INTERACTION OF ATP ANALOGUES WITH MYOSIN

ATP 类似物与肌球蛋白的相互作用

• 批准号: 2856697
• 负责人: RALPH G YOUNT
• 金额: $ 30.29万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2856697
• 关键词: active sites; adenosine triphosphate; affinity chromatography; affinity labeling; chemical cleavage; chemical synthesis; conformation; crosslink; crystallization; enzyme structure; enzyme substrate analog; fluorescence resonance energy transfer; fluorescent dye /probe; myosins; nucleotide analog; photochemistry; photolysis; protein engineering; protein purification; rare earth element

ATP analogs of various types will be prepared to help characterize the nature of nucleotide-protein interactions in contractile proteins with the long-term goal of explaining the molecular basis of muscle contraction. A new class of second generation ATP photoaffinity analogs will be synthesized which carry with them attached reporter groups. These groups will include spin labels, fluorescent and luminescent probes. ATP analogs will be photo-incorporated after first trapping them at the active site by use of the phosphate analogs, vanadate, AlF4, or BeFx. The attached analogs can be displaced from the active site by actin treatment to yield fully active myosin either in solution or in glycerinated muscle fibers. The probes, attached to either side of the active site, will be used to measure distances to other defined parts of myosin (or to actin) by use of the new technique of luminescence resonance energy transfer in collaboration with P. Selvin and R. Cooke. Specifically, changes in the relative orientation of the motor and light chain domains of subfragment 1 as influenced by nucleotides or actin binding will be assessed. Related experiments will utilize a new class of non-nucleoside triphosphate analogs to introduce specific spin probes at myosin's active site in muscle fibers to probe for global orientation changes in heads of myosin during the contraction cycle utilizing EPR spectroscopy. The properties of new thiol mutants of Dictyostelium myosin will be determined with the goal of preparing crystals of thiol-crosslinked subfragment for x-ray structure determination.

各种类型的ATP类似物将准备好有助于表征 收缩蛋白中核苷酸 - 蛋白质相互作用的性质与 解释肌肉分子基础的长期目标 收缩。第二代ATP Phothabrational类似物的新类 将合成随身携带的记者组。 这些组将包括自旋标签，荧光和发光 探针。 ATP类似物将在首次捕获之后被合并 通过使用磷酸盐类似物，钒酸盐，alf4或 befx。附着的类似物可以通过 肌动蛋白治疗以在溶液中或在 甘油纤维纤维。探针，附着在 主动地点将用于测量与其他定义部分的距离 肌球蛋白（或肌动蛋白）通过使用新的发光技术 共振能量转移与P. Selvin和R. Cooke合作。 具体而言，电动机和光的相对方向的变化 核苷酸或肌动蛋白的影响1的链域1 绑定将被评估。相关实验将利用新课程 非核苷三磷酸类似物的引入特定自旋探针 在肌球蛋白在肌肉纤维中的活跃部位，以探测全球取向 利用EPR的收缩周期期间肌球蛋白头部的变化 光谱法。 肌球蛋白肌球蛋白的新硫醇突变体的特性将是 用准备硫醇 - 跨链接的晶体确定 X射线结构确定的亚碎片。