INTERACTION OF ATP ANALOGUES WITH MYOSIN

ATP 类似物与肌球蛋白的相互作用

• 批准号: 2134446
• 负责人: RALPH G YOUNT
• 金额: $ 29.36万
• 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 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2134446
• 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.

将制备各种类型的ATP类似物以帮助表征所述