incorporated into F1Fo ATP synthase (Figure 5-3). The decrease in coupled activity

paralleled the reduction seen in F1-ATP hydrolysis activity (Table 5-1).

Carboxyl Terminal Mutations

 Since the original insertions and deletions constructed by Dr. Paul Sorgen in the in

the tether region, a collaborative effort has been made by other members of the laboratory

to study the remainder of the b subunit (Figure 5-4). Insertions and deletions constructed

in a hydrophobic region of the b subunit in the Fl binding domain bl24-130, WAS

accomplished by Dr. Deepa Bhatt. Four amino acid duplications, scattered throughout

the dimerization and Fl binding domains, were constructed by an undergraduate in the

laboratory Stephanie Cole and the corresponding deletions are currently underway by

another undergraduate, Megan Greenlee. Here, a four amino acid insertion and deletion

was constructed at the extreme carboxyl terminus of the b subunit.

Construction and growth characteristics of mutants

 The C-terminal region of the b dimer is in direct contact with the extreme C-

terminal end of the 6 subunit (3, 185, 200, 219, 220). Small deletions at the extreme C-

terminal end of the b subunit had been shown to inhibit F1Fo ATP synthase function (206,

288). Two plasmids were generated to study the affects of deleting or duplicating the

final four amino acids of the b subunit. Plasmids pTAM5 1 and pTAM52 were expressed

in the E. coli KM2 (Ab) cell line to express bAl534end or b+153-156, TOSpectively (Table 5-

2). Since E. coli strains lacking functional F1Fo ATP synthase cannot derive energy from

nonfermentable sources, growth on succinate minimal medium was used as an initial

determination of enzyme activity in vivo. As expected, KM2 cells complemented with