Mechanisms for GroEL/GroES-mediated Folding of a Large 86-kDa Fusion Polypeptide in Vitro
1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês
10.1074/jbc.274.15.10405
ISSN1083-351X
AutoresYi‐Shuian Huang, David Chuang,
Tópico(s)Protein Structure and Dynamics
ResumoOur understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the α subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain α-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-α, when co-expressed with the β subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-α)2β2 structure, similar to the α2β2 structure in native E1. Reactivation of MBP-E1 denatured in 8 m urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376m−1 s−1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-α fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the α moiety on the same chain with rate constants of 1.9 × 10−3 s−1 and 2.95 × 10−4 s−1, respectively. This explained the occurrence of an MBP-α·GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformedEscherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the α·GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-α polypeptide (incis) or the opposite unoccupied cavity (intrans). In contrast, the MBP-α·GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES. Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the α subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain α-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-α, when co-expressed with the β subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-α)2β2 structure, similar to the α2β2 structure in native E1. Reactivation of MBP-E1 denatured in 8 m urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376m−1 s−1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-α fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the α moiety on the same chain with rate constants of 1.9 × 10−3 s−1 and 2.95 × 10−4 s−1, respectively. This explained the occurrence of an MBP-α·GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformedEscherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the α·GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-α polypeptide (incis) or the opposite unoccupied cavity (intrans). In contrast, the MBP-α·GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES. heat shock proteins trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid branched-chain α-ketoacid decarboxylase dihydrolipoyl transacylase dihydrolipoyl dehydrogenase maltose-binding protein the fusion polypeptide between MBP and the α subunit of E1 Ni-nitrilotriacetic acid proteinase K phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis Molecular chaperones are a class of proteins which assist folding of other proteins in the cell by preventing or reversing aggregation caused by off-pathway folding reactions (1Ellis R.J. Annu. Rev. Biochem. 1991; 60: 321-347Crossref PubMed Google Scholar, 2Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 3Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3130) Google Scholar). Many of the chaperones are heat shock proteins (Hsp),1 named for their induced synthesis in cells during heat-shock stress. Group I chaperonins (4Ellis J. Nature. 1992; 358: 191-192Crossref PubMed Scopus (45) Google Scholar, 5Kim S. Willison K.R. Horwich A.L. Trends Biochem. Sci. 1994; 19: 543-548Abstract Full Text PDF PubMed Scopus (180) Google Scholar) include GroEL/GroES in bacteria, Hsp60/Hsp10 in eukaryotic mitochondria, and ribulose-P2carboxylase-binding protein/cpn21 in plant chloroplasts (6Ostermann J. Horwich A.L. Neupert W. Hartl F.U. Nature. 1989; 341: 125-130Crossref PubMed Scopus (481) Google Scholar, 7Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulous C.P. Hendrix R.W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (934) Google Scholar). These proteins exhibit remarkable structural and functional conservation from bacteria to plants to humans (8Waldinger D.C. Eckerskorn C. Lottspeich F. Cleve H. Biol. Chem. Hoppe-Seyler. 1988; 369: 1185-1189Crossref PubMed Scopus (24) Google Scholar, 9Hartman D.J. Hoogenraad N.J. Condron R. Hoj P.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3394-3398Crossref PubMed Scopus (105) Google Scholar, 10Legname G. Fossati G. Gromo G. Monzini N. Marcucci F. Modena D. FEBS Lett. 1995; 361: 211-214Crossref PubMed Scopus (16) Google Scholar). The conserved function in the chaperonin family is the basis that bacterial GroEL and GroES are widely used in promoting refolding of various proteins, including those from mitochondria and chloroplasts both in vitro and inEscherichia coli (1Ellis R.J. Annu. Rev. Biochem. 