Microsecond time scale rotation measurements of single F1-ATPase molecules

David Spetzler, Justin York, Douglas Daniel, Raimund Fromme, David Lowry, Wayne Frasch

Research output: Contribution to journalArticle

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Abstract

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 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 F1 was 63.3 ± 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F1, resulting in direct implications for its rotational mechanism.

Original languageEnglish (US)
Pages (from-to)3117-3124
Number of pages8
JournalBiochemistry
Volume45
Issue number10
DOIs
StatePublished - Mar 14 2006

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ASJC Scopus subject areas

  • Biochemistry

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