Oxford Molecular Motors

Abstract:

Single-molecule measurements of rotation catalyzed by the F(1)-ATPase or the F(o)F(1) ATP synthase have provided new insights into the molecular mechanisms of the F(1) and F(o) molecular motors. We recently developed a method to record ATPase-driven rotation of F(1) or F(o)F(1) in a manner that solves several technical limitations of earlier approaches that were significantly hampered by time and angular resolution, and restricted the duration of data collection. With our approach it is possible to collect data for hours and obtain statistically significant quantities of data on each molecule examined with a time resolution of up to 5 μs at unprecedented signal-to-noise.

Abstract:

Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F₀F₁-ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F₀ has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F₀ subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli F₀F₁ embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μs to form and 175 μs 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 F₀ motor where c-ring rotational diffusion is limited to 36°.