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.

Date Published: 22 February 2013
PDF: 12 pages
Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 85880Q (22 February 2013);

Published in SPIE Proceedings Vol. 8588:
Multiphoton Microscopy in the Biomedical Sciences XIII
Ammasi Periasamy; Karsten König; Peter T. C. So, Editor(s)