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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)

Cytochrome c/sub 2/ and the detergent-solubilized cytochrome bc/sub 1/ complex, both from Rhodospirillum rubrum, form a tight complex at a low ionic strength that can be isolated by gel permeation chromatography. The dissociation constant of the complex is estimated to be 10/sup -6/ M or less. (/sup 14/C)-labelled and (/sup 3/H)-labelled acetic anhydride were used in the acetylation experiments, and the /sup 3/H//sup 14/C ratio was analyzed. The binding site for the cytochrome bc/sub 1/ complex on cytochrome c/sub 2/ was analyzed by differential acetylation of lysine residues in free and cytochrome bc/sub 1/ complex bound cytochrome c/sub 2/. In bound cytochrome c/sub 2/, three lysine residues at sequence positions 12, 13, and 97 were less reactive toward acetic anhydride. Lys/sup 13/, which is located above the exposed heme edge, was the least reactive, i.e., the most shielded by the cytochrome bc/sub 1/ complex. Correlating this information with the crystal structure of cytochrome c/sub 2/ indicates that the binding site for the cytochrome bc/sub 1/ complex on cytochrome c/sub 2/ involves a surface area above, and probably including, the exposed heme edge. This mode of binding is similar to that observed for horse cytochrome c interacting with the mitochrondrial cytochromemore » bc/sub 1/ complex. A simplified version of the method of differential chemical modification is presented.« less