1991; 60: 321-347Crossref PubMed Google Scholar, 2Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 3Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3130) Google Scholar). The biogenesis of eukaryotic mitochondrial matrix proteins is proposed to follow a complex chaperone-mediated pathway (11Stuart R.A. Cyr D.M. Craig E.A. Neupert W. Trends Biochem. Sci. 1994; 19: 87-92Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 12Schwarz E. Neupert W. Biochim. Biophys. Acta. 1994; 1187: 270-274Crossref PubMed Scopus (38) Google Scholar, 13Hartl F.U. Semin. Immunol. 1991; 3: 5-16PubMed Google Scholar). Unfolded subunit polypeptides are imported into mitochondria as aided by Hsp70 family chaperones, and the final stage of folding and assembly of mitochondrial oligomeric proteins is promoted by chaperonins Hsp60/Hsp10 (11Stuart R.A. Cyr D.M. Craig E.A. Neupert W. Trends Biochem. Sci. 1994; 19: 87-92Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 12Schwarz E. Neupert W. Biochim. Biophys. Acta. 1994; 1187: 270-274Crossref PubMed Scopus (38) Google Scholar, 13Hartl F.U. Semin. Immunol. 1991; 3: 5-16PubMed Google Scholar). Our laboratory has previously shown the obligatory role of GroEL and GroES in facilitating folding and assembly of the decarboxylase (E1) and the transacylase (E2) components of human branched-chain α-ketoacid dehydrogenase complex in vitro (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 15Wynn R.M. Davie J.R. Wang Z. Cox R.P. Chuang D.T. Biochemistry. 1994; 33: 8962-8968Crossref PubMed Scopus (14) Google Scholar) and inE. coli (16Wynn R.M. Davie J.R. Cox R.P. Chuang D.T. J. Biol. Chem. 1992; 267: 12400-12403Abstract Full Text PDF PubMed Google Scholar). E1 is a thiamine pyrophosphate-dependent enzyme, consisting of two 45-kDa α and two 38-kDa β subunits (17Yeaman S.J. Biochem. J. 1989; 257: 625-632Crossref PubMed Scopus (262) Google Scholar). In an earlier study (18Chun S.-Y. Strobel S. Bassford Jr., P.J. Randall L.L. J. Biol. Chem. 1993; 268: 20855-20862Abstract Full Text PDF PubMed Google Scholar), we overexpressed MBP-E1 in which the mature sequence of maltose-binding protein (MBP) was fused to the N terminus of the mature α subunit (abbreviated MBP-α). Although MBP was shown to rapidly and efficiently refold in vitro without chaperonins (18Chun S.-Y. Strobel S. Bassford Jr., P.J. Randall L.L. J. Biol. Chem. 1993; 268: 20855-20862Abstract Full Text PDF PubMed Google Scholar), the overexpression of MBP-E1 in E. coli required co-expression of GroEL/GroES (16Wynn R.M. Davie J.R. Cox R.P. Chuang D.T. J. Biol. Chem. 1992; 267: 12400-12403Abstract Full Text PDF PubMed Google Scholar, 19Davie J.R. Wynn R.M. Cox R.P. Chuang D.T. J. Biol. Chem. 1992; 267: 16601-16606Abstract Full Text PDF PubMed Google Scholar). The results have established that fusion of a spontaneously refolded protein to the α sequence from E1 does not change chaperonin-dependent folding characteristics of the latter sequence. The native conformation of a large polypeptide is often folded into several compact regions or domains (20Wetlaufer D.B. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 697-701Crossref PubMed Scopus (708) Google Scholar). Previous studies with spontaneously refolded proteins, e.g. tryptophan synthetase (21Blond S. Goldberg M.E. Proteins. 1986; 1: 247-255Crossref PubMed Scopus (28) Google Scholar, 22Blond-Elguindi S. Goldberg M.E. Biochemistry. 1990; 29: 2409-2417Crossref PubMed Scopus (42) Google Scholar), dihydrofolate reductase (23Frieden C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4413-4416Crossref PubMed Scopus (62) Google Scholar), and aspartokinase-homoserine-dehydrogenase (24Garel J.-R. Dautry-Varsat A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3379-3383Crossref PubMed Scopus (17) Google Scholar, 25Dautry-Varsat A. Garel J.-R. Biochemistry. 1981; 20: 1396-1401Crossref PubMed Scopus (32) Google Scholar), show that individual domains in the entire polypeptide chain refold differentially and can be expressed as soluble functional units, suggesting that the domain alone is a folding unit. Co-translational independent domain folding in eukaryotes has been recently demonstrated using a Ras-dihydrofolate reductase fusion polypeptide (26Netzer W.J. Hartl F.U. Nature. 