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At least 19 recordsLinked to original sources

PubMed1994

Purification and characterization of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brockii.

Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2-propanol or poly(ethylene glycol) with MgCl2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 +/- 0.4 Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein-folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.

Adenosine Triphosphatases↗

PubMed1991

Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides.

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.

Adenosine Triphosphatases↗

PubMed1990

Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are K+ dependent.

Both the chaperonin- and MgATP-dependent reconstitution of unfolded ribulosebisphosphate carboxylase (Rubisco) and the uncoupled ATPase activity of chaperonin 60 (groEL) require ionic potassium. The spontaneous, chaperonin-independent reconstitution of Rubisco, observed at 15 but not at 25 degrees C, requires no K+ and is actually inhibited by chaperonin 60, with which the unfolded or partly folded Rubisco forms a stable binary complex. The chaperonin-dependent reconstitution of Rubisco involves the formation of a complex between chaperonin 60 and chaperonin 10 (groES). Formation of this complex almost completely inhibits the uncoupled ATPase activity of chaperonin 60. Furthermore, although the formation of the chaperonin 60-chaperonin 10 complex requires the presence of MgATP, hydrolysis of ATP may not be required, since complex formation occurs in the absence of K+. The interaction of chaperonin 60 with unfolded or partly folded Rubisco does not require MgATP, K+, or chaperonin 10. However, discharge of the complex of chaperonin 60-Rubisco, which leads to the formation of active Rubisco dimers, requires chaperonin 10 and a coupled, K(+)-dependent hydrolysis of ATP. We propose that a role of chaperonin 10 is to couple the K(+)-dependent hydrolysis of ATP to the release of the folded monomers of the target protein from chaperonin 60.

Adenosine Triphosphatases↗

PubMed1995

Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8.

The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced. The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins. The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells. The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E. coli. T.th cpn60 was expressed as a tetradecameric form, like GroEL of E. coli. Since chaperonin from T. thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before. T.th cpn60 tetradecamer tended to dissociate into monomers during storage. T.th cpn10 expressed in E. coli was purified as a stable oligomer, most likely a heptamer. The activity as holo-chaperonin was reconstituted by mixing both subunits. T.th cpn60 tetradecamer itself arrested refolding of other proteins. The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography. Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Prevention of in vitro protein thermal aggregation by the Sulfolobus solfataricus chaperonin. Evidence for nonequivalent binding surfaces on the chaperonin molecule.

We have studied the effects of the Sulfolobus solfataricus chaperonin on the aggregation and inactivation upon heating of four model enzymes: chicken egg white lysozyme (one 14.4-kDa chain), yeast alpha-glucosidase (one 68.5-kDa chain), chicken liver malic enzyme (four 65-kDa subunits), and yeast alcohol dehydrogenase (four 37.5-kDa subunits). When the proteins were heated in the presence of an equimolar amount of chaperonin, 1) the aggregation was prevented in all solutions; 2) the inactivation profiles of the single-chain enzymes were comparable with those detected in the absence of the chaperonin, and enzyme activities were regained in the solutions heated in the presence of the chaperonin upon ATP hydrolysis (78 and 55% activity regains for lysozyme and alpha-glucosidase, respectively); 3) the inactivation of the tetrameric enzymes was completely prevented, whereas the activities decreased in the absence of the chaperonin. We demonstrate by gel filtration chromatography that the chaperonin interacted with the structures occurring during thermal denaturation of the model proteins and that the interaction with the single-chain proteins (but not that with the tetrameric proteins) was reversed upon ATP hydrolysis. The chaperonin had nonequivalent surfaces for the binding of the model proteins upon heating: the thermal denaturation intermediates of the single-chain proteins share Surfaces I, while the thermal denaturation intermediates of the tetrameric proteins share Surfaces II. ATP binding to the chaperonin induced a conformation that lacked Surfaces I and carried Surfaces II. These data support the concept that chaperonins protect native proteins against thermal aggregation by two mechanistically distinct strategies (an ATP-dependent strategy and an ATP-independent strategy), and provide the first evidence that a chaperonin molecule bears functionally specialized surfaces for the binding of the protein substrates.

