The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap

F-1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F-1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F-1 (or FoF1) must be minimized in the cell. In bacteria, the epsilon subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F-1. We investigate this proposed mechanism by labeling F-1 specifically with two fluorophores to monitor the C-terminus of the epsilon subunit by Forster resonance energy transfer. Single F-1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F-1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F-1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.