1997; 388: 343-349Crossref PubMed Scopus (353) Google Scholar). Molecular chaperones do not contain information for specifying correct folding. The information for folding into the functional three-dimensional structure of a protein is solely present in its amino acid sequence (27Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5217) Google Scholar). Thus, one can reasonably expect that independent domain folding also occurs on the GroEL scaffold. The crystal structure of unliganded GroEL double-ring complex has indicated that, in the absence of GroES, a polypeptide of up to ∼35 kDa in size can be accommodated within a single ring of GroEL (28Braig 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, 29Chen 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 (325) Google Scholar). Binding of GroES, however, induces a large conformational change in GroEL, leading to an approximate doubling of the volume in the central cavity of that ring. This allows the accommodation of polypeptides of ∼70 kDa in size (30Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1044) Google Scholar). Because of the size constraint of the GroEL cavity, it has been proposed that GroES promotes the productive release of polypeptides larger than 70 kDa from a transconfiguration, in which GroES and the unfolded polypeptide bind to the opposite rings of GroEL (31Weissman 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). However, evidence for the productivetrans release of large polypeptides from GroEL is lacking. In the present study, we investigate GroEL/GroES-assisted refolding of the large 86-kDa MBP-α fusion polypeptide in vitro. The kinetic data show that the MBP moiety folds appreciably more rapidly than the α subunit. This explains the isolation of an MBP-α·GroEL binary complex from the MBP-E1 refolding mixture and E. colilysates with amylose resin. Moreover, we provide evidence that GroES can only cap the MBP-α·GroEL complex in trans in relation to the fusion polypeptide. This is in contrast to that observed in the α·GroEL complex where GroES binds to GroEL in bothcis and trans configurations. These findings provide a paradigm for independent domain folding during chaperonin-assisted folding of large multidomain polypeptides. CG712 (an E. coli EStsstrain) and the expression plasmid pGroESL overexpressing GroEL and GroES (32Goloubinoff P. Gatenby A.A. Lorimer G.H. Nature. 1989; 337: 44-47Crossref PubMed Scopus (527) Google Scholar) were generous gifts from Drs. George Lorimer and Anthony Gatenby of DuPont Experimental Station (Wilmington, DE). The pH1 plasmid carrying MBP-α and -β sequences, the pHisT-hE1 plasmid harboring (His)6-α and -β sequences, and the pMAL-c-hE1α plasmid carrying MBP-α sequence were previously described (19Davie J.R. Wynn R.M. Cox R.P. Chuang D.T. J. Biol. Chem. 1992; 267: 16601-16606Abstract Full Text PDF PubMed Google Scholar, 33Wynn R.M. Davie J.R. Chuang J.L. Cote C.D. Chuang D.T. J. Biol. Chem. 1998; 273: 13110-13118Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The MBP or (His)6 tag was fused to the N terminus of the mature α sequence through a linker containing the factor Xa or tobacco etch virus protease-specific site, respectively. GroEL and GroES were overexpressed in E. coli and purified as described previously (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Amylose resin was prepared according to a previously reported method (34Ferenci T. Klotz U. FEBS Lett. 1978; 94: 213-217Crossref PubMed Scopus (91) Google Scholar). CG-712 cells co-transformed with pGroESL and the pH1, pMAL-c-hE1α, or pHisT-hE1 plasmids were grown overnight at 37 °C in YTGK media containing 2 × YT medium (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), 10 mmKCl, 1% glycerol, and antibiotics (100 μg/ml ampicillin and 50 μg/ml chloramphenicol). The overnight culture was diluted 6:1000 into 1 liter of YTGK medium with added antibiotics. Cultures were grown on a shaker at 220 rpm and 37 °C to a measuredA 600 of 0.6, and expression was induced with 0.75 mm isopropyl-1-thio-β-d-galactosidase. After induction, cultures were grown at 37 °C for 16 h. The extraction of MBP-E1 and MBP-α from the E. coli lysate with amylose resin was described previously (19Davie J.R. Wynn R.M. Cox R.P. Chuang D.T. J. Biol. Chem. 1992; 267: 16601-16606Abstract Full Text PDF PubMed Google Scholar). The purified proteins were separated on a 10–30% sucrose density gradient. Purification of (His)6-E1 on the Ni-NTA column was also