
Proceedings Paper
Monitoring subunit rotation in single FRET-labeled FoF1-ATP synthase in an anti-Brownian electrokinetic trapFormat | Member Price | Non-Member Price |
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Paper Abstract
FoF1-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 Fo. 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 FoF1-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.
Paper Details
Date Published: 22 February 2013
PDF: 12 pages
Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 85880Q (22 February 2013); doi: 10.1117/12.2002966
Published in SPIE Proceedings Vol. 8588:
Multiphoton Microscopy in the Biomedical Sciences XIII
Ammasi Periasamy; Karsten König; Peter T. C. So, Editor(s)
PDF: 12 pages
Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 85880Q (22 February 2013); doi: 10.1117/12.2002966
Show Author Affiliations
Thomas Heitkamp, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Hendrik Sielaff, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Anja Korn, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Hendrik Sielaff, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Anja Korn, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Marc Renz, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Nawid Zarrabi, Univ. of Stuttgart (Germany)
Michael Börsch, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Univ. of Stuttgart (Germany)
Nawid Zarrabi, Univ. of Stuttgart (Germany)
Michael Börsch, Jena Univ. Hospital, Friedrich-Schiller-Univ. Jena (Germany)
Univ. of Stuttgart (Germany)
Published in SPIE Proceedings Vol. 8588:
Multiphoton Microscopy in the Biomedical Sciences XIII
Ammasi Periasamy; Karsten König; Peter T. C. So, Editor(s)
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