Regensburg 2004 – wissenschaftliches Programm
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AKB: Biologische Physik
AKB 50: Poster Session "Biological Physics"
AKB 50.98: Poster
Freitag, 12. März 2004, 10:30–13:00, B
Stepwise rotation of the epsilon-subunit of EFoF1-ATP synthase during ATP synthesis and hydrolysis - a single-molecule FRET approach — •Michael Börsch1, Boris Zimmermann2, Nawid Zarrabi1, Manuel Diez2, and Peter Gräber2 — 13. Physikalisches Institut, Universität Stuttgart — 2Institut für Physikalische Chemie, Universität Freiburg
F-type ATP synthases catalyze the formation of ATP by coupling two rotary motions of subunits within the enzyme. We attached tetramethylrhodamine-maleimide at the rotating epsilon-subunit and Cy5-bismaleimide crosslinking the two b-subunits, and reconstituted the enzymes fully functional into liposomes. Fluorescence resonance energy transfer (FRET) was monitored in photon bursts of freely diffusing ATP synthases with a confocal setup for single-molecule detection. Incubation with non-hydrolysable AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies corresponding to the three possible orientations of the epsilon-subunit.
Upon addition of ATP at mM concentrations, a consecutive order of three distinguishable FRET efficiencies was observed indicating a stepwise movement of the epsilon-subunit, comparable to the stepwise rotation of the gamma-subunit in EFoF1 [1,2]. Under the conditions for ATP synthesis stepwise rotation of the epsilon-subunit was observed in opposite direction. Dwell-time analysis revealed heterogeneity of the three catalytic binding sites.
[1] M. Borsch et al. (2002) FEBS lett. 527, 147-152.
[2] M. Diez et al. (2004) Nat. Struct. Mol. Biol., in press.