Microsecond time scale rotation measurements of single F1-ATPase molecules.
A novel method for detecting F(1)-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 micros (equivalent to 400,000 fps) is reported. This is sufficient for resolving the rate at which the gamma-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating gamma-subunit of an immobilized F(1)-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 F(1)-ATPase gamma-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 Mg(2+)-ATPase k(cat) of 130 s(-)(1) (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determination of the nanorod orientation with respect to the axis of rotation and plane of polarization. This information allowed the drag coefficient to be determined, which implied that the instantaneous torque generated by F(1) was 63.3 +/- 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F(1), resulting in direct implications for its rotational mechanism.
Spetzler
David
D
Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State UniVersity, P.O. Box 874501, Tempe, Arizona 85287-4501.
York
Justin
J
Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State UniVersity, P.O. Box 874501, Tempe, Arizona 85287-4501.
Daniel
Douglas
D
Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State UniVersity, P.O. Box 874501, Tempe, Arizona 85287-4501.
Fromme
Raimund
R
Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State UniVersity, P.O. Box 874501, Tempe, Arizona 85287-4501.
Lowry
David
D
Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State UniVersity, P.O. Box 874501, Tempe, Arizona 85287-4501.
Frasch
Wayne
W
Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State UniVersity, P.O. Box 874501, Tempe, Arizona 85287-4501.
D000255:Adenosine Triphosphate / Q000378:metabolism; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D012399:Rotation; D019415:Torque
D000255:Adenosine Triphosphate; C444660:gamma subunit, F(1) ATPase; D006180:Proton-Translocating ATPases