Alcohol Dehydrogenase↗

PubMed1994

Affinity of chaperonin-60 for a protein substrate and its modulation by nucleotides and chaperonin-10.

The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.

Adenosine Triphosphate↗

PubMed1991

Chemical synthesis of 10 kDa chaperonin. Biological activity suggests chaperonins do not require other molecular chaperones.

Molecular chaperones are required for the correct folding and assembly of certain other polypeptides. It is not known whether molecular chaperones themselves require other chaperones to become functional. A 97-amino acid chaperone, the chaperonin 10 protein was chemically synthesised so that during synthesis and purification there was no contact of the chaperone with any other protein. The purified, synthetic chaperonin 10 protein formed oligomeric structures spontaneously and was biologically active as a chaperonin. This is the first description of a chemically synthesised chaperonin, and suggests that no other chaperones are required for correct folding, polymerisation and biological activity of this chaperone.

Bacterial Proteins↗

PubMed1995

Chaperonin releases the substrate protein in a form with tendency to aggregate and ability to rebind to chaperonin.

To know whether the protein released from chaperonin GroEL/ES is in a form committed to the native state or still an aggregatable non-native one, two experiments were carried out. Dilution of the [GroEL-substrate protein] binary complex prior to ATP addition significantly improved the yield of folding, suggesting that the released protein has a tendency to aggregate. When N-ethylmaleimide treated GroEL, which can form the binary complex but not release the bound protein, was added to the binary complex prior to ATP addition, productive folding was severely inhibited, indicating that the protein released from GroEL/ES can bind to N-ethylmaleimide treated chaperonin. These data favor the 'reservoir' or 'reversion' model, in which GroEL/ES acts as a buffer of folding intermediate or mediates reversion of a misfolded protein to a less folded primitive form, rather than the 'marsupium' model in which folding of the substrate protein proceeds in chaperonin.

Adenosine Triphosphate↗

PubMed1995

Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60.

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.

Adenosine Triphosphatases↗

PubMed1993

Occurrence of chaperonin 60 and chaperonin 10 in primary and secondary bacterial symbionts of aphids: implications for the evolution of an endosymbiotic system in aphids.

All aphids harbor symbiotrophic prokaryotes ("primary symbionts") in a specialized-abdominal cell, the bacteriocyte. Chaperonin 60 (Cpn60, symbionin) and chaperonin 10 (Cpn10), which are high and low molecular weight heatshock proteins, were sought in tissues of more than 60 aphid species. The endosymbionts were compared immunologically and histologically. It was demonstrated that (1) there are two types of aphids in terms of the endosymbiotic system: some with only primary symbionts and others with, in addition, secondary symbionts; (2) the primary symbionts of various aphids are quite similar in morphology whereas the secondary symbionts vary; and (3) irrespective of the aphid species, Cpn60 is abundant in both the primary and secondary symbionts, while Cpn10 is abundant in the secondary symbionts but present in small amounts in the primary ones. Based on these results, we suggest that the primary symbionts have been derived from a prokaryote that was acquired by the common ancestor of aphids whereas the secondary symbionts have been acquired by various aphids independently after divergence of the aphid species. In addition, we point out the possibility that the prokaryotes under intracellular conditions have been subject to some common evolutionary pressures, and as a result, have come to resemble cell organelles.

Animals↗

PubMed1992

Immunochemical localization of a region of chaperonin-60 important for productive interaction with chaperonin-10.

