Subunit rotation in single FRET-labeled F1-ATPase hold in solution by an anti-Brownian electrokinetic trap

Hendrik Sielaff; Thomas Heitkamp; Andrea Zappe; Nawid Zarrabi; Michael Börsch

doi:10.1117/12.2002955

22 February 2013 Subunit rotation in single FRET-labeled F₁-ATPase hold in solution by an anti-Brownian electrokinetic trap

Hendrik Sielaff, Thomas Heitkamp, Andrea Zappe, Nawid Zarrabi, Michael Börsch

Author Affiliations +

Proceedings Volume 8590, Single Molecule Spectroscopy and Superresolution Imaging VI; 859008 (2013) https://doi.org/10.1117/12.2002955
Event: SPIE BiOS, 2013, San Francisco, California, United States

Abstract

F_oF₁-ATP synthase catalyzes the synthesis of adenosine triphosphate (ATP). The F₁ portion can be stripped from the membrane-embedded F_o 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 F_oF₁-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.

Citation Download Citation

Hendrik Sielaff, Thomas Heitkamp, Andrea Zappe, Nawid Zarrabi, and Michael Börsch "Subunit rotation in single FRET-labeled F₁-ATPase hold in solution by an anti-Brownian electrokinetic trap", Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 859008 (22 February 2013); https://doi.org/10.1117/12.2002955

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