Stepwise rotation of the &#947;-subunit of EF<sub>0</sub>F<sub>1</sub>-ATP synthase during ATP synthesis: a single-molecule FRET approach

Michael Borsch; Manuel Diez; Boris Zimmermann; Matthias Trost; Stefan Steigmiller; Peter Graber

doi:10.1117/12.479554

19 June 2003 Stepwise rotation of the γ-subunit of EF_oF₁-ATP synthase during ATP synthesis: a single-molecule FRET approach

Michael Borsch, Manuel Diez, Boris Zimmermann, Matthias Trost, Stefan Steigmiller, Peter Graber

Author Affiliations +

Proceedings Volume 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues; (2003) https://doi.org/10.1117/12.479554
Event: Biomedical Optics, 2003, San Jose, CA, United States

Abstract

F_oF₁-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 F_o 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.

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Michael Borsch, Manuel Diez, Boris Zimmermann, Matthias Trost, Stefan Steigmiller, and Peter Graber "Stepwise rotation of the γ-subunit of EF_oF₁-ATP synthase during ATP synthesis: a single-molecule FRET approach", Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); https://doi.org/10.1117/12.479554

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