| Research Abstract |
The effect of nucleotide binding on the structure of the FィイD21ィエD2-ATPase βsubunit from thermophilic Bacillus PS-3 (TFィイD21ィエD2β) was investigated by monitoring the NMR signals of the twelve tyrosine residues. The 3,5-proton resonances of 12 tyrosine residues could be observed for the specifically deuterated β subunit. The assignment of 3,5-proton resonances of all the tyrosine residues was accomplished using fourteen mutant proteins, in each of which one or two tyrosine residues were substituted 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. Therefore, the intrinsic conformational change in the β subunit induced by the nucleotide binding is proposed to be one of the essential driving forces for the FィイD21ィエD2 rotation.
A solid-state NMR spectrum for the correlation between anisotropic interactions of different spins depends on mutual orientations of anisotropic interactions, which specify dihedral angles. Thus, it can be used to determine the dihedral angle. This was applied to the ψand χ angles in alanine and valine, respectively. In the latter case, the bond lengths, bond angles also alter the spectrum. This enable us experimental determination of all structural parameters, which improves the accuracy of the dihedral angle determination.
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