previously described (33Wynn R.M. Davie J.R. Chuang J.L. Cote C.D. Chuang D.T. J. Biol. Chem. 1998; 273: 13110-13118Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To prepare untagged E1, dialyzed (His)6-E1 was digested with the tobacco etch virus protease. Undigested (His)6-E1 was removed by Ni-NTA extraction. MBP-E1, E1, or MBP was incubated for 1 h at 22 °C in a denaturing buffer (50 mm potassium Pi, pH 7.5, 100 mm KCl, 8 m urea, 0.1% Tween 20, and 1 mm dithiothreitol). Refolding was initiated at 22 °C by a 100-fold dilution of the denatured protein into a refolding buffer (50 mm potassium Pi, pH 7.5, 100 mm KCl, 1 mm thiamine pyrophosphate, 5 mm dithiothreitol, and 5 mm MgCl2) containing GroEL/GroES (molar ratio of GroEL:GroES:monomeric substrate = 2:4:1). Unless otherwise specified, the final concentration of the denatured monomer was 0.5 μm. Rapid mixing was accomplished by vortexing of a denatured protein drop into the refolding buffer, which was immediately followed by an addition of 10 mm ATP. At indicated times, an aliquot of 50 μl was withdrawn and frozen at −20 °C until assays for E1 activity. E1 activity was assayed radiochemically in a reconstituted branched-chain α-ketoacid dehydrogenase system with the addition of excess recombinant E2 and E3 components as described elsewhere (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). An identical concentration of nondenatured E1 or MBP-E1 was also added into the separate refolding buffer and incubated at 22 °C to serve as the 100% activity control. The folding efficiency was defined as the percent recovery of activity compared with the 100% activity control at the final time point. Unless otherwise stated, at indicated times, aliquots (1 ml) of the refolding mixture was withdrawn, and the refolding reaction was terminated by the addition of 50 mmCDTA to chelate Mg2+ ions. The refolding mixture was incubated with 0.5 ml of amylose resin for 10 min. After a brief spinning, the supernatant was removed and the resin was washed three times with 1 ml of the refolding buffer. Proteins bound to the resin were eluted twice with 0.5 ml of the refolding buffer containing 20 mm maltose. The eluted protein sample was loaded on a 10–30% sucrose density gradient, and separated by centrifugation at 210,000 × g for 18 h. To determine rate constants for the folding of MBP and α moieties, the data were fit to the first-order rate equation: ln[A] = ln[A]0-kt, where [A] = molar concentration of the reactant,i.e. the MBP or the α moiety at a given time; [A]0 = initial concentration of the reactant;k = rate constant for the folding reaction, andt = the reaction time. To determine rate constants for the reconstitution of E1 activity from denatured E1 or MBP-E1, the data were fit to second-order rate equation kt = 1/[A] − 1/[A]0, where [A] = α, MBP-α or -β monomers at a given time. The percent of E1 activity recovered was used to calculate the amount of E1 or MBP-E1 tetramers formed. The same amount of E1 or MBP-E1 not treated with the denaturant was incubated in the refolding buffer for 24 h, and the enzyme activity served as a 100% refolding control. The percent of folded E1 or MBP-E1 tetramers at different time points was calculated. The concentration of remaining E1 monomers was derived by subtracting a 2-fold concentration of the E1 tetramer formed from the initial concentration of the monomer (α, MBP-α or -β). The assumption was that the α and β subunits participating in the E1 refolding reaction either stayed as monomers or complexed with each other to form heterodimers or heterotetramers according to the assembly pathway: 2 α + 2 β → 2 αβ → α2β2 (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The concentrations of α and β monomers were identical at any time during the refolding. Therefore, V = k [α][β] was treated as V = k[α]2 =k[β]2, where V = velocity. The second-order equation, kt = 1/[α] − 1/[α]0 = 1/[β] − 1/[β]0 was derived after integration. At indicated times of the refolding reaction, a 100-μl aliquot of the refolding mixture containing 0.5 μm MBP was removed. The sample was digested with 4 μg/ml PK for 10 min at 22 °C, and the digestion then quenched with 5 mm PMSF. The same amounts of native MBP-E1 and MBP were also treated with PK and served as a 100% refolding control. The protease-digested samples were precipitated with 7.5% trichloroacetic acid containing 125 μg/ml sodium deoxycholate, and analyzed by SDS-PAGE and Western