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Chaperonin-mediated Folding of Green Fluorescent Protein

1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês

10.1074/jbc.272.19.12468

1083-351X

Yoshihide Makino, Kei Amada, Hideki Taguchi, Masasuke Yoshida,

Enzyme Structure and Function

Chaperonin-mediated folding of green fluorescent protein (GFP) was examined by real-time monitoring of recovery of fluorescence and by gel filtration high-performance liquid chromatography. Acid-denatured GFP can fold spontaneously upon dilution into the neutral buffer. When Escherichia coli GroEL/ES was present, folding of GFP was arrested. Folding was resumed by subsequent addition of 100 μm or 1 mm ATP, and native GFP was regenerated to 100% yield. When folding was resumed by 10 μM ATP (1.4 mol/mol GroEL subunit), about 60% of GFP recovered native structure, and one-half of them (30%) was found to be still bound to GroEL/ES, indicating the occurrence of folding in the central cavity of the GroEL ring underneath GroES (cis-folding). Because the overall rates of GroEL/ES-, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded thatcis-folding proceeded as fast as spontaneous folding. The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the folding mixture. Holo-chaperonin fromThermus thermophilus, which was purified as a cpn60/10 complex, exhibited the similar cis-folding. Consistently, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed. Chaperonin-mediated folding of green fluorescent protein (GFP) was examined by real-time monitoring of recovery of fluorescence and by gel filtration high-performance liquid chromatography. Acid-denatured GFP can fold spontaneously upon dilution into the neutral buffer. When Escherichia coli GroEL/ES was present, folding of GFP was arrested. Folding was resumed by subsequent addition of 100 μm or 1 mm ATP, and native GFP was regenerated to 100% yield. When folding was resumed by 10 μM ATP (1.4 mol/mol GroEL subunit), about 60% of GFP recovered native structure, and one-half of them (30%) was found to be still bound to GroEL/ES, indicating the occurrence of folding in the central cavity of the GroEL ring underneath GroES (cis-folding). Because the overall rates of GroEL/ES-, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded thatcis-folding proceeded as fast as spontaneous folding. The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the folding mixture. Holo-chaperonin fromThermus thermophilus, which was purified as a cpn60/10 complex, exhibited the similar cis-folding. Consistently, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed. Members of the chaperonin family play an essential role in facilitating folding in the cytosol of both prokaryotes and eukaryotes (1Ellis R.J. Hemmingsen S.M. Trends Biol. Sci. 1989; 14: 339-342Abstract Full Text PDF PubMed Scopus (363) Google Scholar, 2Clarke A.R. Curr. Opin. Struct. Biol. 1996; 6: 43-50Crossref PubMed Scopus (76) Google Scholar, 3Lorimer G.H. FASEB J. 1996; 10: 5-9Crossref PubMed Scopus (196) Google Scholar, 4Buchner J. FASEB J. 1996; 10: 10-19Crossref PubMed Scopus (380) Google Scholar, 5Ellis R.J. Hartl F.U. FASEB J. 1996; 10: 20-26Crossref PubMed Scopus (210) Google Scholar, 6Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3090) Google Scholar). The best studied chaperonin is Escherichia coliGroEL. GroEL is composed of 57-kDa subunits arranged in two seven-membered rings stacked back to back, forming a central cavity ∼45 Å in diameter (7Braig 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, 8Braig K. Adams P.D. Brünger A.T. Nat. Struct. Biol. 1995; 2: 1083-1094Crossref PubMed Scopus (229) Google Scholar, 9Boisvert D.C. Wang J. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar). GroEL binds a variety of substrate polypeptides in nonnative form, and the addition of ATP is sufficient to allow the folding of some proteins in vitro. However, GroEL-mediated folding is dependent on the cochaperonin GroES in many cases, especially under the conditions where only very poor spontaneous folding can occur (10Schmidt M. Buchner J. Todd M.J. Lorimer G.H. Viitanen P.V. J. Biol. Chem. 1994; 269: 10304-10311Abstract Full Text PDF PubMed Google Scholar). GroES is a dome-shaped seven-membered ring of 10-kDa subunits (11Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (397) Google Scholar) that can bind to one or both ends of the GroEL cylinder.Binding of substrate polypeptide occurs exclusively to the GroEL ring not occupied by GroES, as observed by electron micrograph (trans-complex) (12Ishii N. Taguchi H. Sasabe H. Yoshida M. J. Mol. Biol. 1994; 236: 691-696Crossref PubMed Scopus (42) Google Scholar, 13Chen S. Roseman A.M. Hunter A.S. Wood S.P. Burston S.G. Ranson N.A. Clarke A.R. Saibil H.R. Nature. 