MOLECULAR MECHANISM OF ENERGY COUPLING IN CA-ATPASE

CA-ATP酶能量耦合的分子机制

• 批准号: 3280910
• 负责人: TERRENCE L SCOTT
• 金额: $ 16.37万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-15 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280910
• 关键词: adenine nucleotides; adenosinetriphosphatase; affinity labeling; bioenergetics; calcium transporting ATPase; crosslink; enzyme mechanism; enzyme structure; fluorescent dye /probe; gel electrophoresis; high performance liquid chromatography; ion transport; laboratory rabbit; molecular site; photochemistry; point mutation; protein engineering; protein folding; protein structure function; sarcoplasmic reticulum; stop flow technique

The proposed project concerns the molecular mechanism of energy coupling by the Ca++ Mg++ ATPase of skeletal muscle sarcoplasmic reticulum (SR). The aim is to identify the functionally relevant regions of the polypeptide. The catalytic and the regulatory nucleotide sites will be labeled with photoaffinity nucleotide analogs and covalent site-directed probes. The regions of the polypeptide which contain the Ca++ sites will be identified by labeling with specific covalent reagents and Ca. Experiments will be done to determine the relation among the sites of phosphorylation and the catalytic and regulatory nucleotide sites will be determined by fluorescence energy transfer measurements. The functionally relevant polypeptide regions will be dissected by proteolytic enzymes and the resultant fragments separated by polyacrylamide slab gel electrophoresis. A two-dimensional peptide mapping technique will be implemented to allow the proteolytic fragments' alignment within the primary sequence of the ATPase polypeptide. The disposition of these specific regions relative to the internal and external aqueous compartments will be determined. The hydrophobic intramembranous segments of the polypeptide will be identified and placed within the primary sequence. Information concerning the pattern of protein folding within and through the bilayer will be deduced from the above results. Dynamic changes in the protein structure which occur concomitant with energy coupling will be specified by the simultaneous use of flash photoincorporation of probes and rapid mixing techniques. Changes in the structure of the functional sites will be determined at the critical reactions steps in which the enzyme interconverts between its two major conformational states. This will lead to new insights as to the relation between structure and function in the SR ATPase and the molecular basis of energy transduction. It is hoped that these results will enhance our understanding of the function of these systems in both normal and pathological conditions.

拟议的项目涉及能量耦合的分子机制 骨骼肌肌浆网（SR）的Ca ++ mg ++ ATPase。 这 目的是确定多肽与功能相关的区域。 催化和调节核苷酸位点将标记为 光亲和核苷酸类似物和共价定向探针。 这 将识别包含Ca ++位点的多肽区域 通过标记特定的共价试剂和CA。 实验将是 完成的是确定磷酸化部位与 催化和调节核苷酸位点将由 荧光能量传递测量。 功能相关 多肽区域将通过蛋白水解酶和 由聚丙烯酰胺平板凝胶电泳分离的结果片段。 将实施二维肽映射技术以允许 蛋白水解片段的对齐 ATPase多肽。 这些特定区域相对于 将确定内部和外部水隔室。 这 将确定多肽的疏水性膜内段 并放置在主序列中。 有关模式的信息 从双层中和通过双层折叠的蛋白质折叠将从 上述结果。 发生的蛋白质结构的动态变化 与能量耦合同时将通过同时使用指定 探针和快速混合技术的闪光灯企业。 更改 在功能位点的结构中，将在临界处确定 酶在其两个主要之间相互互换的反应步骤 构象状态。 这将导致有关关系的新见解 在SR ATPase中的结构和功能与分子基础之间 能量转导。 希望这些结果能够增强我们的 了解这些系统在正常和 病理状况。