Chapter 4: Development of Cysteine Chemical Modifications of Altered b Subunits

 The work described in Chapter 4 was performed in order to examine the nature of

the apparent flexibility of the tether region. It has been shown by Dr. Paul Sorgen that an

eleven amino acid deletion and a fourteen amino acid insertion in the the b subunit

spanning the tether domain and the beginning of the dimerization domain was

accommodated by the enzyme (Figure 4-5). Assuming co-helical structure, this 21 A+

insertion and 16 A+ deletion corresponds to well over a third of the length spanning from

the top of the membrane to Fl, or right under a quarter of the length spanning towards the

top of Fl. In contrast to the previously accepted role of the b subunit as a rigid "stator",

these observations suggested that the role of the b dimer is more of a flexible or elastic

structural feature during rotational catalysis. In Chapter 4, cysteine chemical

modifications were created in the 6 and b subunits to provide reactive thiols groups for

future labeling and measurement studies. A set of twelve unc operon expression

plasmids that encode amino acid substitutions were developed to void the F1Fo ATP

synthase complex of all known reactive thiols as well as generate strategically placed

cysteines. Cysteines were chosen because the thiol side chain is highly reactive and can

be modified by maleimide reagents. The cysteine mutations did not affect enzyme

assembly or function. Plasmids were designed to express a single cysteine at one of two

locations in the 6 subunit, Scys640T 6cysl40, aS well as one position, either above or below

the site of insertion or deletion in the b subunit, boys84 Or boys43 (Figure 4-5). The idea for

establishing a single reactive cysteine in both Fl and Fo is to use them as targets for

labeling with fluorescence compounds (see "Future Directions"). One major limitation

plagued this line of research. F1Fo ATP synthase is incorporated with a homodimer of b