1994; 371: 261-264Crossref PubMed Scopus (324) Google Scholar). On addition of ATP, either polypeptide or GroES is released. When polypeptide is released, it rebinds to the trans GroEL ring to regenerate trans-complex, or it completes folding by itself in the medium if conditions are suitable for spontaneous folding. When GroES is released, it rebinds to either one of two GroEL rings. If it binds to the GroEL ring not containing polypeptide, trans-complex is regenerated. If it binds to the GroEL ring containing polypeptide (cis-complex), it sequesters polypeptide in the central cavity, and productive folding can proceed there (cis-folding). ATP acts as a set timer (∼15 s) to induce dissociation of the GroES from the GroEL ring, and the substrate protein is released into the medium. How much of the fraction of the substrate protein in the cis-complex has acquired the native conformations before the release differs from one protein to another (14Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (424) Google Scholar, 15Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 16Taguchi H. Yoshida M. FEBS Lett. 1995; 359: 195-198Crossref PubMed Scopus (34) Google Scholar, 17Weissman 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 (387) Google Scholar, 18Mayhew M. da-Silva A.C. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (342) Google Scholar, 19Weissman 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 (356) Google Scholar).Green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; AMP-PNP, 5′-adenylyl imidodiphosphate; BSA, bovine serum albumin: HPLC, high-performance liquid chromatography. 1The abbreviations used are: GFP, green fluorescent protein; AMP-PNP, 5′-adenylyl imidodiphosphate; BSA, bovine serum albumin: HPLC, high-performance liquid chromatography. from the jellyfish Aequorea victoria is a monomeric 238-residue protein that emits 508-nm fluorescent light by excitation light at 395 nm (20Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1193) Google Scholar). The fluorophore results from autocatalytic cyclization of the polypeptide backbone between residues Ser65 and Gly67 and oxidation of the α-β bond of Tyr66 (20Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1193) Google Scholar, 21Ormö M. Cubitt A.B. Kallio K. Gross L.A. Tsien R.Y. Remington S.J. Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1902) Google Scholar, 22Heim R. Prasher D.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12501-12504Crossref PubMed Scopus (1476) Google Scholar, 23Yang F. Moss L.G. Phillips G.N.J. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1286) Google Scholar). Once formed, covalent structure of the fluorophore is stable. Denaturation of GFP by acid, base, or guanidine HCl results in loss of fluorescence, but the denatured GFP restores fluorescence after the shift of pH to neutral or dilution of guanidine HCl (24Bokman S.H. Ward W.W. Biochem. Biophys. Res. Commun. 1981; 101: 1372-1380Crossref PubMed Scopus (263) Google Scholar, 25Ward W.W. Bokman S.H. Biochemistry. 1982; 21: 4535-4540Crossref PubMed Scopus (309) Google Scholar). Structural bases of necessity of native protein structure for fluorescence has been provided from the recently reported crystal structures of GFPs (21Ormö M. Cubitt A.B. Kallio K. Gross L.A. Tsien R.Y. Remington S.J. Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1902) Google Scholar, 23Yang F. Moss L.G. Phillips G.N.J. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1286) Google Scholar) in which the fluorophore interacts with many residues distant in the primary sequence. Therefore, GFP has the advantage for the study of protein folding, that is, one can readily monitor the folding in real time using fluorescence as a marker of recovery of native structure. Indeed, Weissman et al. (19Weissman 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 (356) Google Scholar) used GFP as a substrate protein of the GroEL/ES-mediated protein folding and presented a solid support for the cis-folding; GFP recovered the fluorescence while it remained bound to GroEL/ES.Although cis-folding has been established as a major pathway of chaperonin-mediated protein folding, some of its important characteristics remain unclear. Is the microscopic folding process of substrate proteins in cis-folding the same as that of spontaneous folding, or does it include a different process? Is the rate of cis-folding slower, the same, or faster than spontaneous folding? Does only ATP drive cis-folding, or does ADP also do it as reported (18Mayhew M. da-Silva A.C. