INTERACTION OF ATP ANALOGUES WITH MYOSIN

将准备各种类型的ATP类似物，以帮助表征 肌肉蛋白质中核苷酸-蛋白质相互作用的性质。一个新班级 将准备一系列类似物，利用形成稳定的 与ATP的2‘(3’)羟基连接的氨甲酰基。新的ATP/ADP亲和力 将利用柱子制备不同修饰的肌球蛋白亚片段 L(S1)用于潜在结晶研究的衍生物。这些 衍生物包括通过两个动力学活性半胱氨酸交联化的S1 和S1用三磷酸腺苷光亲和衍生物进行光标记。化学物质 还原甲基化的S1的性质将通过使用[~3H]H_2CO定义为 一种帮助制备更均一的S1制剂的方法 结合核苷酸的结晶研究。光亲和标记 对ATP类似物和扇贝肌球蛋白的研究将探索最近 发现的差异标记发生在存在 钙离子的调节量。突变的轻链将被合并到 扇贝肌球蛋白验证钙离子与人体必需钙结合的假说 轻链以直接方式影响活动站点。它的作用机制 肌球蛋白活性部位的钒酸光裂解和光修饰 被探索。这项研究应该为其他研究提供一个范例 钒酸盐促进结合磷酸化的蛋白质的光解 底物。 其他研究将制备核糖修饰的三磷酸腺苷重金属衍生物 和GTP(Re络合物)，用于单晶X射线研究和ATP 将AU11络合物连接到2‘(3’)核糖羟基上，用于 用电子显微镜和图像确定粗丝结构 重建研究。

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.