DELETIONS IN THE 2ND STALK OF F1F0-ATP SYNTHASE IN ESCHERICHIA-COLI

In Escherichia coli F1F0-ATP synthase, the two b subunits form the sec ond stalk spanning the distance between the membrane F-0 sector and th e bulk of F-1. Current models predict that the stator should be relati vely rigid and engaged in contact with F-1 at fixed points. To test th is hypothesis, we constructed a series of deletion mutations in the un cF(b) gene to remove segments from the middle of the second stalk of t he subunit, Mutants with deletions of 7 amino acids were essentially n ormal, and those with deletions of up to 11 amino acids retained consi derable activity. Membranes prepared from these strains had readily de tectable levels of F-1-ATPase activity and proton pumping activity. Re moval of 12 or more amino acids resulted in loss of oxidative phosphor ylation. Levels of membrane-associated F-1-ATPase dropped precipitousl y for the longer deletions, and immunoblot analysis indicated that red uctions in activity correlated with reduced levels of b subunit in the membranes, Assuming the likely alpha-helical conformation for this ar ea of the b subunit, the 11-amino acid deletion would result in shorte ning the subunit by approximately 16 Angstrom. Since these deletions d id not prevent the b subunit from participating in productive interact ions with F-1, we suggest that the b subunit is not a rigid rodlike st ructure, but has an inherent flexibility compatible with a dynamic rol e in coupling.