(215). Second, deletion of the last four amino acids at the C-terminus has been shown to

affect the ability of the b dimer to form a stable interaction with the 6 subunit Fl resulting

in a maj or defect in F1Fo ATP synthase assembly (205, 206). Thirdly, insertions or

deletions constructed in a cytoplasmic region of the b dimer that contains a stretch of

hydrophobic amino acids, b!24-130 (VAILAVA) has been shown to completely affect the

ability of the b subunit to form a stable dimer and insert into the membrane. Here, we

demonstrate functional activity for F1Fo ATP synthase complexes containing a

heterodimeric peripheral stalk, with maj or defects occurring in the different domains of

each b subunit.

 Materials and Methods

 A thorough account of many of the procedures used in Chapter 4 can be found in

previous chapters. Many of the techniques, including recombinant DNA techniques, site

directed mutagenesis, western blotting, as well as assays of protein concentration and

F1Fo ATP synthase activity have been described in detail in Chapter 2. A detailed

description of purification of F1Fo ATP synthases containing b subunit heterodimers to

homogeneity can be found in Chapter 3.

Materials

 Molecular biology enzymes and mutagenic oligonucleotides were obtained from

Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Grand Island, NY), New England

Biolabs (Beverly, MA) and Stratagene (La Jolla, CA). Reagents were obtained from

Sigma (St. Louis, MO), BioRad Laboratories (Hercules, CA) and Fisher Scientific

(Pittsburgh, PA). Plasmid purification kits were acquired from Qiagen Inc. (Valencia,

CA). The anti-rabbit immunoglobulin horseradish peroxidase-linked whole antibody

(from donkey), anti-mouse immunoglobulin horseradish peroxidase-linked whole