Chaperone Activity of a Chimeric GroEL Protein That Can Exist in a Single or Double Ring Form
1999; Elsevier BV; Volume: 274; Issue: 29 Linguagem: Inglês
10.1074/jbc.274.29.20351
ISSN1083-351X
AutoresAnnette H. Erbse, Ofer Yifrach, Susan R. Jones, Peter A. Lund,
Tópico(s)Protein Structure and Dynamics
ResumoThe molecular chaperone GroEL is a protein complex consisting of two rings each of seven identical subunits. It is thought to act by providing a cavity in which a protein substrate can fold into a form that has no propensity to aggregate. Substrate proteins are sequestered in the cavity while they fold, and prevented from diffusion out of the cavity by the action of the GroES complex, that caps the open end of the cavity. A key step in the mechanism of action of GroEL is the transmission of a conformational change between the two rings, induced by the binding of nucleotides to the GroEL ring opposite to the one containing the polypeptide substrate. This conformational change then leads to the discharge of GroES from GroEL, enabling polypeptide release. Single ring forms of GroEL are thus predicted to be unable to chaperone the folding of GroES-dependent substrates efficiently, since they are unable to discharge the bound GroES and unable to release folded protein. We describe here a detailed functional analysis of a chimeric GroEL protein, which we show to exist in solution in equilibrium between single and double ring forms. We demonstrate that whereas the double ring form of the GroEL chimera functions effectively in refolding of a GroES-dependent substrate, the single ring form does not. The single ring form of the chimera, however, is able to chaperone the folding of a substrate that does not require GroES for its efficient folding. We further demonstrate that the double ring structure of GroEL is likely to be required for its activity in vivo. The molecular chaperone GroEL is a protein complex consisting of two rings each of seven identical subunits. It is thought to act by providing a cavity in which a protein substrate can fold into a form that has no propensity to aggregate. Substrate proteins are sequestered in the cavity while they fold, and prevented from diffusion out of the cavity by the action of the GroES complex, that caps the open end of the cavity. A key step in the mechanism of action of GroEL is the transmission of a conformational change between the two rings, induced by the binding of nucleotides to the GroEL ring opposite to the one containing the polypeptide substrate. This conformational change then leads to the discharge of GroES from GroEL, enabling polypeptide release. Single ring forms of GroEL are thus predicted to be unable to chaperone the folding of GroES-dependent substrates efficiently, since they are unable to discharge the bound GroES and unable to release folded protein. We describe here a detailed functional analysis of a chimeric GroEL protein, which we show to exist in solution in equilibrium between single and double ring forms. We demonstrate that whereas the double ring form of the GroEL chimera functions effectively in refolding of a GroES-dependent substrate, the single ring form does not. The single ring form of the chimera, however, is able to chaperone the folding of a substrate that does not require GroES for its efficient folding. We further demonstrate that the double ring structure of GroEL is likely to be required for its activity in vivo. The chaperonins or HSP60 proteins are essential for cell growth ( 1The abbreviations LDHlactate dehydrogenasemMDHmitochondrial malate dehydrogenaseDTTdithiothreitolPAGEpolyacrylamide gel electrophoresis ). In vivo, they assist in the folding of newly synthesized or stress denatured proteins (for recent reviews, see Refs.2Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2428) Google Scholar, 3Horovitz A. Curr. Opin. Struct. Biol. 1998; 8: 93-100Crossref PubMed Scopus (54) Google Scholar, 4Richardson A. Landry S.J. Georgopoulos C. Trends Biochem. Sci. 1998; 23: 138-143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The chaperonin GroEL from Escherichia coli is the best characterized member of the chaperonin family. It facilitates protein folding in vivo together with its protein co-factor GroES. GroEL has 14 identical 57-kDa subunits, organized in two heptameric rings stacked back to back with a central cavity in each ring. The arrangement of the subunits and the size of the cavity change dramatically in the presence of nucleotide and GroES (5Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.