F
1-ATPase has been shown to be a stepwise molecular motor. Its rotation mechanism has beenexplained by the interaction of the
axis with the open and closed forms of the
subunit. Although NMRshould 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
subunit,and succeeded in assigning 89% of the NH (402/451), 89% of the C
(417/473), 83% of the C
(357/431),and 90% of the CO (425/473) signals of the
subunit monomer. The secondary structures predicted fromthe chemical shifts of the main chain atoms and the relative orientations determined from residual dipolarcouplings indicated that the subunit
monomer takes on the open form in the absence of nucleotide.Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotidebinding show that conformational change from the open to the closed form takes place on nucleotide binding.The intrinsic conformational change of the
subunit monomer induced by nucleotide binding must be oneof the essential driving forces for the rotation of F
1-ATPase.