High Salt-induced Conversion of Escherichia coliGroEL into a Fully Functional Thermophilic Chaperonin
2000; Elsevier BV; Volume: 275; Issue: 43 Linguagem: Inglês
10.1074/jbc.m006256200
ISSN1083-351X
AutoresAndrew R. Kusmierczyk, Jörg Martin,
Tópico(s)Protein Structure and Dynamics
ResumoThe GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5,5′)-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 °C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins. The GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5,5′)-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 °C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins. 3-(N-morpholino) propanesulfonic acid bis-(5,5′)-8-anilino-1-naphthalene sulfonic acid green fluorescent protein N-ethylmaleimide The chaperonin GroEL from Escherichia coli belongs to a class of proteins, termed molecular chaperones, whose collective function is to assist in the folding of newly synthesized proteins and in the refolding of non-native polypeptides generated under conditions of stress (reviewed in Refs. 1Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (163) Google Scholar and 2Fenton W.A. Horwich A.L. Prot. Sci. 1997; 6: 743-760Crossref PubMed Scopus (325) Google Scholar). Like its homologs, CCT (chaperonin containing TCP-1) in eukaryotes and the thermosome in archaea, GroEL forms a multi-subunit assembly arranged into twin rings stacked end-to-end (3Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulos C.P. Hendrix R., W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (919) Google Scholar, 4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar, 5Gutsche I. Essen L.-O. Baumeister W. J. Mol. Biol. 1999; 293: 295-312Crossref PubMed Scopus (182) Google Scholar). The resultant homotetradecamer of 57-kDa subunits provides a deep cavity where non-native protein species may bind and undergo productive folding (6Braig K. Furuya F. Hainfeld J. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3978-3982Crossref PubMed Scopus (152) Google Scholar). GroEL is assisted in its chaperoning function by GroES, a heptamer composed of identical 10-kDa subunits arranged into a single ring (7Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar, 8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar). One of the functions of GroES is to act as a lid on the GroEL cylinder, thereby providing an enclosed environment for the folding polypeptide. However, its binding to GroEL plays other important roles too, such as modulating the low intrinsic ATPase activity of GroEL by coordinating the actions of nucleotide binding and hydrolysis (7Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar, 8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 9Gray T.E. Fersht A.R. FEBS Lett. 1991; 292: 254-258Crossref PubMed Scopus (165) Google Scholar). Crystallographic data have enabled the visualization of the GroEL complex and individual subunit architecture (10Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar, 11Boisvert D.C. Wang J.M. Otwinowski Z. Horwich A.L. Sigler P.B. Nature Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar, 12Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). It has revealed the regions implicated in both nucleotide and substrate binding. It is now known that each subunit of GroEL is arranged into three domains. An equatorial domain forms the majority of intersubunit interactions and is the site of nucleotide binding. The apical domain contains the substrate binding and GroES binding residues (13Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar). The two domains are connected by a hinge region, which transmits information on the status of GroES, polypeptide, and nucleotide binding (14Martin J. J. Biol. Chem. 1998; 273: 7351-7357Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Because GroEL/GroES has been the most widely studied chaperonin system, its mechanism of protein folding is known in some detail. Briefly, GroEL binds substrate protein in one of its two ring cavities. The bound substrate protein is in a molten-globule state characterized by the presence of secondary structure but lacking well defined tertiary structure (8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 15Robinson C.V. Gross M. Eyles S.J. Ewbank J.J. Mayhew M. Hartl F.U. Dobson C.M. Radford S.E. Nature. 1994; 372: 646-651Crossref PubMed Scopus (194) Google Scholar). Cooperative binding of seven molecules of ATP to the same (cis) ring as the bound polypeptide is immediately followed by the binding of GroES, also to the cis ring, which causes the polypeptide to be displaced into the cavity (4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar, 17Martin J. Mayhew M. Langer T. Hartl F.U. Nature. 