Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 100, Issue 23, Pages 13179-13183Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2234364100
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- NIGMS NIH HHS [R01 GM023105, T32 GM008349, 5T32 GM08349, GM23105, R37 GM023105] Funding Source: Medline
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The role of subunit a in promoting proton translocation and rotary motion in the Escherichia coli F1F0 ATP synthase is poorly understood. In the membrane-bound F-0 sector of the enzyme, H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access pathways have previously been mapped to surfaces of aTMH4. Here we have substituted Cys into the second and fifth TMHs of subunit a and carried out chemical modification with Ag+ and N-ethylmaleimide to define the aqueous accessibility of residues along these helices. Access to cAsp-61 at the center of the membrane may be mediated in part by Ag+-sensitive residues 248, 249, 251, and 252 in aTMH5. From the periplasmic surface, aqueous access to cAsp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in aTMH2. The Ag+-sensitive residues in TMH2,-4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane. In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2-5. The aqueous access pathways at the interior of subunit a may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to cAsp-61 during the H+ transport cycle.
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