title: In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex: requirement for chaperonins GroEL and GroES.
abstract: We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain alpha-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 HCl (Gdn.HCl). The denatured E2c monomers (apparent M(r) = 27,000) were diluted 100-fold at 25 degrees 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 chaperonin-to-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 degrees C. The E2c trimers with an apparent M(r) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: R M
authors.initials: RM
authors.lastname: Wynn
authors.forename: J R
authors.initials: JR
authors.lastname: Davie
authors.forename: W
authors.initials: W
authors.lastname: Zhi
authors.forename: R P
authors.initials: RP
authors.lastname: Cox
authors.forename: D T
authors.initials: DT
authors.lastname: Chuang
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D002417:Cattle; D018835:Chaperonin 10; D018834:Chaperonin 60; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry*; D007658:Ketone Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)