1993; 366: 228-233Crossref PubMed Scopus (235) Google Scholar). The released polypeptide now folds in the protective environment of the enclosed chaperonin complex. Hydrolysis of the seven ATP molecules primes the cis complex for disassembly, and the binding of seven ATP molecules to the opposite (trans) ring of GroEL causes the release of both GroES and the folded substrate (18Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (351) Google Scholar). The system is now reset and ready to either accept a new substrate or rebind the just-released but not yet native polypeptide for another round of folding.Folding by GroEL involves a complex interplay of three ligands (i.e. substrate, GroES, and nucleotide), and all three are capable of inducing allosteric conformational changes within GroEL, either individually or in concert (19Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar, 20Yifrach O. Horovitz A. J. Mol. Biol. 1996; 255: 356-361Crossref PubMed Scopus (97) Google Scholar, 21Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Attempts to perturb the GroEL system by mutagenesis (13Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar, 22Horovitz A. Bochkareva E.S. Kovalenko O. Girshovich A.S. J. Mol. Biol. 1993; 231: 58-64Crossref PubMed Scopus (57) Google Scholar, 23Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 24Yifrach O. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1521-1524Crossref PubMed Scopus (55) Google Scholar), chemical modification (14Martin J. J. Biol. Chem. 1998; 273: 7351-7357Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar,25Gibbons D.L. Horowitz P.M. J. Biol. Chem. 1995; 270: 7335-7340Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), substrate modification (26Luo G.-X. Horowitz P.M. J. Biol. Chem. 1994; 269: 32151-32154Abstract Full Text PDF PubMed Google Scholar, 27Mayhew M. da Silva A.C.R. Erdjument-Bromage H. Tempst P. Hartl F.-U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (342) Google Scholar), or solvent manipulation (28Horowitz P.M. Hua S. Gibbons D.L. J. Biol. Chem. 1995; 270: 1535-1542Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 29Perrett S. Zahn R. Stenberg G. Fersht A.R. J. Mol. Biol. 1997; 269: 892-901Crossref PubMed Scopus (57) Google Scholar, 30Brazil B.T. Ybarra J. Horowitz P.M. J. Biol. Chem. 1998; 273: 3257-3263Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) have often resulted in chaperonins with altered functional properties. These can provide a wealth of information on the inner workings of the system as a whole. Here we report on the functional properties of one such altered state, induced by the presence of high concentrations of ammonium sulfate.Our work was prompted by structural data on both GroEL and the thermosome from the archaeon Thermoplasma acidophilum, which have been obtained from the analysis of crystals grown in high concentrations of ammonium sulfate (10Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar, 31Ditzel L. Löwe J. Stock D. Stetter K.-O. Huber H. Huber R. Steinbacher S. Cell. 1998; 93: 125-138Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Furthermore, the in vitro assembly of functional chaperonins from certain species of archaea has required the presence of ammonium sulfate (32Furutani M. Iida T. Yoshida T. Maruyama T. J. Biol. Chem. 1998; 273: 28399-28407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and the ATPase activity of at least one archaeal chaperonin has been shown to be dependent on a relatively high concentration of ammonium ions (33Andrä S. Frey G. Jaenicke R. Stetter K.O. Eur. J. Biochem. 1998; 255: 93-99Crossref PubMed Scopus (32) Google Scholar). We find that high ammonium sulfate concentrations alter the functional properties of GroEL, resulting in, among other things, an increased hydrophobic surface area and increased stringency for protein folding. Most surprisingly, these conditions allow the extension of E. coli chaperonin action to thermophilic conditions.DISCUSSIONIn this study, we have characterized the functional properties of the GroEL chaperonin system in the presence of high salt concentrations. The most surprising result is the ability of GroEL to function as a chaperone under thermophilic conditions in 1.2m (NH4)2SO4. GroEL is able to suppress thermally induced aggregation of citrate synthase. It can bind to intermediates of heat-inactivated α-glucosidase and mediate their refolding in a GroES-dependent manner, and, as at room temperature, it can bind and refold acid-denatured GFP with the same increased substrate stringency. We find that high concentrations of ammonium ions have a stimulatory effect on the ATPase activity of GroEL. At 30 °C, NH4Cl and (NH4)2SO4 are equally effective, even though the former contributes only half the number of ammonium ions on a per mole basis. A higher concentration of ammonium ions may counteract negative effects of sulfate. In fact, Na2SO4 has an inhibitory effect on the ATPase activity of GroEL. At high concentrations, some salts are thought to make a protein more rigid (51Timasheff S.N. Arakawa T. Chapter 14: Stabilization of Protein Structure by Solvents."Protein Structure: A Practical Approach". Oxford University Press Inc., New York1997Google Scholar) and are widely used as protein stabilizers. This is particularly true of SO42−, which has a high charge density and resides high on the Hofmeister series of anions. Sulfate