Stepping rotation of F-1-ATPase visualized through angle-resolved single-fluorophore imaging

Orientation dependence of single-fluorophore intensity was exploited in ord er to videotape conformational changes in a protein machine in real time. T he fluorophore Cy3 attached to the central subunit of F-1-ATPase revealed t hat the subunit rotates in the molecule in discrete 120 degrees steps and t hat each step is driven by the hydrolysis of one ATP molecule. These result s, unlike those from the previous study under a frictional load, show that the 120 degrees stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exe rted on the rotor subunit.