Manipulating the length of the b subunit F1 binding domain in F1F0 ATP synthase from Escherichia coli

The peripheral stalk of F1F0 ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F-0 to the top of the F-1 sector. The stalk serves as the stator necessary for holding F-1 against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F-1. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F1F0 complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F1F0 ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F1F0 complex by a normal b subunit.