ions, by virtue of making the protein more rigid, may hinder the ability of the chaperonin to hydrolyze ATP. Nevertheless, the stabilizing effect of the sulfate ions enables GroEL to function at thermophilic conditions by preventing its denaturation and keeping it soluble. This is evident in the fact that at temperatures of up to 75 °C, only ammonium sulfate is able to support a markedly enhanced chaperonin activity.Several features of the GroEL/GroES chaperonin system can be explained in terms of a Monod-Wyman-Changeux representation (52Monod J. Wyman J. Changeux J.P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6084) Google Scholar). Each of the two rings can either be in a tense acceptor state (T), in which GroEL has high affinity for substrate protein, low affinity for ATP, and high ATP hydrolysis rates, or in a relaxed state (R) with high affinity for ATP and low affinity for protein substrate. With increasing ATP concentrations, the equilibrium of conformations shifts first to the TR state, and when most GroEL subunits are occupied by ATP, the RR state dominates in which ATP hydrolysis rates are slightly decreased and substrate protein is released (19Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar, 20Yifrach O. Horovitz A. J. Mol. Biol. 1996; 255: 356-361Crossref PubMed Scopus (97) Google Scholar, 24Yifrach O. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1521-1524Crossref PubMed Scopus (55) Google Scholar, 53Ma J. Karplus M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8502-8507Crossref PubMed Scopus (165) Google Scholar, 54Cliff M.J. Kad N.M. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar). Our data are consistent with the possibility that (NH4)2SO4, rather than acting as an uncoupler, induces a conformational change in GroEL to a TT-like state in which the ATPase activity is at or near capacity. Unlike in low concentrations of KCl, where the substrate protein αs1-casein stimulates the ATPase activity of GroEL, no such increase is observed in (NH4)2SO4. Indeed, with GroEL already in a TT-like state, substrate protein should have no further effect. It has been established that GroES binding to GroEL regulates the ATPase activity of the chaperonin (4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar, 7Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar, 8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 40Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (310) Google Scholar, 41Jackson G.S. Staniforth R.A. Halsall D.J. Atkinson T. Holbrook J.J. Clarke A.R. Burston S.G. Biochemistry. 1993; 32: 2554-2563Crossref PubMed Scopus (236) Google Scholar). When GroES binds to GroEL, it is able to shift the chaperonin conformation to TR and RR states of submaximal ATP hydrolysis. Consequently, in the presence of GroES, substrate should show more pronounced effects in ammonium sulfate by trying to shift the T/R equilibrium back toward the TT state. This is exactly what we have observed with αs1-casein, which stimulates ATP hydrolysis in 1.2m (NH4)2SO4 in the presence of GroES.GroEL is fully able to fold proteins under these conditions at both ambient and thermophilic temperatures. Interestingly, the presence of the high concentration of ammonium sulfate increases substrate stringency such that GFP, capable of folding in a GroES-independent manner under low salt conditions, now becomes strictly GroES-dependent. A strongly favored TT-like state in 1.2m (NH4)2SO4 would explain this inability of ATP hydrolysis alone to mediate GFP release from GroEL. In the absence of GroES, the nucleotide is not able to induce on its own the conformational shifts toward the TR and RR state that are necessary to dissociate the substrate. The predominantly present TT-like GroEL form can thus be seen as locked in a conformation with high substrate affinity. Moreover, in this conformation GroEL exposes more hydrophobic binding surface than in low salt, which may affect the interaction with substrate protein. GroEL-bound substrates are typically in a molten globule-like state (8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 15Robinson C.V. Gross M. Eyles S.J. Ewbank J.J. Mayhew M. Hartl F.U. Dobson C.M. Radford S.E. Nature. 1994; 372: 646-651Crossref PubMed Scopus (194) Google Scholar). This quasi-ordered condition, in which secondary structure is present but tertiary structure is undefined, is characterized by the exposure of hydrophobic residues that would normally be buried in a native protein. GroEL contains a number of hydrophobic residues in its apical domain that have been demonstrated to be necessary for binding of non-native substrate protein (13Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar). It has been demonstrated that perturbation of the ionic strength of the solvent can increase exposure of hydrophobic residues on GroEL (28Horowitz P.M. Hua S. Gibbons D.L. J. Biol. Chem. 1995; 270: 1535-1542Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 30Brazil B.T. Ybarra J. Horowitz P.M. J. Biol. Chem. 1998; 273: 3257-3263Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Ammonium