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

TL;DR

This study uses single-molecule FRET combined with an anti-Brownian electrokinetic trap to monitor subunit rotation in individual FoF1-ATP synthase enzymes, revealing their conformational dynamics under various conditions.

Contribution

It introduces a method to observe and analyze the rotational dynamics of FoF1-ATP synthase in single proteoliposomes using FRET and ABELtrap, enhancing understanding of enzyme mechanics.

Findings

Successful monitoring of enzyme rotation in trapped proteoliposomes.

Application of Hidden Markov Models to identify enzyme dwells and substeps.

Extended observation times enable detailed kinetic analysis.

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 F\"orster 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.