Abstract
Since 20 years we apply intensity-based and time-resolved single-molecule FRET measurements to study subunit rotation and regulatory conformational changes of individual FoF1-ATP synthases in liposomes, either driven by ATP hydrolysis or during ATP synthesis. However, observation times of freely diffusing proteoliposomes in a confocal microscope are limited by Brownian motion. In addition, arbitrary trajectories through the confocal detection volume cause large intensity fluctuations in smFRET traces resulting in varying precision for the smFRET distance measurement within the data for single enzyme at work. To counteract diffusive motion actively in real time, we have built a fast anti-Brownian electrokinetic trap (ABELtrap, invented by A. E. Cohen and W. E. Moerner) with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. We recorded surprisingly fast subunit rotation of FoF1-ATP synthases at different ATP concentrations, and analyzed Michaelis-Menten kinetics as extracted from single-molecule FRET traces of individual membrane enzymes hold in solution by the ABELtrap. Substeps of the rotor and ADP inhibition were identified by extended single-enzyme observation in the ABELtrap.
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