Résumé
The rotary motor F-1-ATPase from the thermophilic Bacillus PS3 (TF1) is one of the best-studied of all molecular machines. F-1-ATPase is the part of the enzyme F1FO-ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F-1-ATPase from Escherichia coli (EF1) is governed by the same mechanism as TF1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the gamma-subunit of single surface-bound EF1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF1. We also show that the ATP-binding and catalytic states are separated by 35 +/- 3 degrees. At room temperature, chemical processes occur faster in EF1 than in TF1, and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.