Aqueous Access Channels in Subunit a of Rotary ATP Synthase

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

“…Mutagenesis-A two-step PCR method that utilizes a mutagenic primer and two wild type primers (31) was used to generate the cysteine substitutions in subunit c. The template plasmid, pCMA113, codes the eight structural genes of F 1 F 0 wherein all endogenous cysteines are replaced with alanine or serine and subunit a is modified with a C-terminal His tag (25). Mutant PCR fragments were transferred into pCMA113 between the BsrGI and BssHII (or PpuMI) restriction sites.…”

“…The reactivity of a substituted cysteine to thiolate-directed probes provides an indication of aqueous accessibility because the reactive thiolate species is preferentially formed in an aqueous environment. The aqueous accessibility of the five TMHs in subunit a of E. coli F 0 has been probed using Ag ϩ and NEM (19,(25)(26)(27). The results suggest the presence of an aqueous accessible channel in subunit a in the center of TMHs 2-5 extending from the periplasm to the center of the membrane.…”

“…The first model proposes that the ions bound at Asp-61 exit to the cytoplasm via a half-channel composed at least partially by residues in TMH4 of subunit a (25)(26)(27). Chemical modification studies of Cys-substituted subunit a of E. coli revealed an aqueous accessible surface of TMH4 that includes the essential Arg-210 residue, which extended from the center of the membrane to the cytoplasm, suggesting that the ion exit channel may lie at the a-c interface (19,25). Alternatively studies of the c-ring from the I. tartaricus enzyme indicate that Na ϩ can access Glu-65 in the absence of other F 0 subunits, suggesting an intrinsic channel in subunit c (28,29).…”

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F 1 F 0 ATP synthase gate H + transport to the cytoplasm

“…A second set of Ag + -sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18,19), and Ag + -sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19,20). The Ag + -sensitive substitutions on the periplasmic side of TMHs 2-5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11,13,19,20).…”

“…The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag + and other thiolate-reactive agents (18)(19)(20). Two regions of aqueous access were found with distinctly different properties.…”