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Proceedings Paper

Subunit rotation in single FRET-labeled F1-ATPase hold in solution by an anti-Brownian electrokinetic trap
Author(s): Hendrik Sielaff; Thomas Heitkamp; Andrea Zappe; Nawid Zarrabi; Michael Börsch
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Paper Abstract

FoF1-ATP synthase catalyzes the synthesis of adenosine triphosphate (ATP). The F1 portion can be stripped from the membrane-embedded Fo portion of the enzyme. F1 acts as an ATP hydrolyzing enzyme, and ATP hydrolysis is associated with stepwise rotation of the γ and ε subunits of F1. This rotary motion was studied in great detail for the last 15 years using single F1 parts attached to surfaces. Subunit rotation of γ was monitored by videomicroscopy of bound fluorescent actin filaments, nanobeads or nanorods, or single fluorophores. Alternatively, we applied single-molecule Förster resonance energy transfer (FRET) to monitor subunit rotation in the holoenzyme FoF1-ATP synthase which was reconstituted in liposomes. Now we aim to extend the observation times of single FRET-labeled F1 in solution using a modified version of the anti-Brownian electrokinetic trap (ABELtrap) invented by A. E. Cohen and W. E. Moerner. We used Monte Carlo simulations to reveal that stepwise FRET efficiency changes can be analyzed by Hidden Markov Models even at the limit of a low signal-to-background ratio that was expected due to high background count rates caused by the microfluidics of the ABELtrap.

Paper Details

Date Published: 22 February 2013
PDF: 12 pages
Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 859008 (22 February 2013); doi: 10.1117/12.2002955
Show Author Affiliations
Hendrik Sielaff, Friedrich-Schiller-Univ. Hospital Jena (Germany)
Thomas Heitkamp, Friedrich-Schiller-Univ. Hospital Jena (Germany)
Andrea Zappe, Univ. Stuttgart (Germany)
Nawid Zarrabi, Univ. Stuttgart (Germany)
Michael Börsch, Friedrich-Schiller-Univ. Hospital Jena (Germany)
Univ. Stuttgart (Germany)


Published in SPIE Proceedings Vol. 8590:
Single Molecule Spectroscopy and Superresolution Imaging VI
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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