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.

将准备各种类型的 ATP 类似物来帮助表征 肌肉蛋白质中核苷酸-蛋白质相互作用的性质。新班级 将制备类似物，利用其形成稳定的能力 氨基甲酰基与 ATP 的 2'(3') 羟基相连。新的 ATP/ADP 亲和力 柱将用于制备各种修饰的肌球蛋白亚片段 l (S1) 衍生物，用于潜在结晶研究。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 S1用ATP光亲和衍生物进行光标记。化学品 还原甲基化 S1 的性质将通过使用 [3H]H2CO 定义为 一种帮助制备更均匀的 S1 制剂的方法 结合核苷酸的结晶研究。光亲和标记 ATP 类似物和扇贝肌球蛋白的研究将探索最近 发现了存在时发生的差异标记 Ca2+ 的调节量。突变的轻链将被纳入 扇贝肌球蛋白来检验 Ca2+ 与必需物质结合的假设 轻链直接影响活性位点。其机制为 肌球蛋白活性位点的钒酸盐光裂解和光修饰将 被探索。这项研究应该成为其他研究的范例 钒酸盐促进结合磷酸化的蛋白质的光裂解 基材。 其他研究将制备核糖修饰的ATP重金属衍生物 和 GTP（Re 复合物）用于单晶 X 射线研究和 ATP Au11 复合物连接到 2'(3') 核糖羟基上，用于 通过电子显微镜和图像定义粗丝结构 重建研究。