INTERACTIONS OF SKELETAL MUSCLE REGULATORY PROTEINS

骨骼肌调节蛋白的相互作用

• 批准号: 2390520
• 负责人: JOHN H COLLINS
• 金额: $ 20.49万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-15 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390520
• 关键词: Escherichia coli; X ray crystallography; actins; calcium flux; crosslink; crystallization; fluorescence spectrometry; fluorescent dye /probe; intermolecular interaction; microfilaments; muscle contraction; muscle relaxation; myofibrils; site directed mutagenesis; striated muscles; tropomyosin; troponin

The long-term goal of this proposal is to elucidate the molecular mechanism of troponin-linked calcium regulation of skeletal muscle contraction. Several experimental approaches will be used to gain a detailed understanding of Ca2+-dependent interactions among the following thin filament proteins: the Ca2+-binding troponin C (TnC), the inhibitory troponin I (TnI), actin, and the tropomyosin-binding protein troponin T (TnT). The specific aims are to elucidate: (1) the mechanism whereby Ca2+ binding to TnC changes its interaction with TnI, thus triggering the series of events leading to muscle contraction; (2) the mechanism whereby Ca2+- dependent changes in TnC-Tnl interactions are transmitted to actin in the thin filament; (3) the vital but little-characterized interactions of TnT with TnC and TnI. The principal experimental methods to be used are: (1) site-directed mutagenesis of the three Tn subunits in order to provide functionally interesting sites for spectroscopic probes and chemical crosslinkers; (2) covalent crosslinking to identify sites of contact among the proteins: (3) frequency domain measurements of fluorescence resonance energy transfer to measure distance distributions between specific sites in thin filament protein complexes: (5) crystallization studies with the goal of preparing troponin subunits and complexes for three-dimensional structure determination by X-ray diffraction. The results of these studies may help to diagnose, prevent, and perhaps ultimately cure, congenital muscle diseases, some resulting from mutations of single amino acids, in which crucial aspects of Ca2+ regulation are not functioning normally.

该提案的长期目标是阐明分子 骨骼肌肌钙蛋白相关钙调节机制 收缩。将使用多种实验方法来获得 详细了解以下 Ca2+ 依赖性相互作用 细丝蛋白：Ca2+ 结合肌钙蛋白 C (TnC) 肌钙蛋白 I (TnI)、肌动蛋白和原肌球蛋白结合蛋白肌钙蛋白 T （TnT）。 具体目标是阐明：（1）Ca2+结合的机制 TnC 改变了它与 TnI 的相互作用，从而引发了一系列 导致肌肉收缩的事件； (2)Ca2+-的作用机制 TnC-Tnl 相互作用的依赖性变化传递至肌动蛋白 细丝； (3) TnT 重要但鲜为人知的相互作用 与 TnC 和 TnI 一起。 主要采用的实验方法有：（1）定点实验 三个 Tn 亚基的诱变，以提供功能 光谱探针和化学交联剂的有趣场所； (2) 共价交联以确定蛋白质之间的接触位点：(3) 荧光共振能量转移的频域测量 测量细丝中特定位点之间的距离分布 蛋白质复合物：（5）结晶研究，目的是制备 三维结构的肌钙蛋白亚基和复合物 通过X射线衍射测定。 这些研究的结果可能有助于诊断、预防，甚至可能有助于 最终治愈先天性肌肉疾病，其中一些是由突变引起的 单个氨基酸，其中 Ca2+ 调节的关键方面不 功能正常。