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 γ 亚基。 F1的催化功能 来自催化位点的不对称性，而催化位点的不对称性又取决于 γ 亚基和 Mg2+ 辅因子。伽马旋转的驱动力为 据信是由 Mg2+-ATP 复合物的初始结合能产生的 来自磷酸盐的释放，磷酸盐是 Mg2+ 配体。伽玛的能力 使用荧光微球测量要旋转的 F1 亚基 使用 CCD 相机记录的连接到伽马亚基的数据。扭矩 在伽马旋转期间生成以及旋转之间的停留时间 将使用包含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耦合的位置进行处理 伽马旋转。三个位点是调查的目标，包括：(a) 开关3，γ亚基C端和β亚基油脂轴承 靠近 Mg2+ 结合和磷酸盐释放的位点； (b) 开关 2， γ亚基和β亚基之间的界面DELSEED序列； (c) 开关 1，其中伽马亚基和 betaE 亚基 catch 循环。实验将检验 Switch 的可能性 1 是擒纵机构的一部分，仅当 催化位点充满金属-核苷酸复合物。实验还有 旨在识别催化位点金属配体的变化 与伽马旋转相关的构象变化特别相关。