Rotation of the γ-subunit in single membrane-bound H+-ATP synthases from chloroplasts during ATP synthesis

Single-molecule fluorescence resonance energy transfer (smFRET) is used to investigate the movement of the gamma-subunit within the H+-ATPsynthase from chloroplasts during proton transport-coupled ATP synthesis. The FRET donor fluorophore is attached to residue gamma C322 and the FRET acceptor is non-covalently bound to a non-catalytic nucleotide binding site. The labeled enzyme is integrated into liposomes and ATP synthesis is started by an acid-base transition. A custom-built confocal microscope with two spectral detection channels is used to measure smFRET with freely diffusing proteoliposomes. Analysis of the smFRET time traces reveals stepwise distance changes between the two fluorophores. During catalysis the gamma-subunit interacts with equal probability with each alpha beta-pair. In ADP-inhibited enzymes, the gamma-subunit interacts with only one specific alpha beta-pair. Comparison of the smFRET data with cryo-EM data (Hahn, Vonck, Mills, Meier, & Kuhlbrandt, 2018, Science, 360, pp. 620-628) indicated that the transition from the ADP-inhibited state to the active state is accompanied by a large rotational movement of the.-subunit.