-L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1194) Google Scholar, 6Boisvert D.C. Wang J. Otwinowski Z. Horwich A.-L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (245) Google Scholar, 7Xu Z. Horwich A.-L. Sigler P.B. Nature. 1997; 338: 741-750Crossref Scopus (1042) Google Scholar, 8Roseman A.M. Chen S. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-247Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 9White H.E. Chen S. Roseman A.M. Yifrach O. Horovitz A. Saibil H.R. Nat. Struct. Biol. 1997; 4: 690-694Crossref PubMed Scopus (52) Google Scholar). Protein folding is thought to take place within this cavity. Detailed in vitro studies of the ATPase activity of GroEL, and GroEL-mediated protein refolding, suggest that the double ring structure and the transmission of allosteric information between the rings are key features of the mechanism by which the chaperone acts. Unfolded or partially folded substrate protein is thought to bind to the apical region of one of the two rings. The subsequent binding of ATP and GroES to the same ring induces a structural reorientation of the subunits, creating a cis-complex where the bound protein has been discharged into the central cavity, which is sealed by GroES. The protein folds in this cavity, where unfavorable hydrophobic interactions with other unfolded proteins are prevented. The protein remains trapped by the GroES co-factor until the binding of ATP to the opposite (trans) ring releases GroES. This binding cannot take place until the slow hydrolysis of ATP on the cis ring has occurred, which also primes the GroES for release (10Rye 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 (357) Google Scholar). In vitro, GroEL can assist the refolding of some substrate proteins without the aid of GroES. The mechanism of GroES independent refolding is not clearly understood. lactate dehydrogenase mitochondrial malate dehydrogenase dithiothreitol polyacrylamide gel electrophoresis ATP binding and hydrolysis is an intrinsic part of the mechanism by which GroEL functions. GroEL shows two levels of cooperativity for the binding and hydrolysis of ATP: positive cooperativity between the subunits of each ring and negative cooperativity between the two rings (11Gray T.E. Fersht A.R. FEBS Lett. 1991; 292: 254-258Crossref PubMed Scopus (167) Google Scholar, 12Yifrach O. Horovitz A. J. Mol. Boil. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar, 13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (265) Google Scholar, 14Yifrach O. Horovitz A. Biochemistry. 1998; 37: 7083-7088Crossref PubMed Scopus (62) Google Scholar). According to the model of nested cooperativity (12Yifrach O. Horovitz A. J. Mol. Boil. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar, 13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (265) Google Scholar, 14Yifrach O. Horovitz A. Biochemistry. 1998; 37: 7083-7088Crossref PubMed Scopus (62) Google Scholar), the subunits in each ring are in equilibrium between a T-state (with high affinity for unfolded polypeptides, and low affinity for ATP) and an R-state (with low affinity for protein and high affinity for ATP). In the absence of nucleotide the subunits of both rings are mostly in the T-state, forming a symmetricalTT-state. Binding of ATP to one ring induces a concerted transition of all the seven subunits in that ring to anR-state, while those in the other ring remain in theT-state, resulting in an asymmetrical TR form. With increasing ATP concentration the negative cooperativity between the rings is overcome and the binding of ATP to the second ring induces a second transition so that a symmetrical R′R′-state is reached. The designation R′ indicates that the catalytical properties of the R-states in the TR andR′R′ form are