Osnabrück 2002 – wissenschaftliches Programm

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SYBP: Biophotonik

SYBP 4: Postersitzung

SYBP 4.15: Poster

Donnerstag, 7. März 2002, 17:45–20:00, Schloss

Monitoring gamma-subunit movement in reconsituted single EFoF1 ATP synthase by fluorescence resonance energy transfer — •Michael Börsch, Manuel Diez, Boris Zimmermann, and Peter Gräber — Institut für Physikalische Chemie, Universität Freiburg, Albertstr. 23 a, 79104 Freiburg, Germany

The membrane-bound enzymes H+ ATP synthases contain two coupled rotary motors that drive catalysis. We applied a single molecule spectroscopy approach to monitor the internal rotation of the gamma-subunit of the F1 part against its static counterpart, the b-subunits of the Fo part. We specifically attached two fluorophores to H+ ATP synthase from E. coli, namely Cy5 at the gamma-subunit and tetramethylrhodamine at one b-subunit. After reconstitution into liposomes, these enzymes regained their full catalytic activity as measured by ATP synthesis rates. Fluorescence resonance energy transfer (FRET) was monitored in photon bursts of freely diffusing proteoliposomes using a confocal setup for single molecule detection. Incubation with non-hydrolysable AMPPNP resulted in stable intensity ratios within a photon burst. This corresponds to a fixed gamma-subunit orientation. We detected three different FRET efficiencies, i.e. gamma-subunit orientations. After addition of ATP a consecutive order of three distinguishable FRET efficiencies was observed within the bursts, indicating a stepwise unidirectional gamma-subunit movement against the b-subunits.