To monitor inter-subunit rotation during ATP hydrolysis, we attach two fluorophores - in the first approach Cy5 (red) at the rotating g-subunit (blue helices) and tetramethylrhodamine (green) at one of the two b-subunits - and reconstituted the enzymes fully functional into liposomes [1].

 Fluorescence resonance energy transfer (FRET) is monitored in photon bursts of freely diffusing ATP synthases with a confocal setup for single-molecule detection.

 Incubation with AMPPNP results in stable intensity ratios within a burst and three different FRET efficiencies, which correspond to three possible g-subunit orientations with respect to the b-subunits.

 During ATP hydrolysis, a consecutive order of three distinguishable FRET efficiencies (steps in the proximity
factor traces, blue) was observed indicating a stepwise movement of the g-subunit. The dwell times of the FRET levels observed in this FRET approach are correlated to the catalytic rates.

 The sequence of FRET level transitions was reversed as the conditions were changed from ATP hydrolysis to ATP synthesis. This was the confirmation of the opposite direction of rotation  (Nature Structural & Molecular Biology 02/2004).

 The FRET approach was refined and demonstrated an conformational change of the e-subunit associated with the catalytic activity of the enzyme (The EMBO Journal 06/2005).

 Currently we are using a novel FRET approach to observe the rotation of a single membrane-embedded F_o-motor during catalysis, and we are developing an nano-electrochemical tool to control the rotation and conformational state of individual subunits in  a single ATP synthase.