权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

通过新型 NMR 方法研究 H^-ATP 合酶的旋转催化机制

基本信息

批准号：
14208082
负责人：
AKUTSU Hideo
金额：
$ 31.03万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14208082/
关键词：
solution NMR solid-state NMR H^+-ATP synthase segmental isotope labeling β subunit monitor protein structural analysis subunit c 巨大タンパク質サブユニットC F_1βサブユニット区分安定同位体標識法

项目摘要

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.

F_OF_1-ATP合酶是一种多亚基酶，利用质子梯度的电化学势催化ATP合成。 F_1-ATP酶已被证明是一种步进式分子马达。旋转机制已通过γ轴与β亚基的开放和闭合形式的相互作用来解释。尽管 NMR 应该是阐明其机制的有力方法，但分子大小（473 个氨基酸残基，52 kDa）是分析的主要障碍。我们将基于内含肽连接的分段标记应用于β亚基，并成功分配了β亚基单体的89％NH（402/451）、89％C_α（417/473）、83％C_β（357/431）、90％CO（425/473）信号。由主链原子的化学位移预测的二级结构和由残余偶极耦合确定的相对方向表明，亚基β单体在不存在核苷酸的情况下呈开放形式。此外，核苷酸结合引起的化学位移扰动和残余偶极偶联变化表明，核苷酸结合时发生了从开放形式到封闭形式的构象变化。核苷酸结合引起的β亚基单体的内在构象变化必定是F_1-ATPase旋转的重要驱动力之一。在F_OF_1-ATP合酶中，F_Oc亚基的寡聚环充当F_O-质子马达的旋转质子通道。我们确定了有机溶剂中嗜热芽孢杆菌 PS3、TF_Oc 的 F_Oc 的 NMR 结构。我们的结果表明，与 pH 无关，TF_Oc 的必需 Glu56 的羧基突出到发夹的外部，第三个方向不同于 EF_Oc 中的两个方向中的任何一个。还对固体亚基c进行了固态NMR分析。