Functional conformation changes in the TF1-ATPase beta subunit probed by 12 tyrosine residues

The effect of nucleotide binding on the structure of the F-1-ATPase beta su bunit from thermophilic bacillus PS-3 (TF1 beta) was investigated by monito ring 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 tyrosin e residues was accomplished using 14 mutant proteins, in each of which one or two tyrosine residues were replaced by phenylalanine. Binding of Mg . AT P induced an upfield shift of Tyr(341) resonance, suggesting that their aro matic rings are stacked to each other. Besides Tyr(341), the signal shift o bserved on Mg . ATP binding was restricted to the resonances of Tyr(148) Ty r(199), Tyr(238) and Tyr(307), suggesting that Mg . ATP induces a conformat ional change in the hinge region. This can be correlated to the change from the open to closed conformations as implicated in the crystal structure. M g . ADP induced a similar but distinctly different conformational change. T herefore, the intrinsic conformational change in the beta subunit induced b y the nucleotide binding is proposed to be one of the essential driving for ces for the F-1 rotation. Reconstitution experiments showed that Tyr(277), one of the four conserved tyrosines, is essential to the formation of the a lpha(3)beta(3)gamma complex.