COVALENT MODIFICATION OF AN EXPOSED SURFACE TURN ALTERS THE GLOBAL CONFORMATION OF THE BIOTIN CARRIER DOMAIN OF ESCHERICHIA-COLI ACETYL-COACARBOXYLASE

We have studied the apo (unbiotinylated) and hole (biotinylated) forms of BCCP87, an 87-residue COOH-terminal peptide comprising the biotin carrier domain of the biotin carboxyl carrier protein subunit of Esche richia coli acetyl-CoA carboxylase. The apo; protein spontaneously for med disulfide-linked dimers and was modified readily by sulfhydryl rea gents, whereas the hole protein remained monomeric and was unreactive toward sulfhydryl reagents unless a protein denaturant was present, Th ese data indicated that the single cysteine residue of the domain (Cys -116) was much more reactive in the apo form of the protein. Incubatio n of apoBCCP87 with biotin ligase for different times, followed by rea ction with fluorescein-5-maleimide, clearly showed that the loss of Cy s-116 reactivity was the result of modification with biotin, In additi on, reaction of Cys-116 with 5,5'-dithiobis(2-nitrobenzoic acid) showe d that apoBCCP87 denatured at lower urea concentrations than holoBCCP8 7. We also found that apoBCCP87 was at least 10-fold more sensitive th an the hole form to proteolysis by a range of proteases, Identificatio n of the cleavage sites indicated that the differences in protease sen sitivity could not be attributed to shielding of susceptible bonds by the biotin moiety of the hole protein, These data indicate that a conf ormational change accompanies biotinylation of the biotin domain. Thus , modification of a beta-turn protruding from the protein surface resu lts in alteration of the overall structure of this protein domain.