F1 ATPASE Chemical Mechanical Coupling Mechanisms

F1 ATP酶化学机械耦合机制

• 批准号: 6519558
• 负责人: WAYNE D FRASCH
• 金额: $ 25.86万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519558
• 关键词: Chlamydomonas; active sites; adenosine triphosphate; conformation; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; fluorescence microscopy; gene mutation; hydrogen transporting ATP synthase; interferometry; liposomes; magnesium ion; molecular dynamics; oxidative phosphorylation; phosphates; protein biosynthesis; site directed mutagenesis; tyrosine

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.

描述：（申请人的描述）化学之间的关系 ATP水解和导致旋转的机械事件 将检查F1-ATPase的伽马亚基。 F1的催化功能 源自催化位点的不对称性，进而取决于 伽马亚基和MG2+辅因子。伽马旋转的驱动力是 认为是由Mg2+-ATP复合物的初始结合能和 从磷酸盐的释放中，即Mg2+配体。伽玛的能力 F1的亚基将使用荧光微球测量 使用CCD摄像头记录在伽马亚基上。扭矩 在伽马旋转期间以及旋转之间的停留时间产生 将通过F1进行评估，该F1包含位置定向的突变体或其他 可能影响水解和可能影响耦合的位置的处理 伽马旋转。三个基因座的针对调查，其中包括：（a） 开关3，伽马亚基C末端和Beta亚基油腻的轴承 靠近MG2+结合和磷酸盐释放的位点； （b）开关2， 伽马亚基与β亚基序列之间的接口； （C）开关1，其中氢键在伽马亚基和 BETAE亚基捕获循环。实验将检查开关的可能性 1是逃生机制的一部分，仅在 催化位点充满了金属核苷酸复合物。实验也是 旨在识别在催化位点的金属配体变化 与与伽马旋转有关的构象变化特别相关。