not identical. The role of the double ring structure has been examined by several groups, using a single ring GroEL mutant (10Rye 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 (357) Google Scholar, 15Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 16Weissman J.S. Rye H.S. Fenton W.A. Beechem J.M. Horwich A.L. Cell. 1996; 84: 481-490Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 17Hayer-Hartl M.K. Weber F. Hartl F.U. EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (133) Google Scholar) and "mini-chaperones" (consisting of the apical protein-binding domains alone from GroEL) (18Zahn R. Buckle A.M. Perrett S. Johnson C.M. Corrales F.J. Golbik R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15024-15029Crossref PubMed Scopus (136) Google Scholar, 19Buckle A.M. Zahn R. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3571-3575Crossref PubMed Scopus (201) Google Scholar, 20Chatellier J. Hill F. Lund P.A. Fresht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9861-9866Crossref PubMed Scopus (63) Google Scholar, 21Ben-Zvi A.P. Chatellier J. Fersht A.R. Goloubinoff P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15275-15280Crossref PubMed Scopus (45) Google Scholar). According to the model described above, a single ring form of GroEL should not show GroES-dependent chaperone activity, as the bound GroES would never be released and the substrate protein would stay sequestered beneath it. Studies on a single ring mutant of GroEL have indeed shown this to be the case (10Rye 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 (357) Google Scholar). It was shown that although proteins continue to fold in the cavity of the single ring after ATP and GroES binding, they cannot be released except under special experimental conditions like a cold shock or high salt concentrations (15Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 16Weissman J.S. Rye H.S. Fenton W.A. Beechem J.M. Horwich A.L. Cell. 1996; 84: 481-490Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 17Hayer-Hartl M.K. Weber F. Hartl F.U. EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (133) Google Scholar). It was also shown that the single ring GroEL cannot function in vivo (22Weber F. Keppel F. Georgopoulos C. Hayer-Hartl M.K. Hartl F.U. Nat. Struct. Biol. 1998; 5: 977-985Crossref PubMed Scopus (69) Google Scholar). This provides strong evidence for the above model, but it could be argued that because a number of mutations have to be introduced to obtain single rings (15Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (391) Google Scholar), these mutants may be defective in chaperone function for other unknown reasons. It has also been shown that mini-chaperones cannot mediate the refolding of GroES-dependent substrates, again reinforcing the requirement for the complete double ring structure for GroEL/GroES-mediated folding of proteins. Only in the case of mammalian mitochondrial Hsp60 is there convincing evidence that the protein can act as a chaperone when in the single ring form. However, it can only interact with its own Hsp10 and not with GroES, indicating that there may be quite a distinct mechanism for chaperone activity in this case (23Nielsen K.L. Cowan N.J. Molecular Cell. 1998; 2: 93-94Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). We recently described the construction of a series of chimeric GroEL proteins (24Jones S. Wallington E.J. George R. Lund P.A. J. Mol. Biol. 1998; 282: 789-800Crossref PubMed Scopus (7) Google Scholar), and reported that a number of these proteins were able to support growth as the sole GroEL protein in Escherichia coli but nevertheless appeared to exist as single rings when analyzed by native gel electrophoreses. A single amino acid residue crucial for ring-ring interactions in most GroEL homologues was identified, and mutation of this residue enabled us to obtain pairs of GroEL proteins differing only at this position but with very different mobilities on native polyacrylamide gels. We report here the detailed characterization of one of these pairs of chimeras and show that, in further support of the above model, the single ring form of the