REGULATION OF CONTRACTION IN MUSCLE AND NON-MUSCLE CELLS

研究的目的是了解肌肉变瘦的机制 在分子水平上的灯丝调节。为了激活收缩和 在运动事件中，Ca 2+与换能器结合以传递信息 细胞内Ca 2+浓度通过与靶点的相互作用 proteins.在横纹肌中，Ca 2+与肌钙蛋白C（TnC）结合。TnC in 与TnI和TnT的复合物协同激活含肌动蛋白的瘦素， 通过原肌球蛋白（TM）沿沿着细丝的长度结合。 分子遗传学和蛋白质设计将用于解决基本的 关于TnC功能和TM保守的N-末端的问题。 TnC的X射线结构具有两个连接的球形Ca 2+结合结构域 由一个延伸的中央螺旋结构。目的1是检验假设， 中央螺旋的灵活性对功能很重要，正如所提出的那样， 钙调素（CaM）。 中央螺旋将被不同的 结构：α-螺旋、柔性无规卷曲或刚性聚脯氨酸II 螺旋。 虽然TnC和CaM的结构密切相关，但TnC具有N- CaM缺乏的末端α螺旋。目标2：定义 TnC中的N-螺旋，它在激活细胞中的细丝中的作用， Ca 2+的存在，以及它在稳定TnC中的重要性。在目标1和2中， 突变体将使用阿托伐他汀定向诱变制备，突变体 蛋白质将在E.大肠杆菌，纯化的蛋白质将被 使用已建立的功能和构象测定进行研究。 尽管广泛使用体外和原位测定TnC功能， 在活细胞中没有测定。目标3是开发一个系统来分析 TnC在培养的小鼠肌肉C2细胞中的功能。TnC表达将是 使用反义寡核苷酸抑制。细胞将被转染， 编码野生型或突变型TnC的载体。对肌原纤维的影响 将分析组装和收缩性。 横纹α-TM保守的N-末端对肌动蛋白结合至关重要 和调节功能。目的4描述了使用N-末端的实验 卷曲螺旋合成肽，以确定结构的N-末端（NMR 和X射线晶体学），折叠机理（圆二色性， 量热法，NMR和N-末端乙酰化在稳定 盘绕的线圈

The goal of the research is to understand the mechanism of muscle thin filament regulation at the molecular level. To activate contractile and motile events, Ca2+ binds to a transducer in order to transmit information about the intracellular Ca2+ concentration via interaction with target proteins. In striated muscles, Ca2+ binds to troponin C (TnC). TnC in complex with TnI and TnT cooperatively activates the actin-containing thin filament through tropomyosin (TM)bound along the length of the filament. Molecular genetics and protein design will be used to address fundamental questions about TnC function and the conserved N-terminus of TM. The x-ray structure of TnC has two globular Ca2+ binding domains connected by an extended central helix. Aim 1 is to test the hypothesis that the flexibility of the central helix is important for function, as proposed for calmodulin (CaM). The central helix will be replaced with different structures: alpha-helix, flexible random coil, or a rigid polyproline II helix. Though the structures of TnC and CaM are closely related, TnC has a N- terminal alpha-helix that CaM lacks. Aim 2 is to define the function of the N-helix in TnC, its role in activating the thin filament in the presence of Ca2+, and its importance in stabilizing TnC. In Aims 1 and 2, mutants will be made using oligonucleotide-directed mutagenesis, mutant proteins will be expressed in E. coli, and purified proteins will be studied using established assays for function and conformation. Though there are widely used in vitro and in situ assays for TnC function, there is no assay in living cells. Aim 3 is to develop a system to analyze TnC function in cultured mouse muscle C2 cells. TnC expression will be inhibited using antisense oligonucleotides. Cells will be transfected with vectors encoding wildtype or mutant TnCs. The consequences on myofibril assembly and contractility will be analyzed. The conserved N-terminus of striated alpha-TM is crucial for actin binding and regulatory function. Aim 4 describes experiments to use N-terminal coiled coil synthetic peptides to determine the structure N-terminus (NMR and x-ray crystallography), folding mechanism (circular dichroism, calorimetry, NMR and role of N-terminal acetylation in stabilizing the coiled coil.