Single-Molecule Observation of Rotation of F1-ATPase Through Microbeads
F0F1-ATP synthase catalyzes the synthesis of ATP using proton-motive force across a membrane. When isolated, the F1 sector, composed of five polypeptide chains with a stoichiometry of α3β3γδε, solely hydrolyzes ATP into ADP and phosphate, and is thus called F1-ATPase. Rotation of a shaft domain in F0F1-ATP synthase has been hypothesized by Paul Boyer, and ultimately was confirmed by direct observation as rotation of the γ-subunit in an isolated α3β3γ subcomplex. Unitary turnover of ATP induces 120° steps, consistent with the configuration of three catalytic sites arranged 120° apart around γ. We have shown the relationships between chemical and mechanical events by imaging individual F1 molecules under an optical microscope. A new scheme emerges: as soon as a catalytic site binds ATP, the γ-subunit always turns the same face (interaction surface) to the β hosting that site; ∼80° rotation is driven by ATP binding; ∼40° rotation is induced by completion of hydrolysis [and/or phosphate release] in the site that bound ATP one step earlier.
Key WordsF1-ATPase motor protein single molecule biophysics rotary molecular motor streptavidin bead
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