chimeric protein cannot chaperone the folding of a GroES-dependent substrate, whereas the double ring form can. The folding of a GroES independent substrate, however, is not affected by the state of oligomerization of the two rings. E. coli strains were grown in L-broth with kanamycin (50 μg/ml), carbenicillin (200 μg/ml), and (unless otherwise indicated) 0.2% (w/v) arabinose. Chimeric groELgenes were created and expressed from the pBAD promoter as described (24Jones S. Wallington E.J. George R. Lund P.A. J. Mol. Biol. 1998; 282: 789-800Crossref PubMed Scopus (7) Google Scholar). Chromosomal groEL genes were deleted by P1 transduction from a strain that had a precise replacement of thegroEL gene with the nptII gene from Tn5 (25Ivic A. Olden D. Wallington E.J. Lund P.A. Gene (Amst.). 1996; 195: 1-8Google Scholar). Strains carrying ΔgroEL::nptII are referred to as AI90 plus the name of the complementing plasmid. Complementation was tested in these strains and in SF103, which was made by P1 transduction of the groEL44 allele from CG2241 (a gift from C. Georgopoulos) into TG1. SF103 does not grow at 42 °C unless functional GroEL is expressed from a plasmid. To assay complementation, overnight cultures of strains with a groEL44 or a ΔgroEL genotype, containing plasmids expressing wild type or chimeric groEL genes, were diluted, spotted onto plates, and incubated at 37 and 42 °C (groEL44 derivatives) or on plates containing dilutions of arabinose (ΔgroELderivatives). Growth was scored after 24 h. incubation. Bovine lactate dehydrogenase (LDH)1 and porcine heart mitochondrial malate dehydrogenase (mMDH) were obtained from Sigma.E. coli GroES was a generous gift from A. R. Clarke (University of Bristol). GroEL and GroEL chimeric proteins were isolated as described by Yifrach and Horovitz (12Yifrach O. Horovitz A. J. Mol. Boil. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar) from strains expressing E. coli GroEL or the different chimeras from the pBAD promoter in a ΔgroEL background (24Jones S. Wallington E.J. George R. Lund P.A. J. Mol. Biol. 1998; 282: 789-800Crossref PubMed Scopus (7) Google Scholar). 15% methanol was added to samples before fractionation on a Q-Sepharose high performance column (Pharmacia), equilibrated with buffer B (50 mm Tris, pH 7.5, 1 mm DTT, 0.1 mmEDTA, 15% methanol w/v) (26Todd M.J. Lorimer G.H. Methods Enzymol. 1998; 290: 135-140Crossref PubMed Scopus (20) Google Scholar). Pure GroEL or chimeric chaperone protein was eluted with a linear gradient from 0.1 to 0.6 m NaCl in buffer B. Pure fractions as judged from SDS-PAGE were combined and concentrated using a Centriplus concentrator (Amicon, cutoffM r = 30,000), rebuffered by passing over a Bio-Rad Econo-PacI0 DG column equilibrated with storage buffer (50 mm Tris, pH 7.5, 60 mm KCl, 20 mm MgCl2, 0.1 mm EDTA, and 5 mm DTT) and snap frozen in liquid nitrogen. Aliquots were stored at −80 °C. Chaperonin concentration was determined according to Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216391) Google Scholar) using defatted bovine serum albumin as standard. GroES concentration was measured using the extinction coefficient of 3440m−1 cm−1 for protomers (28Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (317) Google Scholar). Concentrations of GroEL chimeras are given with respect to tetradecamers, the concentrations of mMDH and LDH with respect to monomers. Refolding of mMDH was performed as in Ranson et al. (29Ranson N.A. Duster N.J. Burston S.G. Clarke A.R. J. Mol. Biol. 1995; 250: 581-586Crossref PubMed Scopus (134) Google Scholar) with minor modifications. 