F₁-ATPase is a motor protein. The TF₁β subunit, which carries the catalytic site of this enzyme, consists of 473 amino acids (52 kD). We completed more than 90% assignments of the backbone NMR signals by the segmental isotope labeling method and TROSY. A new insight into the mechanism of the F₁ rotation has been provided by the assignments of the backbone NMR signals. On adding MgADP, signals located only in the nucleotide binding domain shifted, suggesting that the β subunit’s conformation changes even as a monomer on nucleotide binding. The confirmed conformational change from the open to closed forms is realized through two steps, namely, the first step induced by F420 and the second step induced by K164, T165 and D252. The flexibility in the nucleotide binding domain also plays an important role. This conformational change can be an important driving force for the F₁ rotation. The conformational analysis of β subunit-ATP complex by solid-state NMR under magic angle spinning was also carried out.