2001 Volume 130 Issue 4 Pages 527-533
The C2 proton signals of all (twelve) histidine residues of the TF1 β subunit in the 1H-NMR spectrum have been identified and assigned by means of pH change experimentsand site-directed substitution of histidines by glutamines. pH and ligand titration experiments were carried out for these signals. Furthermore, the ATPase activity of the reconstituted α3β3γ complex was examined for the twelve mutant β subunits. Two of threeconserved histidines, namely, His-119 and 324, were found to be important for expression of the ATPase activity. The former fixes the N-terminal domain to the central domain. His-324 is involved in the formation of the interface essential for the α3β3γ complex assembly. The other conserved residue, His-363, showed a very low pKa, suggestingthat it is involved in the tertiary structure formation. On the binding of a nucleotide, only the signals of His-173, 179, 200, and 324 shifted. These histidines are located in thehinge region, and its proximity, of the β subunit. This observation provided further sup-port for the conformational change of the β monomer from the open to the closed formon the binding of a nucleotide proposed by us [Yagi et al. (1999) Biophys. J. 77, 21762183]. This conformational change should be one of the essential driving forces in therotation of the α3β3γ complex.
