Activation and Stiffness of the Inhibited States of F1-ATPase Probed by Single-molecule Manipulation

F-1-ATPase (F-1), a soluble portion of FoF1-ATP synthase (FoF1), is an ATP-driven motor in which gamma epsilon subunits rotate in the alpha(3)beta(3) cylinder. Activity of F1 and FoF1 from Bacillus PS3 is attenuated by the epsilon subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of epsilon in single F-1 molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F-1 with hairpin epsilon directly determines the fraction of active F-1 at any ATP concentration. Next, mechanical activation and stiffness of epsilon-inhibited F-1 were examined by the forced rotation of magnetic beads attached to epsilon. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from epsilon inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results imply that if FoF1 is resting in the epsilon-inhibited state, F-o motor must transmit to gamma a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.