Regulation of the Chloroplast ATP Synthase Rotary Motor

叶绿体 ATP 合酶旋转电机的调节

• 批准号: 0212908
• 负责人: Mark Richter
• 金额: $ 42万
• 依托单位: University of Kansas Center for Research Inc
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212908&HistoricalAwards=false
• 关键词: Regulation; Chloroplast; ATP; Synthase; Rotary

The F1-ATPase is a tiny molecular rotary motor driven by binding and hydrolysis of ATP in one direction and by trans-membrane proton flux in the other direction. Preliminary studies using single molecule fluorescence microspectroscopy demonstrate that a hybrid ATPase assembled from recombinant F1 subunits from photosynthetic organisms is capable of generating sufficient torque to propel large (1-2 micrometer) actin filaments through solution in a manner similar in most respects to its bacterial counterpart. Remarkably, rotation can be driven either by hydrolysis of CaATP or MgATP yet calcium, unlike magnesium, is known to be an ineffective co-substrate for proton coupled ATP hydrolysis or ATP synthesis. The fact that CaATP hydrolysis can drive the fully cooperative catalytic process without being coupled to proton movement has suggested a new catalytic model in which MgATP/ADP binding to the enzyme induces a special conformation of the enzyme which is required for proton coupling but not ATP hydrolysis. This project has the following two specific aims to test the new model and to identify the dynamic events leading to torque generation: 1. To identify the specific interactions between the gamma and epsilon subunits and the core hexameric alpha3 beta3 structure of the F1-ATPase that define the intermediate states of the catalytic sites involved in the catalytic cycle and its regulation. This will be addressed using a combination of site-directed mutagenesis and biochemical approaches; 2. To determine the magnitude and direction of relative movements of the gamma and epsilon subunits during catalysis and regulation of the F1-ATPase using real-time single molecule fluorescence microspectroscopy. The photosynthetic ATP synthase has several unique properties which separate it from its mitochondrial and bacterial counterparts and that offer new inroads to examine the remarkable rotary mechanism of the enzyme. One such property is the presence of a special regulatory segment located in the gamma subunit that has evolved in higher plant species to provide a molecular "switch" mechanism that tightly controls the catalytic activity of the enzyme. The switch is designed to block futile energy loss during photosynthesis. A major goal of this research is to identify the productive binding interactions between the gamma subunit and other subunits within the enzyme structure that are involved in the molecular switch mechanism. The information to be gained from this work is likely to prove seminal in understanding natural processes that have evolved to regulate the rotary motor, identifying different intermediate structural states that occur during rotation and the molecular steps involved in achieving these structural states. This in turn will provide the template to design gated "nanomachines" for a wide variety of important future practical applications. The research will also provide training in critical research skills for two doctoral students and at least two semesters of laboratory research experience for a minimum of eight undergraduate students, most of whom are likely to pursue graduate studies and to develop into independent scientists.

F1-ATP酶是一个微小的分子旋转马达，在一个方向上由ATP的结合和水解驱动，在另一个方向上由跨膜质子通量驱动。使用单分子荧光显微光谱的初步研究表明，从光合生物的重组F1亚基组装的混合ATP酶能够产生足够的扭矩，推动大（1-2微米）肌动蛋白丝通过溶液中的方式在大多数方面类似于其细菌对应物。 值得注意的是，旋转可以由CaATP或MgATP的水解驱动，但与镁不同，已知钙是质子偶联ATP水解或ATP合成的无效共底物。 事实上，CaATP水解可以驱动完全合作的催化过程，而不耦合到质子运动提出了一个新的催化模型，其中MgATP/ADP结合到酶诱导的特殊构象的酶，这是所需的质子耦合，但不是ATP水解。 该项目有以下两个具体目标来测试新模型并识别导致扭矩产生的动态事件：1。确定F1-ATP酶的γ和β亚基与核心六聚体α 3 β 3结构之间的特异性相互作用，这些结构定义了参与催化循环及其调节的催化位点的中间状态。 这将使用定点诱变和生物化学方法的组合来解决; 2.采用实时单分子荧光显微光谱法测定F1-ATP酶催化和调节过程中γ亚基和γ亚基相对运动的幅度和方向。光合ATP合酶具有几种独特的性质，将其与线粒体和细菌的对应物分开，并为研究该酶的显着旋转机制提供了新的进展。 一个这样的性质是存在一个特殊的调节片段位于γ亚基，已在高等植物物种中进化，以提供分子“开关”机制，严格控制酶的催化活性。 这个开关的设计是为了阻止光合作用过程中无用的能量损失。 本研究的一个主要目标是确定γ亚基和酶结构中参与分子开关机制的其他亚基之间的有效结合相互作用。 从这项工作中获得的信息很可能证明是开创性的理解自然过程，已经演变为调节旋转电机，确定不同的中间结构状态，在旋转过程中发生的分子步骤，在实现这些结构状态。 这反过来将提供模板来设计门控“纳米机器”，用于各种重要的未来实际应用。 该研究还将为两名博士生提供关键研究技能培训，并为至少八名本科生提供至少两个学期的实验室研究经验，其中大多数人可能会继续研究生学习并发展成为独立的科学家。