F1 ATPASE Chemical Mechanical Coupling Mechanisms

描述：(申请人的描述)化学与化学之间的关系 三磷酸腺苷的水解和机械事件导致的旋转 将检查F1-ATPase的伽马亚基。F1的催化作用 源于催化位置的不对称性，而催化位置的不对称性又取决于 伽马亚基和镁离子辅因子。伽马旋转的驱动力是 被认为是由镁离子-三磷酸腺苷复合体的初始结合能和 由于磷酸盐的释放，这是一种镁离子配体。伽马的能力 将使用荧光微球测量F1亚基的旋转 附着在伽马亚单位上，如使用CCD相机记录的。扭矩 在伽马旋转期间生成以及旋转之间的驻留时间 将与含有定点突变或其他基因的F1进行评估 在可能影响水解液和水解液之间的耦合的位置进行处理 伽马旋转。有三个基因座是调查目标，包括：(A) 开关3，伽马亚基C端和贝塔亚基光滑轴承 其靠近镁离子结合和磷酸盐释放的部位；(B)开关2， 伽马亚基和β亚基脱色序列之间的界面； 和(C)开关1，其中在伽马亚单位和 BetaE亚基捕获环。实验将检验开关是否有可能 1是擒纵机构的一部分，该机构只允许在 催化中心充满了金属-核苷酸络合物。实验也是 旨在识别催化位置上的金属配体的变化 特别是与伽马旋转有关的构象变化。

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.