sulfate seems to elicit a similar change in the chaperonin. Titration data presented here suggest that GroEL can bind more bisANS per tetradecamer in 1.2 m(NH4)2SO4 than in low salt. Moreover, the hydrophobic nature of the binding sites is comparable, because the emission λmax is the same in both salts. There may well be additional reasons for the inability of ATP alone to mediate GFP release in (NH4)2SO4. For example, increased GFP stringency could be the result of some change within GFP itself induced by the high salt. The fluorescence of native GFP is virtually the same in both 50 mm KCl and 1.2m (NH4)2SO4, and the spontaneous recovery of fluorescence of acid-denatured GFP is essentially complete in both buffers. This suggests that GFP behaves similarly in both buffers. Nevertheless, it is conceivable that non-native GFP intermediate(s) bound by GroEL upon dilution from denaturant are different in nature such that those in 50 mmKCl are more amenable to release from GroEL by ATP alone than those in 1.2 m (NH4)2SO4.The presence of high concentrations of ammonium ions and ammonium sulfate pertaining to chaperonin structure and function has surfaced a few times in recent literature (10Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar, 31Ditzel L. Löwe J. Stock D. Stetter K.-O. Huber H. Huber R. Steinbacher S. Cell. 1998; 93: 125-138Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 32Furutani M. Iida T. Yoshida T. Maruyama T. J. Biol. Chem. 1998; 273: 28399-28407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 33Andrä S. Frey G. Jaenicke R. Stetter K.O. Eur. J. Biochem. 1998; 255: 93-99Crossref PubMed Scopus (32) Google Scholar). Notably, high ammonium sulfate concentrations were used to obtain crystals for the determination of the structures of both E. coli GroEL and the thermosome from T. acidophilum. Although the structures represent well the overall architecture of the chaperonins, questions have arisen as to the nature of the actual state, in terms of functional properties, that these structures represent. For instance, the unliganded thermosome from T. acidophilum was crystallized in 2 m(NH4)2SO4 in a "closed" conformation said to represent the Mg-ATP bound form (31Ditzel L. Löwe J. Stock D. Stetter K.-O. Huber H. Huber R. Steinbacher S. Cell. 1998; 93: 125-138Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). However, Gutsche et al. (55Gutsche I. Holzinger J. Rössle M. Heumann H. Baumeister W. May R.P. Curr. Biol. 2000; 10: 405-408Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) have recently demonstrated by small angle neutron scattering that in solution, the Mg-ATP bound thermosome favors the "open" conformation in low salt buffer. The closed conformation occurs only after ATP hydrolysis, but before release of Pi. Surprisingly, Gutsche et al. (55Gutsche I. Holzinger J. Rössle M. Heumann H. Baumeister W. May R.P. Curr. Biol. 2000; 10: 405-408Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) also showed that the crystallization buffer can induce the closed conformation in solution. The crystals for the unliganded structure of GroEL were grown in similarly high (NH4)2SO4 concentrations as those employed in this study (10Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar). Based on our results it is conceivable that the GroEL represented in that crystal structure has solution properties similar to the functional state observed here. Although these salt conditions are not physiologically relevant for E. coli in vivo, the changes in chaperonin function that they induce are nevertheless informative. For example, it was noted that high concentrations of sodium sulfate resulted in a stimulation of the ATPase activity of the archaeal chaperonin because of the aforementioned induction of the "closed" conformation, which occurs after ATP hydrolysis (55Gutsche I. Holzinger J. Rössle M. Heumann H. Baumeister W. May R.P. Curr. Biol. 2000; 10: 405-408Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The situation is different with bacterial chaperonins, because our findings show that sulfate inhibits the ATPase of GroEL; an effect that is in turn counterbalanced by high concentrations of ammonium ions. This difference serves to underscore the likelihood that despite having structurally conserved ATP-binding domains, the molecular basis of ATP hydrolysis in the two chaperonin systems may differ in some respects.It appears that examination of the solution properties of chaperonins, under the solvent conditions used for crystallization, is a worthwhile endeavor to better assign the functional state that the respective structures represent. Moreover, the ability to convert a mesophilic chaperonin into a thermophilic chaperonin opens interesting possibilities for direct comparison with and study of homologs from naturally occurring thermophiles. Some methanogenic archaea use increased intracellular ion concentrations to stabilize their proteins in vivo (16Hensel R. König H. FEMS Microbiol. Lett. 1988; 49: 75-79Crossref Scopus (162) Google