An IgG1 monoclonal antibody (mAb 54G8) which binds to both Bordetella pertussis chaperonin-60 (cpn60) and Escherichia coli cpn60 (GroEL) was produced. mAb 54G8 as well as Fab fragments prepared from this antibody were found to abolish the ability of chaperonin-10 (cpn10, GroES) to inhibit the ATPase activity of both B. pertussis cpn60 and E. coli cpn60. Electron microscopy was used to localize the binding site of the monoclonal antibody on the B. pertussis cpn60 molecule. In the absence of the antibody, the B. pertussis molecule exhibited the tetradecameric structure typical of cpn60. Both end views (showing 7-fold symmetry of the face of the molecule) and side views were evident. When mAb 54G8 was bound, B. pertussis cpn60 molecules appeared to be cross-linked so that they formed long chains. Only side views of the molecules were seen in these long chains. When B. pertussis cpn60 complexed with Fab fragments of mAb 54G8 was examined, chains were no longer observed. Instead, side views of B. pertussis cpn60 were often seen with Fab fragments extending from the ends of the molecule. These data indicate that mAb 54G8 appears to bind at or near the end of the B. pertussis cpn60 molecule and that binding of mAb 54G8 at this location affects the ability of cpn10 to productively interact with cpn60, most likely either by sterically blocking the binding of cpn10, by affecting the conformation of cpn60 in such a way that it no longer binds cpn10, or by inhibiting proper transduction of the effects of cpn10 binding.

Adenosine Triphosphatases↗

PubMed1994

Alteration of the quaternary structure of cpn60 modulates chaperonin-assisted folding. Implications for the mechanism of chaperonin action.

Chaperonin-mediated, in vitro folding of rhodanese by the intact protein cpn60 has previously been shown to require cpn10 and ATP hydrolysis (Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F.-U. (1991) Nature 352, 36-42; Mendoza, J. A., Rogers, E., Lorimer, G. H., and Horowitz, P. M. (1991) J. Biol. Chem. 266, 13044-13049). The present work demonstrates that the rhodanese-cpn60 complex can be dissociated by urea to allow folding to proceed, thus removing the obligatory requirement for cpn10 and ATP. Analytical ultracentrifugation and circular dichroism show that tetradecameric cpn60 can be disassembled into monomers that retain substantial secondary structure. Unfolded rhodanese induces the reassembly of tetradecameric cpn60 from monomers, and binding of rhodanese stabilizes cpn60 quaternary structure. Intermediate cpn60 species, possibly heptamers, are detected at intermediate urea concentrations after addition of unfolded rhodanese. The use of urea has demonstrated a functionally related loosening of subunit interactions in cpn60 that is not detectable under usual solution conditions. Our data suggest a highly dynamic role for the quaternary structure of cpn60 in chaperonin-mediated protein folding.

Adenosine Triphosphatases↗

PubMed1997

Differential induction of the chaperonin GroEL and the Co-chaperonin GroES by heat, acid, and UV-irradiation in Lactococcus lactis subsp. lactis.

Microsequencing of a polypeptide with MW of 14.5 and pI of 5.0 induced by heat treatment at 42 degrees C and 50 degrees C in Lactococcus lactis subsp. lactis revealed that it corresponds to the co-chaperonin GroES. Quantitative analysis of analytical 2-D gels showed a relative induction of 12- and 11-fold after 30 min of heat adaptation at 42 degrees C and 50 degrees C, respectively. GroES is also induced by an acid shift from pH 7 to pH 5.5 and by UV254 nm-irradiation, with relative induction factors of 3.8 and 2.3, respectively. To our knowledge this is the first report showing induction of GroES by mild acid treatment. Contrasting to the relative induction of the groEL gene product, the second protein encoded by the groESL operon, GroES shows significantly higher induction under all stress situations.

Amino Acid Sequence↗

PubMed1994

Identification of the major chromaffin granule-binding protein, chromobindin A, as the cytosolic chaperonin CCT (chaperonin containing TCP-1).

