Activation and stiffness of the inhibited states of F₁-ATPase probed by single-molecule manipulation

F₁-ATPase (F₁), a soluble portion of F _oF₁-ATP synthase (FoF1), is an ATP-driven motor in which γε subunits rotate in the α₃β₃ cylinder. Activity of F₁ and F_oF₁ from Bacillus PS3 is attenuated by the ε subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of ε in single F₁ molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F₁ with hairpin ε directly determines the fraction of active F₁ at any ATP concentration. Next, mechanical activation and stiffness of ε-inhibited F₁ were examined by the forced rotation of magnetic beads attached to γ. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from ε 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 F_oF₁ is resting in the ε-inhibited state, F_o motor must transmit to γ a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.