Monitoring γ-Subunit Movement in Reconstituted Single EF°F1 ATP Synthase by Fluorescence Resonance Energy Transfer

摘要: The membrane-bound enzymes H+ ATP synthases contain two coupled rotary motors that drive catalysis. We applied a single molecule spectroscopy approach to monitor the internal rotation of γ-subunit F1 part against its static counterpart, b-subunits F0 part. specifically attached fluorophores synthase from E. coli, namely Cy5 at and tetramethylrhodamine one b-subunit. After reconstitution into liposomes, these regained their full catalytic activity as measured by synthesis rates. Fluorescence resonance energy transfer (FRET) was monitored in photon bursts freely diffusing proteoliposomes using confocal setup for detection. Incubation with non-hydrolyzable AMPPNP resulted stable intensity ratios within burst. This corresponds fixed orientation. detected three different FRET efficiencies, i.e., orientations. addition consecutive order distinguishable efficiencies observed bursts, indicating stepwise unidirectional movement b-subunits.