blotting. MBP-E1 (3 mg) was denatured in 400 μl of the denaturation buffer (50 mm sodium acetate, pH 4.5, and 8 m urea) for 15 min. The denatured protein was incubated with 200 μl of the carboxymethyl-Sepharose CL-6B resin (Pharmacia) equilibrated with the denaturation buffer for 15 min. The resin was washed with the denaturation buffer containing 60 mm KCl. Denatured MBP-α bound to the resin was eluted with the denaturation buffer containing 300 mm KCl. The eluted MBP-α was repeatedly diluted in 50 mm potassium Pi, pH 7.5, containing 100 mm KCl and 8m urea and concentrated in a Microcon-30 concentrator (Amicon). The denatured protein was diluted into 50 mmpotassium Pi, pH 7.5, containing 100 mm KCl and 1:1 molar ratio of GroEL to make 1:1 stoichiometric substrate-GroEL complexes. The mixture was separated on a 10–25% sucrose density gradient and the MBP-α·GroEL complex collected. The preparation of (His)6-α·GroEL and β·GroEL complexes was described (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The refolding of untagged E1 or MBP-E1 denatured in 8 m urea was initiated by dilution of the denatured protein into a refolding buffer at 22 °C containing GroEL/GroES and Mg2+-ATP. Fig. 1 shows the time course for reactivation of untagged E1. No E1 activity was recovered when GroEL, GroES, or Mg2+-ATP was omitted from the refolding mixture. The folding kinetics of E1 at concentrations of 0.125 μmand 0.0625 μm were fit to the second-order equation, with rate constants of 512 and 417 m−1s−1, respectively. The folding of MBP-E1 also showed an absolute requirement for GroEL/GroES and Mg2+-ATP (Fig.2). The kinetic data at 0.2, 0.1, and 0.05 μm denatured MBP-E1 were fit to the second-order equation. This resulted in rate constants of 324, 397, and 407m−1 s−1, respectively, for the reactivation of E1 activity. The average rate constant for untagged E1 was 465 m−1 s−1 and for MBP-E1 was 376 m−1 s−1.Figure 2Time course for chaperonin-mediated reactivation of urea-denatured MBP-E1. The denaturation and reconstitution at 22 °C of the MBP-E1 fusion protein were as described in the legend to Fig. 1. The concentrations of the denatured MBP-E1 tetramer for refolding were 0.2 μm (■), 0.1 μm (⋄), and 0.05 μm (○). The data at all three protein concentrations were fit to the second-order function to produce folding rate constants. Folding efficiencies at 0.2, 0.1, and 0.05 μm denatured MBP-E1 were 67, 99, and 84%, respectively. Δ, GroEL, GroES, or Mg2+-ATP absent.View Large Image Figure ViewerDownload (PPT) The urea-denatured MBP was refolded for 1 h at 22 °C in the absence or presence of GroEL·GroES·Mg2+-ATP. The refolded MBP was digested with PK (4 μg/ml) and separated on a sucrose density gradient, and the fractions were analyzed by SDS-PAGE. The appearance of MBP on the top of the sucrose density gradient either without (panel A) or with (panel B) chaperonins indicated that the refolded MBP was soluble and resistant to PK digestion (Fig.3). The results showed that limited proteolysis with PK was a feasible approach to monitor MBP refolding. The GroEL 14-mer sedimented to the bottom of the gradient (panel B). The presence of GroEL monomers at the top of the gradient resulted from a partial dissociation of the GroEL 14-mer during centrifugation in the presence of Mg2+-ATP (38Mendoza J.A. Demeler B. Horowitz P.M. J. Biol. Chem. 1994; 269: 2447-2451Abstract Full Text PDF PubMed Google Scholar). The refolding kinetics of unfused and fused forms of MBP denatured in 8m urea was studied. Aliquots taken at different times during the refolding at 22 °C were treated with PK, followed by analysis by SDS-PAGE. To increase sensitivity of the folding assay, the PK-resistant MBP moiety was probed with the antibody to MBP by Western blotting. Radioactivity associated with the PK-resistant folded MBP moiety was compared with the intensity of the PK-treated native MBP or MBP-E1 control present at the same level as that used in the refolding reaction. Either control was expressed as 100% folded MBP. As shown in Fig. 4, refolding of unconjugated MBP reached 99.8 and 99.5% of the native MBP control in the absence or presence of GroEL·GroES·Mg2+-ATP, respectively. The plateau levels for refolding of the MBP moiety on the MBP-α fusion polypeptide from the denatured MBP-E1 were 68.3 and 27.2% in the presence or absence of GroEL·GroES·Mg2+-ATP, respectively (Fig. 4). The rate constants for the first-order reaction for MBP folding