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

摘要

F_oF_1-ATP synthase catalyzes the synthesis of adenosine triphosphate (ATP). The F_1 portion can be stripped from the membrane-embedded F_o portion of the enzyme. F_1 acts as an ATP hydrolyzing enzyme, and ATP hydrolysis is associated with stepwise rotation of the γ and ε subunits of F_1. This rotary motion was studied in great detail for the last 15 years using single F_1 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 F?rster resonance energy transfer (FRET) to monitor subunit rotation in the holoenzyme F_oF_1-ATP synthase which was reconstituted in liposomes. Now we aim to extend the observation times of single FRET-labeled F_1 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.