Robert Ishmukhametov

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°.

Abstract:

Increases in the power stroke and dwell durations of single molecules of Escherichia coli F(1)-ATPase were measured in response to viscous loads applied to the motor and inhibition of ATP hydrolysis. The load was varied using different sizes of gold nanorods attached to the rotating gamma subunit and/or by increasing the viscosity of the medium using PEG-400, a noncompetitive inhibitor of ATPase activity. Conditions that increase the duration of the power stroke were found to cause 20-fold increases in the length of the dwell. These results suggest that the order of hydrolysis, product release, and substrate binding may change as the result of external load on the motor or inhibition of hydrolysis.

Abstract:

The torque generated by the power stroke of Escherichia coli F(1)-ATPase was determined as a function of the load from measurements of the velocity of the gamma-subunit obtained using a 0.25 micros time resolution and direct measurements of the drag from 45 to 91 nm gold nanorods. This result was compared to values of torque calculated using four different drag models. Although the gamma-subunit was able to rotate with a 20x increase in viscosity, the transition time decreased from 0.4 ms to 5.26 ms. The torque was measured to be 63+/-8 pN nm, independent of the load on the enzyme.