= Single-molecule observation of F1 rotation
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In 1997, <Hiroyuki Noji> and colleagues attached a fluorescent <actin> filament to the central $\gamma$ shaft of isolated $F_1$. With <ATP> supplied, the marker made sustained rotations. This exposed rotation directly, but used <ATP> <hydrolysis>: there was no membrane $F_0$ motor producing <ATP> from a <proton-motive force> in this assay.<Direct observation of the rotation of F1-ATPase by Hiroyuki Noji and others>[Noji, Yasuda, Yoshida and Kinosita Jr., Direct observation of the rotation of F1-ATPase, Nature 386 (1997), 299–302, accessible primary abstract]{ref}
A subsequent primary experiment made the optical readout much faster. Purified <Escherichia coli> $F_1$ was immobilized on glass in a $30\,\mathrm{\mu m}$-deep flow cell, with a $60\,\mathrm{nm}$ <gold> bead attached to $\gamma$. With $2\,\mathrm{mM}$ <ATP> and <magnesium> supplied, laser illumination and <dark-field microscopy> recorded bead positions at $4000$ or $8000$ frames per second. The position trajectory resolved turns and intervening dwells. This apparatus follows a shaft-attached marker, rather than imaging the shaft's <chemical atoms>.<Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell by Mizuki Sekiya and others>[Sekiya et al., 2009, Experimental Procedures: Direct Observation of gamma Subunit Rotation; Results, Figure 1]{ref}
Controls addressed perturbation and chemical activity: comparison of $40$–$200\,\mathrm{nm}$ probes found similar rates for the two smallest sizes, supporting low <drag>; a separate coupled-<enzyme> assay followed <ATP> <hydrolysis> through <NADH> absorbance. But the rotational analysis excluded pauses longer than $0.1\,\mathrm s$, so it characterizes continuously active intervals, not total output including long inhibited periods. It neither counts transported <protons> nor measures simultaneous single-<molecule> <ATP> synthesis.<Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell by Mizuki Sekiya and others>[Sekiya et al., 2009, Experimental Procedures: Assay methods and data selection; Results: Direct Observation of Rotation Using a 60-nm Gold Bead]{ref}
The experimental distinction is essential. Sustained directional marker motion tests a rotary mechanism. Demonstrating <proton>-driven synthesis additionally requires intact $F_0F_1$ in a membrane with a controlled <proton-motive force> and a chemical <ATP>-production readout; dependence on that driving force and inhibition or <respiratory uncoupling> controls then test the proposed <energy coupling>. A rotation movie alone cannot establish the complete material and <free energy> ledger.
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