Abstract
The effect of nucleotide binding on the structure of the F(1)-ATPase beta subunit from thermophilic bacillus PS-3 (TF(1)beta) was investigated by monitoring the NMR signals of the 12 tyrosine residues. The 3,5-proton resonances of 12 tyrosine residues could be observed for the specifically deuterated beta subunit. The assignment of 3,5-proton resonances of all of the tyrosine residues was accomplished using 14 mutant proteins, in each of which one or two tyrosine residues were replaced by phenylalanine. Binding of Mg. ATP induced an upfield shift of Tyr(341) resonance, suggesting that their aromatic rings are stacked to each other. Besides Tyr(341), the signal shift observed on Mg.ATP binding was restricted to the resonances of Tyr(148), Tyr(199), Tyr(238), and Tyr(307), suggesting that Mg.ATP induces a conformational change in the hinge region. This can be correlated to the change from the open to closed conformations as implicated in the crystal structure. Mg.ADP induced a similar but distinctly different conformational change. Therefore, the intrinsic conformational change in the beta subunit induced by the nucleotide binding is proposed to be one of the essential driving forces for the F(1) rotation. Reconstitution experiments showed that Tyr(277), one of the four conserved tyrosines, is essential to the formation of the alpha(3)beta(3)gamma complex.