REGULATION OF CONTRACTION IN MUSCLE AND NON-MUSCLE CELLS

这项研究的目的是了解肌肉变瘦的机制 分子水平上的微丝调控。激活收缩和收缩 动态事件，Ca~(2+)与传感器结合以传递信息 与靶细胞相互作用引起的细胞内钙离子浓度变化 蛋白质。在横纹肌中，钙离子与肌钙蛋白C(TNC)结合。TNC输入 TnI与TnT的复合体协同激活含肌动蛋白的Thin 细丝通过原肌球蛋白(TM)沿细丝的长度结合。 将使用分子遗传学和蛋白质设计来解决基本问题 关于TNC功能和TM保守N末端的问题。 TNC的x射线结构具有两个连接的球状钙结合域 通过一个延伸的中央螺旋。目标1是测试假设 正如所提出的，中央螺旋的灵活性对于功能是重要的 钙调素(CaM)。中央螺旋将被不同的 结构：α-螺旋，柔性随机卷曲，或刚性多聚脯氨酸II 螺旋。 虽然TNC和CaM的结构密切相关，但TNC有一个N- 末端α-螺旋是Cam所缺乏的。目标2是定义 TNC中的N-螺旋，它在激活细丝中的作用 Ca~(2+)的存在及其对稳定TNC的重要性。在目标1和目标2中， 突变人将使用寡核苷酸定向突变，突变人 蛋白质将在大肠杆菌中表达，纯化的蛋白质将被 使用已建立的功能和构象分析方法进行研究。 尽管有广泛使用的体外和原位TNC功能检测方法， 在活细胞中没有化验。目标3是开发一个分析系统 TNC在培养的小鼠肌肉C2细胞中的作用TNC表达式将为 使用反义寡核苷酸被抑制。细胞将被转基因 编码野生型或突变型TNCs的载体。对肌原纤维的影响 将分析组件和伸缩性。 纹状α-TM保守的N末端对肌动蛋白结合是至关重要的 和监管职能。目标4描述了使用N-末端的实验 盘绕合成多肽测定N-末端结构(核磁共振 和X射线结晶学)、折叠机制(圆二向色性、 量热法、核磁共振及N-端乙酰化在稳定化反应中的作用 盘绕线圈。

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.