Scholar). Whether or not a similar method of thermoadaptation is used by some extremophilic bacteria remains to be seen, but the results presented here suggest that this is a distinct possibility. The chaperonin GroEL from Escherichia coli belongs to a class of proteins, termed molecular chaperones, whose collective function is to assist in the folding of newly synthesized proteins and in the refolding of non-native polypeptides generated under conditions of stress (reviewed in Refs. 1Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (163) Google Scholar and 2Fenton W.A. Horwich A.L. Prot. Sci. 1997; 6: 743-760Crossref PubMed Scopus (325) Google Scholar). Like its homologs, CCT (chaperonin containing TCP-1) in eukaryotes and the thermosome in archaea, GroEL forms a multi-subunit assembly arranged into twin rings stacked end-to-end (3Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulos C.P. Hendrix R., W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (919) Google Scholar, 4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar, 5Gutsche I. Essen L.-O. Baumeister W. J. Mol. Biol. 1999; 293: 295-312Crossref PubMed Scopus (182) Google Scholar). The resultant homotetradecamer of 57-kDa subunits provides a deep cavity where non-native protein species may bind and undergo productive folding (6Braig K. Furuya F. Hainfeld J. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3978-3982Crossref PubMed Scopus (152) Google Scholar). GroEL is assisted in its chaperoning function by GroES, a heptamer composed of identical 10-kDa subunits arranged into a single ring (7Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar, 8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar). One of the functions of GroES is to act as a lid on the GroEL cylinder, thereby providing an enclosed environment for the folding polypeptide. However, its binding to GroEL plays other important roles too, such as modulating the low intrinsic ATPase activity of GroEL by coordinating the actions of nucleotide binding and hydrolysis (7Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar, 8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 9Gray T.E. Fersht A.R. FEBS Lett. 1991; 292: 254-258Crossref PubMed Scopus (165) Google Scholar). Crystallographic data have enabled the visualization of the GroEL complex and individual subunit architecture (10Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar, 11Boisvert D.C. Wang J.M. Otwinowski Z. Horwich A.L. Sigler P.B. Nature Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar, 12Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). It has revealed the regions implicated in both nucleotide and substrate binding. It is now known that each subunit of GroEL is arranged into three domains. An equatorial domain forms the majority of intersubunit interactions and is the site of nucleotide binding. The apical domain contains the substrate binding and GroES binding residues (13Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar). The two domains are connected by a hinge region, which transmits information on the status of GroES, polypeptide, and nucleotide binding (14Martin J. J. Biol. Chem. 1998; 273: 7351-7357Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Because GroEL/GroES has been the most widely studied chaperonin system, its mechanism of protein folding is known in some detail. Briefly, GroEL binds substrate protein in one of its two ring cavities. The bound substrate protein is in a molten-globule state characterized by the presence of secondary structure but lacking well defined tertiary structure (8Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 15Robinson C.V. Gross M. Eyles S.J. Ewbank J.J. Mayhew M. Hartl F.U. Dobson C.M. Radford S.E. Nature. 1994; 372: 646-651Crossref PubMed Scopus (194) Google Scholar). Cooperative binding of seven molecules of ATP to the same (cis) ring as the bound polypeptide is immediately followed by the binding of GroES, also to the cis ring, which causes the polypeptide to be displaced into the cavity (4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar, 17Martin J. Mayhew M. Langer T. Hartl F.U. Nature. 1993; 366: 228-233Crossref PubMed Scopus (235) Google Scholar). The released polypeptide now folds in the protective environment of the enclosed chaperonin complex. Hydrolysis of the seven ATP molecules primes the cis complex for disassembly, and the binding of seven ATP molecules to the opposite (trans) ring of GroEL causes the release of both GroES and the folded substrate (18Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (351) Google Scholar). The system is now reset and ready to either accept a new substrate or rebind the just-released but not yet native polypeptide for another round of folding. Folding by GroEL involves a complex interplay of three ligands (i.e. substrate, GroES, and nucleotide), and all three are capable of inducing allosteric conformational changes within GroEL, either individually or in concert (19Yifrach O. Horovitz A. Bioche
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