INTERACTION OF ATP ANALOGUES WITH MYOSIN

ATP 类似物与肌球蛋白的相互作用

• 批准号: 2879360
• 负责人: RALPH G YOUNT
• 金额: $ 10.59万
• 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 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2879360
• 关键词: active sites; adenosine triphosphate; affinity chromatography; affinity labeling; calcium; chemical cleavage; chemical synthesis; conformation; crosslink; crystallization; divalent cations; enzyme inhibitors; enzyme substrate analog; high performance liquid chromatography; laboratory rabbit; myosins; nucleotide analog; oxidation; photochemistry; photolysis; protein purification; protein structure; scanning transmission electron microscopy; vanadium

ATP analogues of various types will be prepared to help characterize the nature of nucleotide-protein interactions in muscle proteins. A new class of analogues will be prepared which exploit the ability to form a stable carbamoyl linkage to the 2'(3')hydroxyls of ATP. New ATP/ADP affinity columns will be utilized to prepare variously modified myosin subfragment l (S1) derivatives for potential crystallization studies. These derivatives include S1 cross-linked via two kinetically reactive cysteines and S1 photolabeled with ATP photoaffinity derivatives. The chemical nature of reductively methylated S1 will be defined by use of [3H]H2CO as a means to help prepare more homogeneous S1 preparations for crystallization studies with bound nucleotide. Photoaffinity labeling studies with ATP analogues and scallop myosin will explore the recently discovered differential labeling which occurs in the presence of regulatory amounts of Ca2+. Mutant light chains will be incorporated into scallop myosin to test the hypothesis that Ca2+ binding to the essential light chains affects the active site in a direct manner. The mechanism of vanadate photocleavage and photomodification of myosins active site will be explored. This study should serve as a paradigm for other studies of vanadate promoted photocleavage of proteins which bind phosphorylated substrates. Other studies will prepare ribose-modified heavy metal derivatives of ATP and GTP (Re complexes) for use in single crystal x-ray studies and ATP with Au11 complexes attached to the 2'(3') ribose hydroxyls to be used to define thick filament structure by electron microscopy and image reconstruction studies.

各种类型的ATP类似物将准备好有助于表征 肌肉蛋白中核苷酸 - 蛋白质相互作用的性质。一个新课 将准备类似物，以利用形成稳定的能力 与ATP的2'（3'）羟基的卡巴氨基链接。新的ATP/ADP亲和力 列将用于准备各种修改的肌球蛋白亚纹码 L（S1）衍生物用于潜在的结晶研究。这些 衍生物包括通过两个动力学反应性半胱氨酸交联的S1 和S1具有ATP光相位衍生物的光标记。化学物质 还原性甲基化S1的性质将通过使用[3H] H2CO定义 一种帮助准备更均匀的S1准备工作的方法 结晶核苷酸的结晶研究。光性标签 使用ATP类似物和扇贝肌球蛋白的研究将探索最近 发现的差异标记是在存在的情况下发生的 CA2+的调节量。突变轻链将被合并到 扇贝肌球蛋白测试Ca2+与必不可少的假设 光链以直接方式影响活跃位点。机制 蛋白活性位点的钒酸盐光裂解和光疗法将 被探索。这项研究应作为其他研究的范式 钒酸盐促进了蛋白质的光丝分裂，这些蛋白质结合了磷酸化的蛋白质 基材。 其他研究将准备ATP的核糖改性重金属衍生物 在单晶X射线研究和ATP中使用的GTP（RE复合物） Au11复合物附着在2'（3'）核糖羟基上 通过电子显微镜和图像定义厚细丝结构 重建研究。