Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trap

Thomas Heitkamp; Hendrik Sielaff; Anja Korn; Marc Renz; Nawid Zarrabi; Michael Börsch

doi:10.1117/12.2002966

22 February 2013 Monitoring subunit rotation in single FRET-labeled F_oF₁-ATP synthase in an anti-Brownian electrokinetic trap

Thomas Heitkamp, Hendrik Sielaff, Anja Korn, Marc Renz, Nawid Zarrabi, Michael Börsch

Author Affiliations +

Proceedings Volume 8588, Multiphoton Microscopy in the Biomedical Sciences XIII; 85880Q (2013) https://doi.org/10.1117/12.2002966
Event: SPIE BiOS, 2013, San Francisco, California, United States

Abstract

F_oF₁-ATP synthase is the membrane protein catalyzing the synthesis of the 'biological energy currency' adenosine triphosphate (ATP). The enzyme uses internal subunit rotation for the mechanochemical conversion of a proton motive force to the chemical bond. We apply single-molecule Förster resonance energy transfer (FRET) to monitor subunit rotation in the two coupled motors F₁ and F_o. Therefore, enzymes have to be isolated from the plasma membranes of Escherichia coli, fluorescently labeled and reconstituted into 120-nm sized lipid vesicles to yield proteoliposomes. These freely diffusing proteoliposomes occasionally traverse the confocal detection volume resulting in a burst of photons. Conformational dynamics of the enzyme are identified by sequential changes of FRET efficiencies within a single photon burst. The observation times can be extended by capturing single proteoliposomes in an anti-Brownian electrokinetic trap (ABELtrap, invented by A. E. Cohen and W. E. Moerner). Here we describe the preparation procedures of F_oF₁-ATP synthase and simulate FRET efficiency trajectories for 'trapped' proteoliposomes. Hidden Markov Models are applied at signal-to-background ratio limits for identifying the dwells and substeps of the rotary enzyme when running at low ATP concentrations, excited by low laser power, and confined by the ABELtrap.

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Thomas Heitkamp, Hendrik Sielaff, Anja Korn, Marc Renz, Nawid Zarrabi, and Michael Börsch "Monitoring subunit rotation in single FRET-labeled F_oF₁-ATP synthase in an anti-Brownian electrokinetic trap", Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 85880Q (22 February 2013); https://doi.org/10.1117/12.2002966

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