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

F₁-ATPase is the soluble portion of the membrane-embedded enzyme F_oF₁-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F₁ part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F₁ (or F_oF₁) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F₁. We investigate this proposed mechanism by labeling F₁ specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F₁ molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F₁ in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F₁ and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.