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Proceedings Paper

Stepwise rotation of the γ-subunit of EF0F1-ATP synthase during ATP synthesis: a single-molecule FRET approach
Author(s): Michael Borsch; Manuel Diez; Boris Zimmermann; Matthias Trost; Stefan Steigmiller; Peter Graber
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Paper Abstract

FoF1-ATP synthases couple proton translocation with the synthesis of ATP using two rotary motors within the enzyme. To monitor inter-subunit movements during catalysis, we selectively attached two fluorophores to the F1 part, sulforhodamine B at one of three β-subunits and Cy5 at the γ-subunit. Reassembly with Fo parts embedded in liposomes yielded functional holoenzymes. Fluorescence resonance energy transfer (FRET) was investigated in photon bursts of freely diffusing liposomes with reconstituted ATP synthases using a confocal set-up for single-molecule detection. Incubation with AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies. Upon ATP addition, a repeating sequence of three distinct FRET efficiencies was observed, indicating the stepwise movement of the γ-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the γ-subunit under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition). The direction of rotation is opposite to the direction observed during ATP hydrolysis.

Paper Details

Date Published: 19 June 2003
PDF: 11 pages
Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); doi: 10.1117/12.479554
Show Author Affiliations
Michael Borsch, Univ. Stuttgart (Germany)
Manuel Diez, Albert-Ludwigs-Univ. Freiburg (Germany)
Boris Zimmermann, Albert-Ludwigs-Univ. Freiburg (Germany)
Matthias Trost, Albert-Ludwigs-Univ. Freiburg (Germany)
Stefan Steigmiller, Albert-Ludwigs-Univ. Freiburg (Germany)
Peter Graber, Albert-Ludwigs-Univ. Freiburg (Germany)

Published in SPIE Proceedings Vol. 4962:
Manipulation and Analysis of Biomolecules, Cells, and Tissues
Dan V. Nicolau; Joerg Enderlein; Robert C. Leif; Daniel L. Farkas, Editor(s)

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