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.