tailieunhanh - Báo cáo khoa học: Rotary F1-ATPase Is the C-terminus of subunit c fixed or mobile?

F-ATP synthase synthesizes ATP at the expense of ion motive forcebyarotarycouplingmechanism. Acentral shaft, subunitc, functionally connects the ion-driven rotary motor, FO, with the rotary chemical reactor, polarized spectrophotometry we have demonstrated previ-ously the functional rotation of the C-terminala-helical portion ofcin the supposed ‘hydrophobic bearing’ formed by the (ab)3hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between thea-helixofcand subunitadidnot impair enzyme activity | Eur. J. Biochem. 271 3914-3922 2004 FEBS 2004 doi Rotary F1-ATPase Is the C-terminus of subunit c fixed or mobile Martin Miiller Karin Gumbiowski Dmitry A. Cherepanov Stephanie Winkler Wolfgang Junge Siegfried Engelbrecht and Oliver Panke Universitat Osnabríick FB BiologieịChemie Abt. Biophysik OsnabrUck Germany F-ATP synthase synthesizes ATP at the expense of ion motive force by a rotary coupling mechanism. A central shaft subunit c functionally connects the ion-driven rotary motor FO with the rotary chemical reactor F1. Using polarized spectrophotometry we have demonstrated previously the functional rotation of the C-terminal a-helical portion of c in the supposed hydrophobic bearing formed by the ab 3 hexagon. In apparent contradiction with these spectroscopic results an engineered disulfide bridge between the a-helix of c and subunit a did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a functional unwinding of the a-helix to form a swivel joint. Furthermore they suggested a firm clamping of that part of c even without the engineered cross-link . in the wild-type enzyme. Here we rechecked the rotational mobility of the C-terminal portion of c relative to ab 3. Non-fluorescent engineered F1 aP280C cA285C was oxidized to form a nonfluorescent ac heterodimer. In a second mutant containing just the point mutation within a all subunits were labelled with a fluorescent dye. Following disassembly and reassembly of the combined preparations and cystine reduction the enzyme was exposed to ATP or 5 -adenylyl-imidodiphosphate AMP-PNP . After reoxidation we found fluorescent ac dimers in all cases in accordance with rotary motion of the entire c subunit under these conditions. Molecular dynamics simulations covering a time range of nanoseconds therefore do not necessarily account for motional freedom in microseconds. The rotation of c within hours is compatible with the spectroscopically detected .

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