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pubmed:abstractText |
Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag(+) and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag(+) sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag(+) sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd(2+), a membrane-impermeant soft metal ion with properties similar to Ag(+). Cd(2+) inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.
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pubmed:affiliation |
Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706, USA.
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