FIGURE 3. Single-molecule FRET reveals ordered, stepwise rotation of F₀F₁-ATP synthase on the millisecond timescale during ATP hydrolysis and synthesis.

a, Model of F₀F₁-ATP synthase embedded in a lipid bilayer. The rotor subunits are shown in blue, and the stator subunits are shown in orange. The FRET donor (green) is bound to the -subunit, and the FRET acceptor (red) is bound to the b-subunits. b, Cross-section at the level of the fluorophore, as viewed from the membrane. The change in position of the donor is shown relative to the acceptor on rotation of the rotor subunits (blue) in 120 ° steps. c, Single-molecule time traces of a single F₀F₁-ATP synthase molecule in liposomes during ATP hydrolysis. The fluorescence intensities (lower panel) of the donor (F_D, green) and the acceptor (F_A, red), as well as the corrected intensity ratio (F_D/F_A, upper panel, orange), uncover stepping between three states, with unique donor–acceptor distances in the order 1321 (which correspond to the numbers in part b). Data were collected over 1 ms intervals. (Figure reproduced, with permission, from ref. 46.)