A mutation in which alanine 128 is replaced by aspartic acid abolishes dimerization of the b-subunit of the F0F1-ATPase from Escherichia coli

Abstract

Site-directed mutagenesis was used to investigate the roles of a short series of hydrophobic amino acids in the b-subunit of the Escherichia coli F0F1-ATPase. A mutation affecting one of these, G131D, had been previously characterized and was found to interrupt assembly of the F0F1-ATPase (Jans, D. A., Hatch, L., Fimmel, A. L., Gibson, D., and Cox, G. B. (1985) J. Bacteriol. 162, 420426). To extend this work, aspartic acid was substituted for each one of the residues from positions 124 to 132. The properties of mutants in this series are consistent with the region from Va1124 to Gly131 forming an α-helix. Two of the mutations, VI24D and A128D, resulted in a similar phenotype to the G131D mutation. This suggested that Va1124, Ala128, and Gly131 form a helical face which may have a role in inter- or intrasubunit interactions. This was tested by overexpressing and purifying the cytoplasmic domains of the wild type and A128D mutant b- subunits. Sedimentation equilibrium centrifugation indicated that the wild type domain formed a dimer whereas the mutant was present as a monomer. Show more