F1 ATPASE Chemical Mechanical Coupling Mechanisms

描述：（申请人的描述）化学性质之间的关系 ATP水解和机械事件，导致旋转的 将检查F1-ATP酶的γ亚基。F1的催化功能 来源于催化位点的不对称性，反过来，取决于 γ亚基和Mg 2+辅因子。伽马旋转的驱动力是 据信是由Mg 2 +-ATP复合物的初始结合能引起的， 从磷酸盐的释放，磷酸盐是Mg 2+配体。伽马的能力 将使用荧光微球测量F1的旋转亚基 附接到伽马子单元，如使用CCD相机记录的。扭矩 以及旋转之间的停留时间 将使用含有定点突变体或其他突变体的F1进行评估 在可能影响水解和水解之间偶联的位置进行处理， 伽玛旋转三个基因座作为研究的目标，包括： 开关3，γ亚单位C端和β亚单位润滑轴承 其靠近Mg 2+结合和磷酸盐释放的位点;（B）开关2， γ亚基和β亚基DELSEED序列之间的界面; 和（c）开关1，其中在γ亚基和β亚基之间形成氢键。 β E亚基捕获环。实验将检验Switch 1是擒纵机构的一部分，该擒纵机构仅允许伽马旋转， 催化位点填充有金属-核苷酸络合物。实验也 旨在确定催化位点处金属配体的变化， 特别是与伽马旋转相关的构象变化。

DESCRIPTION: (Applicant's Description) The relationship between the chemistry of ATP hydrolysis and the mechanical events that result in rotation of the gamma subunit of the F1-ATPase will be examined. Catalytic function of F1 derives from the asymmetry of the catalytic sites that, in turn, depends on the gamma subunit and the Mg2+ cofactor. The driving force for gamma rotation is believed to result from the initial binding energy of the Mg2+-ATP complex and from the release of phosphate which is a Mg2+ ligand. The ability of the gamma subunit of F1 to rotate will be measured using a fluorescent microsphere attached to the gamma subunit as recorded using a CCD camera. The torque generated during gamma rotation as well as the dwell time between rotations will be as assessed with F1 that contains site-directed mutants or other treatments at locations that may affect the coupling between hydrolysis and gamma rotation. Three loci are targeted for investigation that include: (a) Switch 3, the gamma subunit C-terminus and the beta subunit greasy bearing which is close to the site of Mg2+ binding and phosphate release; (b) Switch 2, the interface between the gamma subunit and the beta subunit DELSEED sequence; and (c) Switch 1, where hydrogen bonds form between the gamma subunit and the betaE subunit catch loop. Experiments will examine the possibility that Switch 1 is part of an escapement mechanism that only allows gamma rotation when the catalytic sites are filled with metal-nucleotide complex. Experiments are also designed to identify changes in metal ligands at the catalytic sites that are specifically associated with conformational changes linked to gamma rotation.