150 μm mMDH was denatured in 3 m guanidine hydrochloride containing 20 mm DTT at 20 °C for 2 h. Refolding was initiated by dilution into refolding buffer (50 mm triethylammonium, pH 7.5, 50 mm KCl, 20 mm MgCl2, 5 mm DTT) to a final concentration of 150 nm. If refolding was measured in the presence of cofactors, the final concentrations were 150 nmchaperonin oligomer (unless otherwise indicated), 2 mm ATP, and a 4-fold excess GroES oligomer to GroEL oligomer. Chaperonins, GroES, and ATP were added to the standard buffer directly before initiating refolding. To measure refolding, aliquots were removed from the refolding mixture and mMDH activity was determined by adding them to a solution containing 0.2 mm NADH and 1 mmketomalonate. Consumption of NADH by mMDH was assayed by observing the decrease in absorption at 340 nm over 120 s in a Shimadzu UV1601 spectrophotometer. All assays were carried out at 30 °C. Porcine LDH refolding was measured as described (30Clarke A.R. Waldman A.D.B. Munro I. Holbrook J.J. Biochim. Biophys. Acta. 1985; 828: 375-379Crossref PubMed Scopus (44) Google Scholar) with minor modifications. LDH was denatured in 6 m guanidine hydrochloride (pH 3.2) for 15 min at room temperature. If not indicated otherwise, renaturation was initiated by dilution into buffer (0.1 mNaH2PO4, pH 7, 1 mm EDTA, 1 mm DTT, 10 mm KCl, and 10 mmMgCl2) to a final concentration of 150 nm LDH. If LDH refolding was measured in the presence of chaperonins and ATP the final concentrations of chaperonin and ATP were 150 nm(if not indicated otherwise) and 2 mm, respectively. Chaperonins and ATP were added to the buffer directly before the refolding was initiated. Aliquots were taken and added to a solution of 0.2 mm NADH and 2.25 mm pyruvate in 0.1m phosphate buffer, pH 7. NADH oxidation by active LDH was observed by monitoring the decrease in absorption at 340 nm over 120 s in a Shimadzu UV16010 spectrophotometer. The ATPase activity of GroEL and the chimeric proteins were measured as described by Horovitz et al.(31Horovitz A. Bochkareva E.S. Kovalenko O. Girshovich A.S. J. Mol. Biol. 1993; 231: 58-64Crossref PubMed Scopus (58) Google Scholar). Initial velocity of ATP hydrolysis as a function of the ATP concentration by wild type GroEL and Cpn60-1 was fitted to Equation 1 derived by Yifrach and Horovitz (13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (265) Google Scholar) for a three-state nested allosteric model, VO=[0.5Vmax(1)L1([S]/KR)(1+[S]/KR)N1+Vmax(2)L1L2([S]/KR)(1+[S]/KR)2N1]/[1+L1(1+[S]/KR)N+L1L2(1+[S]/KR)2N]Equation 1 where L 1 and L 2are the apparent allosteric constants for the transitions TT→ TR and TR → R′R′,K R is the dissociation constant of ATP for rings in the R form, and V 1 andV 2 are the maximal initial velocities of theTR- and R′R′-states. [S] is the ATP concentration. Cooperativity in ATP hydrolysis by P211 at high protein concentration (150 nm) and by P211T101R were analyzed by directly fitting initial ATPase velocity to the simplified nested cooperativiy equation below (12Yifrach O. Horovitz A. J. Mol. Boil. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar) assuming that the TT-state can be ignored, VO=[0.5Vmax(1)[S]+Vmax(2)L2[S](1+[S]/KR)N]/[KR+KRL2(1+[S]/KR)N+1]Equation 2 with the same parameters as before. Cooperativity in ATP hydrolysis by P211 at low enzyme concentration (50 nm) was analyzed by directly fitting initial ATPase velocity to the Hill equation, VO=VmaxK[S]n/(1+K[S]n)Equation 3 where V o and V max are the initial and maximal initial ATPase velocity, [S] is the ATP concentration, K is the apparent ATP binding constant, andn is the Hill coefficient. We have used homologous recombination in vivo to generate chimeric GroEL proteins between the GroEL protein of E. coli and the GroEL homologue Cpn60-1 from the root nodulating bacterium Rhizobium leguminosarum. One such chimeric chaperone, referred to as P211, contains the amino acid sequence from GroEL from position 1 to 364, and the sequence from Cpn60-1 from position 365 to 547. As we reported earlier (24Jones S. Wallington E.J. George R. Lund P.A. J. Mol. Biol. 1998; 282: 789-800Crossref PubMed Scopus (7) Google Scholar), purified P211 protein migrates on native gradient PAGE gels with an apparent molecular mass of 380 kDa (by comparison with native molecular mass markers), unlike GroEL or Cpn60-1, which run with an apparent molecular mass of approximately 800 kDa (Fig.1). This is consistent with P211 running as a single ring. We also purified the mutant P211T101R, which is identical with P211 but with an arginine replacing threonine at position 101 (24Jones S. Wallington E.J. George R. Lund P.A. J. Mol. Biol. 1998; 282: 789-800Crossref PubMed Scopus (7) Google Scholar). This