Rotary catalysis mechanism of H^+-ATP synthase investigated by novel NMR methodology

F_OF_1-ATP合成酶是一种利用质子梯度的电化学电势催化ATP合成的多亚基酶。F_1-ATP酶是一种步进式分子马达。旋转机制可以通过γ轴与β亚基的开放和封闭形式的相互作用来解释。虽然NMR应该是一个强大的方法来阐明其机制，分子大小（473个氨基酸残基，52 kDa）是分析的主要障碍。我们将内含肽连接的片段标记技术应用于β亚基，成功地将89%的NH（402/451），89%的C_α（417/473），83%的C_β（357/431），90%的CO（425/473）信号归属于β亚基单体。由主链原子的化学位移预测的二级结构和由残余偶极偶联确定的相对取向表明，在不存在核苷酸的情况下，亚基β单体呈开放形式。此外，由核苷酸结合引起的化学位移扰动和残基偶极偶联变化表明，核苷酸结合时发生了从开放到封闭的构象变化。核苷酸结合引起的β亚基单体的内在构象变化可能是F_1-ATP酶旋转的重要驱动力之一。在F_OF_1-ATP合酶中，F_Oc亚基的寡聚环作为F_O-质子马达的旋转质子通道。本文测定了嗜热芽孢杆菌PS 3的F_Oc，TF_Oc在有机溶剂中的NMR结构。结果表明，TF_Oc的Glu 56的羧基不受pH的影响而突出到发夹结构的外侧，这是不同于EF_Oc的两种取向的第三种取向。还进行了固体亚基c的固态NMR分析。

F_OF_1-ATP synthase is a multisubunit enzyme that catalyzes ATP synthesis using the electrochemical potential of proton gradient. F_1-ATPase has been shown to be a step-wise molecular motor. The rotation mechanism has been explained by the interaction of the γ axis with the open and closed forms of the β subunit. Although NMR should be a powerful method to elucidate its mechanism, the molecular size (473 amino acid residues, 52 kDa) was the major obstacle to analyze. We have applied the segmental labeling based on intein ligation to the β subunit, and succeeded in assigning 89% NH (402/451),89% C_α (417/473),83% C_β(357/431),90% CO (425/473) signals of the β subunit monomer. The secondary structures predicted by the chemical shifts of the main chain atoms and the relative orientations determined by residual dipolar couplings indicated that the subunit β monomer takes the open form in the absence of nucleotide. Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotide binding have shown the conformational change from the open to closed forms takes place on nucleotide binding. The intrinsic conformational change of the β subunit monomer induced by nucleotide binding must be one of essential driving forces of the rotation of F_1-ATPase. In F_OF_1-ATP synthase, an oligomer ring of F_Oc subunits acts as a rotary proton channel of F_O-proton motor. We have determined the NMR structure of F_Oc from thermophilic Bacillus PS3, TF_Oc in an organic solvent. Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF_Oc protrudes to the outside of the hairpin, a third orientation differed from either of two orientations in EF_Oc. A solid-state NMR analysis of solid subunit c was also carried out.