in the absence or presence of chaperonins were 0.020 and 0.036 s−1, respectively. They were in the same order of magnitude as previously reported value of 0.025 s−1 for spontaneous MBP refolding (39Sparrer H. Lilie H. Buchner J. J. Mol. Biol. 1996; 258: 74-87Crossref PubMed Scopus (63) Google Scholar), as determined by tryptophan fluorescence measurements. The rate constants for refolding of the MBP moiety on MBP-α in the absence or presence of chaperonins are 1.1 × 10−3 and 1.9 × 10−3s−1, respectively (Fig. 4). These values are an order of magnitude lower than those measured with unlinked MBP. Since the folded α subunit does not possess enzyme activity, the refolding of the α moiety on MBP-α was monitored by the release of MBP-α monomers from GroEL and subsequent assembly of the folded α moiety with β to form the (MBP-α)β dimer or the (MBP-α)2β2 tetramer (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Aliquots collected at different times during refolding were separated on a sucrose density gradient, and fractions analyzed by SDS-PAGE and Coomassie Blue staining. The reaction mixture was not extracted with amylose resin so that measurement of the released MBP-α fusion was not dependent on the prefolded MBP moiety. Fractions 3 to 8 corresponding to MBP-α monomers, (MBP-α)β dimers, and (MBP-α)2β2 tetramers were quantified by densitometry scanning (Fig.5 A). As shown in Fig.5 A, at the 0-min time point, all of MBP-α or -β was complexed with GroEL and sedimented close to the bottom of the gradient (fractions 13–15). The bands in fractions 3 and 4 were dissociated GroEL monomers. At the earlier time points (5 min to 1 h), the majority of MBP-α was present as unassembled MBP-α monomers (fractions 3 and 4) and assembled (MBP-α)β dimers (fractions 4–6). At later time points (2 to 24 h), the dimers were gradually converted to (MBP-α)2β2 tetramers, which peaked in fractions 6–8. The identities of these E1 folding intermediates as separated by the sucrose density gradient were confirmed by fast protein liquid chromatography gel filtration (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The densities of all folded MBP-α species (fractions 3–8) were summed and the maximum at the 4-h time point was expressed as 100%. The percent of folded MBP-α species was plotted against the folding time to depict the refolding kinetics of the α moiety. The folding of the α moiety reached a plateau in about 4 h (Fig.5 B), which was much slower than the MBP (15 min) on the same fusion polypeptide (Fig. 4). The rate constant for folding of the α moiety fit to the first-order reaction was 2.95 × 10−4 s−1. This rate constant is independent of assembly with the β subunit, since both unassembled and assembled MBP-α species were counted. To measure the percent formation of dimers and tetramers during the refolding, subtotals of the normalized MBP-α signal in fractions 4–5 (in the dimeric state) and fractions 7–8 (in the tetrameric state) were expressed as percents of the maximal intensity and plotted against the folding time (Fig.5 C). No significant E1 activity was recovered (about 10%) until 2 h when an appreciable amount of tetramers accumulated. The time course for recovery of E1 activity was in approximate parallel with that for the formation of MBP-E1 tetramers (Fig. 5 C). The slight disconcordance between the two rates was the result of two separate experiments. The data show that the tetramer, but not the dimer, is the enzymatically active form, and confirm that the slow process in E1 refolding is the formation of tetramers from dimers (14Chuang J.L. Wynn R.M. Song J.-L. Chuang D.T. J. Biol. Chem. 1999; 274: 10395-10404Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). During the overexpression of the MBP-α fusion polypeptide with GroEL·GroES in E. coli, a significant amount (10 mg/liter) of the MBP-α·GroEL complex (Fig.6, lanes 11–14) and aggregated MBP-α (Fig. 6, lane 15) were isolated with the amylose resin when analyzed by sucrose density gradient centrifugation. The data were consistent with the notion that the MBP moiety was folded and capable of binding to the resin. The α moiety on the MBP-α fusion was apparently unfolded and associated with GroEL, resulting in the formation of the MBP-α·GroEL complex. The MBP-α* polypeptide was a preterminated translation product. The MBP-α·GroEL complex isolated from the E. coli lysate produced enzymaticall
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