amino acid is crucial for the formation of a double ring structure in Cpn60-1 and re-establishes a double ring structure in the chimera (24Jones S. Wallington E.J. George R. Lund P.A. J. Mol. Biol. 1998; 282: 789-800Crossref PubMed Scopus (7) Google Scholar). Consequently purified P211T101R runs on native gels with the same molecular mass as wild type GroEL (Fig.1) To investigate the properties of the chimeric proteins in vivo, we deleted the chromosomal groEL gene from strains where P211, P211T101R, or GroEL were expressed on plasmids from the pBAD promoter, which is activated in the presence of arabinose by the AraC protein (32Guzman A. Beli D. Carson M.J. Bechwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3959) Google Scholar). The strains expressing P211 and P211T101R grew in liquid media in the presence of arabinose at both 37 and 43 °C (Ref. 24Jones S. Wallington E.J. George R. Lund P.A. J. Mol. Biol. 1998; 282: 789-800Crossref PubMed Scopus (7) Google Scholar and data not shown). However, at 43 °C strains expressing P211 grew at approximately half the rate of those expressing P211T101R or wild type GroEL. We carried out further studies monitoring growth of the strains on solid media at different concentrations of inducer. The results (Table I) show that at lower arabinose concentrations (< 0.01%) the growth of the groELdeletion strain expressing P211 (AI90/pBAD211) is reduced and at an arabinose concentration of 0.005% growth is completely abolished, in contrast to the same strains expressing GroEL (AI90/pBAD50) or P211T101R (AI90/pBAD211T101R), which still grow under these conditions. These results indicate that cells require a higher concentration of P211 than they do of GroEL or P211T101R for growth, even under non-heat shock conditions. We also examined the ability of the strains to plate the GroEL-dependent bacteriophage λ using strain SF103 (carrying the groELts44 allele) expressing GroEL, P211, or P211T101R from the groE promoter, together with GroES, on derivatives of the plasmid pMa5.8 (33Stanssens P. Opsomer C. McKeown Y.M. Kramer W. Zabeau M. Fritz H.-J. Nucleic Acids Res. 1989; 17: 4441-4454Crossref PubMed Scopus (260) Google Scholar). Bacteriophage λ plated on SF103/pMa211 with an efficiency of 0.16 relative to the wild type control SF103/pMa50, whereas λ plated on SF103/pma211T101R with approximately the same efficiency as the wild type control. From these in vivo results we conclude that P211 can function in vivo, but is somewhat less effective than wild type E. coli GroEL protein or P211T101R.Table IComparison of the abilities of GroEL and the chimeric chaperones to support growth in E. coliStrain/plasmidCell growth at 37 °C in the presence of arabinose at0.2 %0.02 %0.01 %0.005 %AI90/pBAD501111AI90/pBAD21 T101R0.890.830.950.73AI90/pBAD2110.850.600.08<0.01Overnight cultures were diluted and plated on different combinations of arabinose as described under "Materials and Methods," and the number of colonies scored after overnight incubation. Figures are expressed as a proportion of the number of colonies of AI90/pBAD50 at 0.2% arabinose. Open table in a new tab Overnight cultures were diluted and plated on different combinations of arabinose as described under "Materials and Methods," and the number of colonies scored after overnight incubation. Figures are expressed as a proportion of the number of colonies of AI90/pBAD50 at 0.2% arabinose. The results from the in vivoexperiments led us to ask whether the P211 chimera is indeed able to act as a single ring, or whether double rings are required for chaperone activity despite its appearance as a single ring on native gels. We used analysis of the kinetics of ATP hydrolysis by P211 and P211T101R to probe for possible inter-ring communication in the proteins under the same conditions as those used for carrying out protein refolding assays. The initial velocity of ATP hydrolysis by wild type GroEL as a function of the ATP concentration shows a very characteristic curve (Fig.2 a, inset). Two transitions are observed in this curve, which correspond to the two levels of cooperativity, one between subunits in the same ring and the second between the two rings (12Yifrach O. Horovitz A. J. Mol. Boil. