Stepwise rotation of the γ-subunit of EFoF1-ATP synthase during ATP synthesis: a single-molecule FRET approach

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

    Research output: Chapter in Book/Report/Conference proceedingConference contribution

    35 Citations (Scopus)

    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 F 1 part, sulforhodamine B at one of three ß-subunits and Cy5 at the ?-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 ?-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the ?-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.

    Original languageEnglish
    Title of host publicationManipulation and Analysis of Biomolecules, Cells, and Tissues
    EditorsDan V. Nicolau, Joerg Enderlein, Robert C. Leif, Daniel L. Farkas
    Place of PublicationBellingham
    PublisherSPIE-International Society for Optical Engineering
    Pages11-21
    Number of pages11
    ISBN (Print)0819447625
    DOIs
    Publication statusPublished - 2003
    EventSPIE Photonics West 2003: Manipulation and Analysis of Biomolecules, Cells and Tissues - San Jose, United States
    Duration: 28 Jan 200329 Jan 2003

    Publication series

    NameProgress in Biomedical Optics
    Number14
    Volume4
    NameProceedings of SPIE
    PublisherSPIE
    Volume4962

    Conference

    ConferenceSPIE Photonics West 2003: Manipulation and Analysis of Biomolecules, Cells and Tissues
    Country/TerritoryUnited States
    CitySan Jose
    Period28/01/0329/01/03

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