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.

将制备各种类型的ATP类似物以帮助表征 收缩蛋白质中核苷酸-蛋白质相互作用的性质 解释肌肉分子基础的长期目标 收缩。一类新的第二代ATP光亲和类似物 将被合成，其带有连接的报告基团。 这些组将包括自旋标记，荧光和发光 probes. ATP类似物在第一次捕获它们后将被光结合 通过使用磷酸盐类似物、钒酸盐、AlF 4或 BeFx。连接的类似物可以通过以下方式从活性位点置换： 肌动蛋白处理以在溶液或溶液中产生完全活性的肌球蛋白， 甘油化的肌肉纤维探针，连接到任何一侧的 活动站点，将用于测量到其他定义部分的距离 肌球蛋白（或肌动蛋白）的发光新技术 共振能量转移，与P. Selvin和R.库克 具体来说，电机和光的相对方向的变化 受核苷酸或肌动蛋白影响的亚片段1的链结构域 将评估约束力。相关实验将利用一个新的类 非核苷三磷酸类似物引入特异性自旋探针 在肌纤维中肌球蛋白的活性部位， 利用EPR在收缩周期中肌球蛋白头部的变化 谱 新的网囊藻肌球蛋白巯基突变体的性质将是 目的是制备硫醇交联的晶体 用于X射线结构测定的亚片段。