Covalent modification of an exposed surface turn alters the global conformation of the biotin carrier domain of Escherichia coli acetyl-CoA carboxylase.
We have studied the apo (unbiotinylated) and holo (biotinylated) forms of BCCP87, an 87-residue COOH-terminal peptide comprising the biotin carrier domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The apo protein spontaneously formed disulfide-linked dimers and was modified readily by sulfhydryl reagents, whereas the holo protein remained monomeric and was unreactive toward sulfhydryl reagents unless a protein denaturant was present. These data indicated that the single cysteine residue of the domain (Cys-116) was much more reactive in the apo form of the protein. Incubation of apoBCCP87 with biotin ligase for different times, followed by reaction with fluorescein-5-maleimide, clearly showed that the loss of Cys-116 reactivity was the result of modification with biotin. In addition, reaction of Cys-116 with 5,5'-dithiobis(2-nitrobenzoic acid) showed that apoBCCP87 denatured at lower urea concentrations than holoBCCP87. We also found that apoBCCP87 was at least 10-fold more sensitive than the holo form to proteolysis by a range of proteases. Identification of the cleavage sites indicated that the differences in protease sensitivity could not be attributed to shielding of susceptible bonds by the biotin moiety of the holo protein. These data indicate that a conformational change accompanies biotinylation of the biotin domain. Thus, modification of a beta-turn protruding from the protein surface results in alteration of the overall structure of this protein domain.
Chapman-Smith
A
A
Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5005, Australia.
Forbes
B E
BE
Wallace
J C
JC
Cronan
J E
JE
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D019921:Biotinylation; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D054889:Fatty Acid Synthase, Type II; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013499:Surface Properties
D002352:Carrier Proteins; D010446:Peptide Fragments; C108671:biotin carboxyl carrier protein (70-156); D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein