subunits, hence two b subunits, each with a reactive cysteine. Since only one b subunit

was desired to be labeled, a system necessarily had to be developed to allow purification

of F1Fo ATP synthase complexes with two genetically different b subunits: one cysteine-

less and one with a single cysteine. With the epitope-tag system developed in Chapter 2,

this should not be a problem for future studies.

Chapter 5: Mutagenesis of the Amino and Carboxyl Termini of the b subunit in
F1Fo ATP Synthase

 In a collaborative effort, members of the laboratory have conducted an extensive

mutational study of the entire length of the b subunit (Figure 5-5). The work illustrated

in Chapter 5 was achieved in order to contribute to two other proj ects in the lab. The

chapter focuses on the extreme amino- and carboxyl-termini of the b subunit. At the

amino end, a systematic mutational analysis of the membrane domain was performed by

Andrew Hardy in our laboratory. His work established that there were specific sequence

requirements in the b subunit membrane spanning domain and supported a model in

which the extreme amino-termini of the two b subunits are in close contact with one

another, accounting for most of the important b-b interactions in the membrane domain,

and then flares apart as they cross the membrane (202). Three amino acids that have

been shown to exhibit the strongest crosslinking efficiency in the membrane-spanning

domain were replaced with alanines (basn24ala,thr64ala, glnl04ala), yielding a defecting F1Fo

ATP synthase complex. It appeared that there were adequate b-b interactions to support

assembly of the enzyme, but the proton channel of Fo was not functional, suggesting that

the b subunit membrane spanning domain may have a role in allowing the proton channel

of the a and c subunits to align correctly (202). Work performed in contribution to this

dissertation demonstrated that mutation of only a single amino acid, at positions 2, 6 or