The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector.
A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.
Dunn
S D
SD
Department of Biochemistry, University of Western Ontario, London, Canada.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011796:Quinacrine / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D002918:Chymotrypsin; D006180:Proton-Translocating ATPases; D011796:Quinacrine