Conformational change of H+-ATPase beta monomer revealed on segmental isotope labeling NMR spectroscopy.

F1-ATPase has been shown to be a stepwise molecular motor. Its rotation mechanism has been explained by the interaction of the gamma axis with the open and closed forms of the beta subunit. Although NMR should be a powerful method for elucidating its mechanism, its molecular size (473 amino acid residues, 52 kDa) is a major obstacle. We have applied segmental labeling based on intein ligation to the beta subunit, and succeeded in assigning 89% of the NH (402/451), 89% of the Calpha (417/473), 83% of the Cbeta (357/431), and 90% of the CO (425/473) signals of the beta subunit monomer. The secondary structures predicted from the chemical shifts of the main chain atoms and the relative orientations determined from residual dipolar couplings indicated that the subunit beta monomer takes on the open form in the absence of nucleotide. Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotide binding show that conformational change from the open to the closed form takes place on nucleotide binding. The intrinsic conformational change of the beta subunit monomer induced by nucleotide binding must be one of the essential driving forces for the rotation of F1-ATPase.