Transmembrane helix interactions and mechanism of gating hydrogen transport in the rotary motor of ATP synthase

This thesis addresses the function and structure of the H+ translocating Fo sector of the rotary ATP Synthase in Escherichia coli. Subunit a of Fo plays a key role in promoting H+ transport and the coupled rotary motion of the subunit c ring in ATP synthase. H+ binding and release occur at Asp61 in the middle of the second transmembrane helix (TMH) of Fo subunit c. H+ are thought to reach Asp61 via aqueous pathways mapping to surfaces of TMHs 2--5 of subunit a based upon the chemical reactivity of Cys substituted into these helices. Chapter 2 describes a rigid domain composed of Ag +-sensitive loop residues clustered together near the surface of the membrane from cross-linking of Cys substitutions introduced into the two domains. We conclude that the Ag+-sensitive regions of loops 1--2 and 3--4 interact in a single domain that packs at the ends of TMHs 3 and 4.;In Chapter 3, I propose an arrangemnt for TMH4 and TMH5 of subunit a and TMH2 of subunit c based upon disulfide cross-link formation between Cys substitutions introduced into the two subunits. I conclude that both aTMH4 and aTMH5 pack in proximity to cTMH2 of the c-ring. Five mutants showed pH dependent cross-linking consistent with aTMH5 changing conformation at lower pHs to facilitate cross-linking. I suggest that the pH dependent conformational change may be related to the proposed role of aTMH5 in gating H+ access from the periplasm to the cAsp61 residue in cTMH2.;The functional importance of the rigid loop-loop domain proposed in Chapter 2 is tested and the cross-linked form of subunit a is determined to accurately reflect the functional state of the protein. The swiveling of TMHs 4 and 5 in subunit a proposed in Chapter 3, which facilitates protonation of the cAsp6l from the periplasm, is shown to occur since cross-linking in this region of subunit a inhibited function of the enzyme. Chapter 4 will conclude that, based on the cross-linking observed between subunit a and a genetically fused c2 dimer, aTMH4 packs in parallel to the c-ring. Other experiments will show that, aTMH5 can be cross-linked to two subunit c monomers simultaneously, an event that could only take place if aTMH5 packs at an angle to the c-ring from the middle of the membrane to the cytoplasmic side.