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Structure-Function Analysis of HィイD1+ィエD1-ATP Synthase by Solution and Solid State NMR

H-D1+H-D1-ATP 合成酶的溶液和固态 NMR 结构-功能分析

基本信息

批准号：
09480173
负责人：
AKUTSU Hideo
金额：
$ 8.06万
依托单位：
Yokohama National University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

通过监测12个酪氨酸残基的核磁共振信号，研究了核苷酸结合对嗜热芽孢杆菌PS-3 (TF γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ) atp酶β亚基结构的影响。12个酪氨酸残基的3,5-质子共振可以观察到特定氘化β亚基。使用14个突变蛋白完成了所有酪氨酸残基的3,5质子共振的分配，每个突变蛋白中有一个或两个酪氨酸残基被苯丙氨酸取代。Mg·ATP的结合诱导了tyr341共振的前场移位，表明它们的芳香环相互堆叠。除了tyr1 -341外，在Mg·ATP结合上观察到的信号移位仅限于tyr1 -148、tyr1 -199、tyr1 -238和tyr1 -307的共振，这表明Mg·ATP诱导了铰链区构象的变化。这可能与晶体结构中从开放构象到封闭构象的变化有关。因此，由核苷酸结合引起的β亚基的内在构象变化被认为是F _(_) _(_) _(_) _() _() _() _() _() _() _() _() _() _() _() _旋转的基本驱动力之一。不同自旋各向异性相互作用之间的相互关系取决于各向异性相互作用的相互取向，即指定的二面角。因此，它可以用来确定二面角。将此方法分别应用于丙氨酸和缬氨酸的ψ角和χ角。在后一种情况下，键长、键角也会改变光谱。这使得我们可以实验确定所有的结构参数，提高了二面角确定的精度。