Chromobindin A is a multisubunit complex ATPase that binds to chromaffin granule membranes in a calcium-dependent manner and requires ATP for release from the membrane (Martin, W. H., and Creutz, C. E. (1987) J. Biol. Chem. 262, 2803-2810). Here we report that the seven previously characterized subunits of chromobindin A cross react with antisera specific to subunits of CCT, the chaperonin containing TCP-1 (Kubota, H., Hynes, G., Carne, A., Ashworth, A., and Willison, K. (1994) Curr. Biol. 4, 89-99). The chromobindin A subunits previously called chromobindins 12, 13, 14, 15, 16, 18, and 19 cross-react specifically with subunits beta, delta, theta, alpha, zeta, xi, and gamma, respectively, of CCT. Additional similarities in subunit molecular weights, isoelectric points, and the morphologies of the two protein complexes as determined by electron microscopy support identification of chromobindin A as an adrenal medullary form of CCT. The chromobindin A/CCT complex was found to bind at least 7-fold more efficiently to affinity columns of chromaffin granule membranes than of adrenal medullary cytosol proteins, suggesting a specific interaction occurs between the complex and membrane components. The results indicate that the previously described characteristics of chromobindin A are likely to be relevant to the functions of CCT and suggest that the adrenal medullary form of CCT may play a role in the activities of secretory vesicle membranes.

Animals↗

PubMed1991

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.

Adenosine Triphosphate↗

PubMed1993

Chaperonin from Thermus thermophilus can protect several enzymes from irreversible heat denaturation by capturing denaturation intermediate.

Chaperonin isolated from Thermus thermophilus is stable up to 80 degrees C. Taking advantage of this heat stability, we have studied the effects of chaperonin on heat denaturation of several relatively heat labile enzymes. When the enzymes are incubated at their denaturating temperatures, the presence of T. thermophilus chaperonin in the solution has little effect on the rate of apparent inactivation of the enzyme. However, this inactivation is not irreversible since most activity is recovered when the solution is shifted to the second incubation at a moderate temperature with concomitant addition of MgATP. When the chaperonin is omitted from the solution, no recovery is observed. Recovery of the activity is also dependent on MgATP in the second incubation and 50% of recovery is attained at 5 microM MgATP. When the chaperonin is added after starting the incubation at a denaturing temperature, recovery of the activity becomes poorer as the delay of chaperonin addition increases. The critical temperature of the incubation at which irreversible denaturation occurs to the enzymes is elevated 8-15 degrees C by inclusion of T. thermophilus chaperonin in the solution. The heat stability of captured proteins by the chaperonin, assessed as retention of the ability to resume productive folding under optimal conditions, is measured more accurately using chemically produced folding intermediate-chaperonin complexes. The ability is lost at about 78 degrees C being irrespective of variable heat stability of individual enzymes. These results indicate that during heat denaturation proteins assume a common structure which is recognizable by the chaperonin. Once a protein with this structure is captured by T. thermophilus chaperonin, it retains the ability to resume productive folding even after exposure to the otherwise denaturing high temperature. Its heat stability seems to be limited solely by heat stability of chaperonin.

Adenosine Triphosphate↗

PubMed1996

A simple model of chaperonin-mediated protein folding.

Chaperonins are oligomeric proteins that help other proteins fold. They act, according to the "Anfinsen cage" or "box of infinite dilution" model, to provide private space, protected from aggregation, where a protein can fold. Recent evidence indicates, however, that proteins are often ejected from the GroEL chaperonin in nonnative conformations, and repeated cycles of binding and ejection are needed for successful folding. Some experimental evidence suggests that GroEL chaperonins can act as folding "catalysts" in an ATP-dependent manner even when no aggregation takes place. This implies that chaperonins must somehow recognize the kinetically trapped intermediate states of a protein. A central puzzle is how a chaperonin can catalyze the folding reaction of a broad spectrum of different proteins. We propose a physical mechanism by which chaperonins can flatten the energy barriers to folding in a nonspecific way. Using a lattice model, we illustrate how a chaperonin could provide a sticky surface that helps pull apart an incorrectly folded protein so it can try again to fold. Depending on the relative sizes of the protein and the chaperonin cavity, folding can proceed both inside and outside the chaperonin. Consistent with experiments, we find that the folding rate and amount of native protein can be considerably enhanced, or sometimes reduced, depending on the amino acid sequence, the chaperonin size, and the binding and ejection rates from the chaperonin.

Catalysis↗

PubMed1992

Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.

Adenosine Triphosphate↗