Direct observation of stepped proteolipid ring rotation in E. coli F o F 1-ATP synthase

Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F_o F₁-ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F_o has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F_o subunits a and c by improving the time resolution to 10 μ at unprecedented S/N, and by using Escherichia coli F_o F 1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μ to form and 175 μ to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c-ring to a limit of ∼36 ° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F_o motor where c-ring rotational diffusion is limited to 36 °.