Microsecond Time Scale Rotation Measurements of Single F1-ATPase Molecules

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• 作者：David Spetzler ; Justin York ; Douglas Daniel ; Raimund Fromme ; David Lowry ; and Wayne Frasch
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：March 14, 2006
• 年：2006
• 卷：45
• 期：10
• 页码：3117 - 3124
• 全文大小：477K
• 年卷期：v.45,no.10(March 14, 2006)
• ISSN：1520-4995

文摘

A novel method for detecting F₁-ATPase rotation in a manner sufficiently sensitive to achieveacquisition rates with a time resolution of 2.5 s (equivalent to 400 000 fps) is reported. This is sufficientfor resolving the rate at which the -subunit travels from one dwell state to another (transition time).Rotation is detected via a gold nanorod attached to the rotating -subunit of an immobilized F₁-ATPase.Variations in scattered light intensity allow precise measurement of changes in the angular position of therod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F₁-ATPase -subunit rotation was determined to be 7.62 ± 0.15 (standard deviation) rad/ms. The averagerate-limiting dwell time between rotation events observed at the saturating substrate concentration was8.03 ms, comparable to the observed Mg²⁺-ATPase k_cat of 130 s^-1 (7.7 ms). Histograms of scattered lightintensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determinationof the nanorod orientation with respect to the axis of rotation and plane of polarization. This informationallowed the drag coefficient to be determined, which implied that the instantaneous torque generated byF₁ was 63.3 ± 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of theinstantaneous torque of F₁, resulting in direct implications for its rotational mechanism.