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (342) Google Scholar)? It has been known that chaperonin of a thermophilic bacterium, Thermus thermophilus, is purified as an apparently stable complex (holo-chaperonin) made up of chaperonin60 (cpn60, a GroEL homolog) and chaperonin10 (cpn10, a GroES homolog) (26Taguchi H. Konishi J. Ishii N. Yoshida M. J. Biol. Chem. 1991; 266: 22411-22418Abstract Full Text PDF PubMed Google Scholar, 35Ishii N. Taguchi H. Sumi M. Yoshida M. FEBS Lett. 1992; 299: 169-174Crossref PubMed Scopus (56) Google Scholar). Thermus holo-chaperonin is corresponding to E. coli GroEL/ES asymmetric complex. Forcis-folding to occur, cpn10 should be released from one cpn60 ring and rebind to the cpn60 ring of the opposite side where substrate polypeptide is already bound. Can Thermusholo-chaperonin mediate cis-folding, and does the release-rebinding of cpn10 really happen? Here, taking advantage of GFP as a substrate protein, we have tried to answer some of these questions. Members of the chaperonin family play an essential role in facilitating folding in the cytosol of both prokaryotes and eukaryotes (1Ellis R.J. Hemmingsen S.M. Trends Biol. Sci. 1989; 14: 339-342Abstract Full Text PDF PubMed Scopus (363) Google Scholar, 2Clarke A.R. Curr. Opin. Struct. Biol. 1996; 6: 43-50Crossref PubMed Scopus (76) Google Scholar, 3Lorimer G.H. FASEB J. 1996; 10: 5-9Crossref PubMed Scopus (196) Google Scholar, 4Buchner J. FASEB J. 1996; 10: 10-19Crossref PubMed Scopus (380) Google Scholar, 5Ellis R.J. Hartl F.U. FASEB J. 1996; 10: 20-26Crossref PubMed Scopus (210) Google Scholar, 6Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3090) Google Scholar). The best studied chaperonin is Escherichia coliGroEL. GroEL is composed of 57-kDa subunits arranged in two seven-membered rings stacked back to back, forming a central cavity ∼45 Å in diameter (7Braig 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, 8Braig K. Adams P.D. Brünger A.T. Nat. Struct. Biol. 1995; 2: 1083-1094Crossref PubMed Scopus (229) Google Scholar, 9Boisvert D.C. Wang J. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar). GroEL binds a variety of substrate polypeptides in nonnative form, and the addition of ATP is sufficient to allow the folding of some proteins in vitro. However, GroEL-mediated folding is dependent on the cochaperonin GroES in many cases, especially under the conditions where only very poor spontaneous folding can occur (10Schmidt M. Buchner J. Todd M.J. Lorimer G.H. Viitanen P.V. J. Biol. Chem. 1994; 269: 10304-10311Abstract Full Text PDF PubMed Google Scholar). GroES is a dome-shaped seven-membered ring of 10-kDa subunits (11Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (397) Google Scholar) that can bind to one or both ends of the GroEL cylinder. Binding of substrate polypeptide occurs exclusively to the GroEL ring not occupied by GroES, as observed by electron micrograph (trans-complex) (12Ishii N. Taguchi H. Sasabe H. Yoshida M. J. Mol. Biol. 1994; 236: 691-696Crossref PubMed Scopus (42) Google Scholar, 13Chen S. Roseman A.M. Hunter A.S. Wood S.P. Burston S.G. Ranson N.A. Clarke A.R. Saibil H.R. Nature. 1994; 371: 261-264Crossref PubMed Scopus (324) Google Scholar). On addition of ATP, either polypeptide or GroES is released. When polypeptide is released, it rebinds to the trans GroEL ring to regenerate trans-complex, or it completes folding by itself in the medium if conditions are suitable for spontaneous folding. When GroES is released, it rebinds to either one of two GroEL rings. If it binds to the GroEL ring not containing polypeptide, trans-complex is regenerated. If it binds to the GroEL ring containing polypeptide (cis-complex), it sequesters polypeptide in the central cavity, and productive folding can proceed there (cis-folding). ATP acts as a set timer (∼15 s) to induce dissociation of the GroES from the GroEL ring, and the substrate protein is released into the medium. How much of the fraction of the substrate protein in the cis-complex has acquired the native conformations before the release differs from one protein to another (14Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (424) Google Scholar, 15Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 16Taguchi H. Yoshida M. FEBS Lett. 1995; 359: 195-198Crossref PubMed Scopus (34) Google Scholar, 17Weissman 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 (387) Google Scholar, 18Mayhew M. da-Silva A.C. