Discussion In the present chapter, we have developed an expression system that facilitates production, purification and detection of b subunits of unequal lengths within E. coli. The experiments involved an epitope tag system that allowed us to determine if the different b subunits segregated into homodimers, or alternatively, if a heterodimer of long and short b subunits can be incorporated into an F1Fo ATP synthase complex. The histidine and V5 epitope tags introduced into the b subunits did not appreciably affect enzyme assembly or function. Expression of two different wild-type length b subunits led to three distinct F1Fo ATP synthase complexes in the same cell; 1) a homodimer of histidine-tagged b subunits, 2) a homodimer of V5-tagged b subunits and 3) a heterodimer consisting of a histidine-tagged b and a V5-tagged b subunit. More importantly, three different F1Fo ATP synthase complexes were present even when the b subunits were not of identical length. We observed dimerization of b subunits between b+7-his and both b .vs and ba7~vs. This demonstrates that b subunits that differ in length by at least 14 amino acids can be incorporated into an enzyme complex. Given that the tether domain is likely an co-helix, the difference in length between the b subunits would be approximately 21 A+. Dimerization could occur in two ways. First, assuming that the transmembrane domains were in parallel, the hydrophilic domains could be out of register. Alternatively, we favor a conformation in which both the transmembrane domains and the dimerization domains as defined by Dunn and coworkers (13) exist in parallel. This would require that a section of the tether domain in the longer b subunit be out of contact with the shorter b subunit (Figure 2-14). It is likely that a parallel alignment of the dimerization domain is required for enzyme assembly.