Structure and Dynamics of the Chloroplast ATP Synthase

叶绿体 ATP 合酶的结构和动力学

• 批准号: 9506255
• 负责人: Mark Richter
• 金额: $ 24万
• 依托单位: University of Kansas Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506255&HistoricalAwards=false
• 关键词: Structure; Dynamics; Chloroplast; ATP; Synthase

; R o o t E n t r y F (& !? C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l !? !? F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT 9506255 cynthia m cox cynthia m cox @ + r e = e j j j j j j j 1 # Y 8 T 5 j j j j j ~ j j j j 9506255 Richter This research addresses the relationship between the structure, function and regulation of the ATP synthase enzyme. This allosteric enzyme reversibly utilizes the energy of a transmembrane proton gradient to synthesize ATP. The long term goal of the research is to contribute toward an understanding of the mechanism of the energy transduction process and to understand how this enzyme is regulated under physiological conditions in plants and animals. Prior NSF funding has led to: 1) Cloning and over-expression of all five subunits of the catalytic F1 portion of the chloroplast ATP synthase (CF1); 2) Recovery of the subunits from bacterial inclusion bodies and refolding them into their native, active forms; and 3) Reconstituting them into a functional F1 complex. Further studies will utilize this system to genetically engineer CF1 subunits for analyzing regions of functional importance, and to examine functional dynamics of subunits during the catalytic process using biochemical and biophysical techniques. This proposal describes a collaborative multidiciplinary approach focusing on the structure and function of the three smaller CF1 subunits, ( ( and (. Genetic engineering experiments will involve: 1) Site-directed mutagenesis studies to probe the functional and structural importance of different regions of the polypeptide chains via replacement or deletion of specific amino acids or sequences of amino acids; 2) Studies of subunit dynamics through attachment of intrinsic (tryptophans) and extrinsic (via cysteine residues) fluorescent probes to specific sites on the ( ( and ( subunits. Labeled subunits will be reconstituted with CF1 deficient in these subunits for fluorescence studies aimed at (a) structural mapping of subunits within the CF1 complex using fluorescence resonance energy transfer measurements and (b) monitoring subunit dynamics during catalytic turnover by the enzyme by monitoring the time-dependent anisotropy of intrinsic and extrinsic fluorescent probes; 3) Chemical crosslinking studies involving attachment of bifunctional chemical crosslinking agents at various engineered sites within the ( and ( subunits. Mutant subunits will be reconstituted with the other CF1 subunits and with the membrane-bound proton channel portion (CF0) of the enzyme for chemical crosslinking studies aimed at identifying the sites of interaction between CF1 and CF0 subunits. %%% This research addresses the relationship between the chemical structure and biological function of the ATP synthase enzyme of plant chloroplast membranes. This enzyme is responsible for the conversion of the energy from sunlight into the chemical storage form. ATP (adenosine triphosphate). ATP in turn supplies the energy for the conversion of carbon dioxide into sugar in plants. Analogous enzymes supply more than 90% of the ATP required for energy-dependent metabolic processes in animals and bacteria. The long term goal of the research described in this proposal is to identify, at the molecular level, how the ATP synthase enzymes capture energy and convert it into ATP. We intend to approach this goal by genetically engineering the ATP synthase so as to probe the importance of specific sites within the enzyme for the energy conversion (catalytic) process. Some of the genetically engineered sites will provide sites for attachment of chemical probes which will be used to identify specific events involving changes in the structure of the enzyme which occur during the catalytic process. Attachment of other chemical probes to genetically engineered sites will allow us to cross-link (tether) pieces of the enzyme together so that we can identify specific sites of protein-protein interaction which occur within the enzyme and which are important for catalysis. A thorough understanding of this very efficient natural energy conserving process will potentially lead to the design and implementation of vastly improved energy storage and utilization processes or human use. *** ; S u m m a r y I n f o r m a t i o n (

; Root Entry F (& !? Comp O b j b Word 文档对象池 !? !? F Microsoft Word 6.0 文档 MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT 9506255 cynthia m考克斯 辛西娅·米·考克斯 @ + re = e j j j j j j j 1 # Y 8 T 5 j j j j j ~ j j j j 9506255 Richter 这项研究探讨了 ATP 合成酶的结构、功能和调节之间的关系。 这种变构酶可逆地利用跨膜质子梯度的能量来合成 ATP。 该研究的长期目标是促进对能量机制的理解 转导过程并了解该酶在植物和动物的生理条件下如何受到调节。 之前 NSF 的资助已导致： 1) 叶绿体 ATP 合酶 (CF1) 催化 F1 部分的所有五个亚基的克隆和过度表达； 2) 从细菌包涵体中回收亚基并将其重新折叠成天然的活性形式； 3) 将它们重构为功能性 F1 复合物。 进一步的研究将利用该系统对 CF1 亚基进行基因改造，以分析具有重要功能的区域，并使用生化和生物物理技术检查催化过程中亚基的功能动态。 该提案描述了一种协作性多学科方法，重点关注三个较小 CF1 亚基的结构和功能，（（和（。基因工程实验将涉及：1）定点诱变研究，通过替换或删除特定氨基酸或氨基酸序列来探测多肽链不同区域的功能和结构重要性；2）通过附着进行亚基动力学研究 将内在（色氨酸）和外在（通过半胱氨酸残基）荧光探针定位到 ( 和 ( 亚基上的特定位点。标记的亚基将用这些亚基中缺乏的 CF1 进行重建，用于荧光研究，目的是 (a) 使用荧光共振能量转移测量对 CF1 复合物内的亚基进行结构映射，以及 (b) 在催化过程中监测亚基动态 通过监测内在和外在荧光探针的时间依赖性各向异性来检测酶的周转； 3) 化学交联研究，涉及在 ( 和 ( 亚基内的各个工程位点连接双功能化学交联剂。突变亚基将与其他 CF1 亚基以及酶的膜结合质子通道部分 (CF0) 重建 用于化学交联研究，旨在确定 CF1 和 CF0 亚基之间的相互作用位点。 %%% 这项研究探讨了植物叶绿体膜 ATP 合酶的化学结构和生物功能之间的关系。 这种酶负责将阳光中的能量转化为化学储存形式。 ATP（三磷酸腺苷）。 ATP 反过来为转化提供能量 二氧化碳在植物中转化为糖。 类似的酶提供了动物和细菌中能量依赖性代谢过程所需 90% 以上的 ATP。 该提案中描述的研究的长期目标是在分子水平上确定 ATP 合酶如何捕获能量并将其转化为 ATP。 我们打算通过对 ATP 合酶进行基因改造来实现这一目标，从而探讨 酶内用于能量转换（催化）过程的特定位点。 一些基因工程位点将提供化学探针的附着位点，这些化学探针将用于识别涉及催化过程中发生的酶结构变化的特定事件。 将其他化学探针附着在基因工程位点上将使我们能够将酶的片段交联（系链）在一起，以便我们能够识别特定的位点 酶内发生的蛋白质-蛋白质相互作用对于催化很重要。 对这种非常有效的自然节能过程的透彻理解将有可能导致设计和实施大大改进的能量存储和利用过程或人类使用。 ***; 摘要信息（