Conformational Changes in the β Subunits of F1-Atpase Revealed by Fret Measurements During the Rotation of the γ Subunit

Abstract

To uncover conformational changes in biomolecules accompanied with biochemical events remains challenging, even though the atomic structures and the kinetics of the biomolecules are revealed. F1-ATPase is a rotary molecular motor in which a central γ subunit rotates against hexagonally arranged subunits α3β3, hydrolyzing ATP sequentially in three β subunits. Previous study using a single-molecule fluorescence polarization method has proposed sets of the β subunit conformations during the rotation (Masaike et al., Nat. Struct. Mol. Biol. 2008). However, further information is indispensable for identifying the ATP-waiting form of which the atomic structure has not been revealed. Here we performed single-pair FRET measurement to detect distance changes between two β subunits. Time trajectories of FRET efficiency showed two-state transitions between high (0.8) and low (0.5). Given the crystal structures, low FRET efficiency indicates one β subunit in the open form but another in the closed form. On the other hand, high FRET efficiency indicates both of the β subunits in the closed form. Next, we performed a simultaneous measurement of FRET between two fluorescently labeled β subunits and the rotation of the γ subunit. High FRET efficiency was occurred in one of the three catalytic dwells. In the remaining five dwells, other two catalytic dwells and three ATP-waiting dwells, FRET efficiency was lower. These results suggest that in the ATP-waiting dwell two of three β subunits would not take the closed form as in the catalytic dwell. We are performing further experiments with a magnetic tweezers.