A MUTATION IN WHICH ALANINE-128 IS REPLACED BY ASPARTIC-ACID ABOLISHES DIMERIZATION OF THE B-SUBUNIT OF THE F0F1-ATPASE FROM ESCHERICHIA-COLI

Site-directed mutagenesis was used to investigate the roles of a short , series of hydrophobic amino acids in the b-subunit of the Escherichi a coli F0F1-ATPase. A mutation affecting one of these, G131D, had been previously characterized and was found to interrupt assembly of the F 0F1-ATPase (Jans, D. A., Hatch, L., Fimmel, A. L., Gibson, D., and Cox , G. B. (1985) J. Bacteriol. 162, 420-426). To extend this work, aspar tic acid was substituted for each one of the residues from positions 1 24 to 132. The properties of mutants in this series are consistent wit h the region hom Val(124) to Gly(131) forming an alpha-helix. Two of t he mutations, V124D and A128D, resulted in a similar phenotype to the G131D mutation. This suggested that Val(124), Ala(128), and Gly(131) f orm a helical face which may have a role in inter- or intrasubunit int eractions, This was tested by overexpressing and purifying the cytopla smic domains of the wild type and A128D mutant b-subunits. Sedimentati on equilibrium centrifugation indicated that the wild type domain form ed a dimer whereas the mutant was present as a monomer.