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar, 13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (265) Google Scholar, 14Yifrach O. Horovitz A. Biochemistry. 1998; 37: 7083-7088Crossref PubMed Scopus (62) Google Scholar). A very similar curve was seen for Cpn60-1. 2R. George and P. A. Lund, unpublished data. Initial rates of ATP hydrolysis by P211 at a concentration of 50 nm with respect to tetradecamer were measured over a range of ATP concentrations, from 0 to 0.8 mm (Fig. 2 A). In contrast to GroEL, only a simple sigmoid curve was seen, and no indication of a second transition was detected even when ATP hydrolysis was measured at 2.5 mMATP (data not shown). The data were directly fitted to the Hill equation. The value of the Hill coefficient for the allosteric transition was found to be 2.04 (± 0.134) indicating positive cooperativity between the seven subunits in a single ring. Thus, at 50 nm P211, no indication of any ring-ring interaction was detectable indicating that P211 exists predominately in a single ring form at this concentration. Dramatically different results were obtained if the initial velocity of ATP hydrolysis was measured using 150 nm P211 over an ATP concentration range from 0 to 2.5 mm. The resulting curve (Fig. 2 B) has two transition points, indicating two levels of cooperativity, even though the overall shape of the curve is very different from the curve for GroEL. The reappearance of a double transition indicates that at high protein concentrations the double ring structure of P211 is restored and allosteric communication between the rings can take place. These findings suggest strongly that P211 exits in equilibrium between a single and a double ring structure, with the double ring form predominating at higher protein concentrations. The data were then fitted to Equation 2. The allosteric transition constant L 2 for the TR to theR′R′-state for P211 was found to be 2 (± 0.5) × 10−5. This is significantly higher than for GroEL whereL 2 is found to be 6 (± 3.2) × 10−9; this indicates weaker inter-ring communication in the P211 double ring structure compared with GroEL. The data for ATP hydrolysis by P211 (150 nm) at ATP concentrations from 0 to 600 nm were fitted to the Hill equation. The Hill coefficient was 1.21 (± 0.141) in contrast to a Hill coefficient of 2.04 found for the single transition observed at 50 nmP211. Thus the formation of the double ring structure not only restores inter-ring communication but also leads to a decrease of the cooperativity between the seven subunits of each ring. The values ofk cat for the TR andR′R′-state obtained from this fit and calculated for 7 and 14 sites, respectively, are 0.0298 (± 0.0015) s−1 and 0.0224 (± 0.0012) s−1. These differ from thek cat values for GroEL which we found to be 0.141 (± 0.0024) s−1 for the TR and 0.016 (± 0.00056) s−1 for the R′R′-state, in good agreement with published data (13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (265) Google Scholar). k cat for theTR-state of P211 is significantly lower than for GroEL and of the same order of magnitude as k cat for theR′R′-state, which accounts for the different overall shape of the curve. For comparison, the initial rate of ATP hydrolysis by the P211T101R protein was measured using 25 nm protein and ATP concentrations from 0 to 800 nm. This protein is expected to have a stable double ring structure, on the basis of its behavior on native gels. The resulting curve (Fig. 3) shows two transitions, indicating that P211T101R has a double ring structure even at this low protein concentration. The overall shape of the curve is very similar to that seen for P211 when this protein is in the double ring form. By fitting the data to Equation 2,L 2 was estimated to be 3.1(± 1.1) × 10−5. As for P211, this is significantly higher than for GroEL, indicating that the cooperativity between the rings in P211T101R is weaker than in GroEL. The values of k cat for the TR and R′R′-state obtained from this fit and calculated for 7 and 14 sites, respec
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