We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain α-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161–421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HC1 (Gdn-HCl). The denatured E2c monomers (apparent Mr = 27 000) were diluted 100-fold at 25 °C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperoninto-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn HCl at 25 °C. The E2c trimers with an apparent Mr of 84 000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins. Our results indicate that in vitro refolding of bovine E2c is a chaperonin-mediated process and that spontaneous assembly of the 24-meric structure of E2 proceeds through active trimeric intermediates.
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