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (342) Google Scholar, 19Weissman 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 (356) Google Scholar). Green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; AMP-PNP, 5′-adenylyl imidodiphosphate; BSA, bovine serum albumin: HPLC, high-performance liquid chromatography. 1The abbreviations used are: GFP, green fluorescent protein; AMP-PNP, 5′-adenylyl imidodiphosphate; BSA, bovine serum albumin: HPLC, high-performance liquid chromatography. from the jellyfish Aequorea victoria is a monomeric 238-residue protein that emits 508-nm fluorescent light by excitation light at 395 nm (20Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1193) Google Scholar). The fluorophore results from autocatalytic cyclization of the polypeptide backbone between residues Ser65 and Gly67 and oxidation of the α-β bond of Tyr66 (20Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1193) Google Scholar, 21Ormö M. Cubitt A.B. Kallio K. Gross L.A. Tsien R.Y. Remington S.J. Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1902) Google Scholar, 22Heim R. Prasher D.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12501-12504Crossref PubMed Scopus (1476) Google Scholar, 23Yang F. Moss L.G. Phillips G.N.J. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1286) Google Scholar). Once formed, covalent structure of the fluorophore is stable. Denaturation of GFP by acid, base, or guanidine HCl results in loss of fluorescence, but the denatured GFP restores fluorescence after the shift of pH to neutral or dilution of guanidine HCl (24Bokman S.H. Ward W.W. Biochem. Biophys. Res. Commun. 1981; 101: 1372-1380Crossref PubMed Scopus (263) Google Scholar, 25Ward W.W. Bokman S.H. Biochemistry. 1982; 21: 4535-4540Crossref PubMed Scopus (309) Google Scholar). Structural bases of necessity of native protein structure for fluorescence has been provided from the recently reported crystal structures of GFPs (21Ormö M. Cubitt A.B. Kallio K. Gross L.A. Tsien R.Y. Remington S.J. Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1902) Google Scholar, 23Yang F. Moss L.G. Phillips G.N.J. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1286) Google Scholar) in which the fluorophore interacts with many residues distant in the primary sequence. Therefore, GFP has the advantage for the study of protein folding, that is, one can readily monitor the folding in real time using fluorescence as a marker of recovery of native structure. Indeed, Weissman et al. (19Weissman 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 (356) Google Scholar) used GFP as a substrate protein of the GroEL/ES-mediated protein folding and presented a solid support for the cis-folding; GFP recovered the fluorescence while it remained bound to GroEL/ES. Although cis-folding has been established as a major pathway of chaperonin-mediated protein folding, some of its important characteristics remain unclear. Is the microscopic folding process of substrate proteins in cis-folding the same as that of spontaneous folding, or does it include a different process? Is the rate of cis-folding slower, the same, or faster than spontaneous folding? Does only ATP drive cis-folding, or does ADP also do it as reported (18Mayhew M. da-Silva A.C. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (342) Google Scholar)? It has been known that chaperonin of a thermophilic bacterium, Thermus thermophilus, is purified as an apparently stable complex (holo-chaperonin) made up of chaperonin60 (cpn60, a GroEL homolog) and chaperonin10 (cpn10, a GroES homolog) (26Taguchi H. Konishi J. Ishii N. Yoshida M. J. Biol. Chem. 1991; 266: 22411-22418Abstract Full Text PDF PubMed Google Scholar, 35Ishii N. Taguchi H. Sumi M. Yoshida M. FEBS Lett. 1992; 299: 169-174Crossref PubMed Scopus (56) Google Scholar). Thermus holo-chaperonin is corresponding to E. coli GroEL/ES asymmetric complex. Forcis-folding to occur, cpn10 should be released from one cpn60 ring and rebind to the cpn60 ring of the opposite side where substrate polypeptide is already bound. Can Thermusholo-chaperonin mediate cis-folding, and does the release-rebinding of cpn10 really happen? Here, taking advantage of GFP as a substrate protein, we have tried to answer some of these questions. We acknowledge Dr. Martin Chalfie for the kind gift of GFP expression plasmid TU#58. We are grateful to Drs. Koreaki Ito and Yoshinori Akiyama for providing us with plasmid pKY206. We thank Dr. Eiro Muneyuki for instruction in nucleotide analysis and discussion on the kinetic analysis, and we thank Noriyuki Murai and Chisa Sakikawa for protein purification.