Microsecond time scale rotation measurements of single F1-ATPase molecules

A novel method for detecting F1-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 μs (equivalent to 400 000 fps) is reported. This is sufficient for resolving the rate at which the γ-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating γ-subunit of an immobilized F1-ATPase. Variations in scattered light intensity allow precise measurement of changes in the angular position of the rod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F1-ATPase γ-subunit rotation was determined to be 7.62 ± 0.15 (standard deviation) rad/ms. The average rate-limiting dwell time between rotation events observed at the saturating substrate concentration was 8.03 ms, comparable to the observed Mg2+-ATPase kcat of 130 s-1 (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle al...