enzyme complexes incorporated with b+153-156 displayed a specific activity similar to the

wild type strain.

 An indication of coupled activity was shown by F1Fo ATP synthase-mediated ATP-

driven proton pumping activity in membrane vesicles prepared from the mutants (data not

shown, work accomplished by Stephanie Cole). Membranes containing the bAl534end

subunit exhibited no coupled activity as expected. Membranes isolated from cells

expressing pTAM52 (b+153-156) exhibited coupled activity comparable to the wild type

strain. The levels ofNADH-driven fluorescence quenching were strong and comparable

in every case (data not shown). The coupled activities observed reflected the

observations from the F1-ATP hydrolysis activities (Table 5-2).

 Discussion

 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). At the amino terminal

end of the b subunit, Andrew Hardy conducted a systematic mutational analysis of the

membrane-spanning domain. 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). Here, an alanine scan was

performed on three amino acids that had been shown to exhibit the strongest crosslinking

efficiency in the membrane-spanning domain (basn24ala, thr64ala, glnl04ala), yielding a

defecting yet intact F1Fo ATP synthase complex (202). It appears that there were

adequate b-b interactions to support assembly of the enzyme, but the proton channel of Fo