which the concentration of exchange-competent states is increased by the law of mass action, can explain the enhancement of the fast refolding rates. It is not necessary to invoke a catalytic role for GroEL, whereby either the intrinsic refolding rate of a productive folding transition or the unfolding rate of a kinetically trapped off-pathway intermediate is increased by the chaperonin.     

Productive and nonproductive intermediates in the folding of denatured rhodanese

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.     

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.     

The detection of kinetic intermediate(s) during refolding of rhodanese

Recent studies showed that the enzyme rhodanese could be reversibly unfolded in guanidinium chloride (GdmCl) if aggregation and oxidation were minimized. Further, these equilibrium studies suggested the presence of intermediate(s) during refolding (Tandon, S., and Horowitz, P. (1989) J. Biol. Chem. 264, 9859-9866). The present work shows that native and refolded enzymes are very similar in structural and functional characteristics. Kinetics of denaturation/renaturation were used to detect the folding intermediate(s). The shift in fluorescence wavelength maximum was used to monitor the structural changes during the process. First order plots of the structural changes during unfolding and refolding show nonlinear curves. The refolding occurs in at least two phases. The first phase is very fast (t1/2 much less than 30 s) and accounts for the partial regain in the structure but not in the activity. The second phase is slow (t1/2 = 2.9 h) during which the enzyme fully regains its structure along with the activity. The fractional renaturation of rhodanese due to the fast phase, monitored in various concentrations of GdmCl, describes a transition centered at 3.5 M GdmCl which is very similar to the higher of the two transitions observed in the reversible refolding. All of these findings support the presence of detectable intermediate(s) during folding of rhodanese.     

Chaperones GroEL/GroES Accelerate the Refolding of a Multidomain Protein through Modulating On-pathway Intermediates

Despite a vast amount information on the interplay of GroEL, GroES, and ATP in chaperone-assisted folding, the molecular details on the conformational dynamics of folding polypeptide during its GroEL/GroES-assisted folding cycle is quite limited. Practically no such studies have been reported to date on large proteins, which often have difficulty folding in vitro. The effect of the GroEL/GroES chaperonin system on the folding pathway of an 82-kDa slow folding protein, malate synthase G (MSG), was investigated. GroEL bound to the burst phase intermediate of MSG and accelerated the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which was then transformed into a more folding-compatible form. Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of the native state via a compact intermediate with the rate several times faster than that of spontaneous refolding. The presence of GroES doubled the ATP-dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Because GroES bound to the trans side of GroEL-MSG complex, it may be anticipated that confinement of the substrate underneath the co-chaperone is not required for accelerating the rate in the assisted folding pathway. The potential role of GroEL/GroES in assisted folding is most likely to modulate the conformation of MSG intermediates that can fold faster and thereby eliminate the possibility of partial aggregation caused by the slow folding intermediates during its spontaneous refolding pathway.     

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.     

Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement

One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.     

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.     

NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions.

The NH2-terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and non-native conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that Delta1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones.     

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.     

Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent

We have established the generality of using detergents for facilitating the reactivation of 6 M guanidinium chloride-denatured rhodanese that was recently described for the nonionic detergent lauryl maltoside (LM) (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). We report here that not only LM but other nonionic as well as ionic and zwitterionic detergents also have favorable effects in reactivating the denatured enzyme. Not all detergents are useful, and the favorable effects occur over a limited concentration range. Above and below that range there is little or no effect. Zwittergents, which represent a homologous series with varying critical micelle concentrations (CMCs) are effective only above their CMCs. Induction phases occur in the progress curves of rhodanese refolded in the presence of the effective detergents, suggesting the presence of refolding intermediates that are apparently stabilized by detergent interactions. Gel filtration chromatography of rhodanese with and without LM suggests that even though the renaturation of the denatured enzyme requires detergent at concentrations above its CMC, the enzyme does not bind an amount of detergent equivalent to a micelle. It is suggested that renaturation of other proteins might also be assisted by inclusion of "nondenaturing" detergents, although the optimal conditions will have to be determined for each individual case.     

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.     

GroEL interacts transiently with oxidatively inactivated rhodanese facilitating its reactivation

When the enzyme rhodanese was inactivated with hydrogen peroxide (H(2)O(2)), it underwent significant conformational changes, leading to an increased exposure of hydrophobic surfaces. Thus, this protein seemed to be an ideal substrate for GroEL, since GroEL uses hydrophobic interactions to bind to its substrate polypeptides. Here, we report on the facilitated reactivation (86%) of H(2)O(2)-inactivated rhodanese by GroEL alone. Reactivation by GroEL required a reductant and the enzyme substrate, but not GroES or ATP. Further, we found that GroEL interacted weakly and/or transiently with H(2)O(2)-inactivated rhodanese. A strong interaction with rhodanese was obtained when the enzyme was pre-incubated with urea, indicating that exposure of hydrophobic surfaces alone on oxidized rhodanese was not sufficient for the formation of a strong complex and that a more unfolded structure of rhodanese was required to interact strongly with GroEL. Unlike prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that GroEL can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein. Additionally, the mechanism for the GroEL-facilitated reactivation of rhodanese shown here appears to be different than that for the chaperonin-assisted folding of chemically unfolded polypeptides in which a nucleotide and sometimes GroES is required.     

GroEL-assisted dehydrogenase folding mediated by coenzyme is ATP-independent.

It has been commonly accepted that GroEL functions as a chaperone by modulation of its affinity for folding intermediates through binding and hydrolysis of ATP. However, we have found that NAD, as a coenzyme of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), also stimulates the discharge of GAPDH folding intermediate from its stable complex with GroEL formed in the absence of ATP and assists refolding with the same yield as ATP/Mg(2+) does. The reactivation further increases when ATP is also present, but addition of Mg(2+) has no more effect. NADP, a coenzyme of glucose-6-phosphate dehydrogenase, also releases its folding intermediates from GroEL and increases reactivation. Different from ATP, NAD triggers the release of GAPDH intermediates bound by GroEL via binding with GAPDH itself but not with GroEL, and the released intermediates all folded to native molecules without the formation of aggregation. The collaborative effects of coenzyme and GroEL mediate GroEL-assisted dehydrogenase folding in an ATP-independent way.     

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

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

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.     

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.     

Molecular chaperones: Inside and outside the Anfinsen cage

The GroEL/GroES chaperonin system acts as a passive anti-aggregation cage for refolding rubisco and rhodanese, and not as an active unfolding device. Refolding aconitase is too large to enter the cage but reversible binding to GroEL reduces its aggregration. Unexpectedly, confinement in the cage increases the rate of refolding of rubisco, but not rhodanese.