REGULATION OF CONTRACTION IN MUSCLE AND NON-MUSCLE CELLS

该实验室的长期目标是了解机制 肌肉和非肌肉细胞收缩的调节。 肌钙蛋白和肌钙蛋白参与的横纹肌细丝调节 原肌球蛋白尤其令人感兴趣。实验概述如下 本研究提出了利用定点突变技术进行基因突变的研究。 肌钙蛋白C的结构与功能关系 钙并通过与肌钙蛋白I的相互作用调节收缩 和肌钙蛋白T的结晶学分析 研究表明，肌钙蛋白C和钙调蛋白一样，有两个钙结合 由一个长的中心螺旋连接的区域。结构性的 蛋白质的一些特性，这些特性对其作为一种 钙结合调节蛋白与钙离子的传递 有关监管机构中钙结合状态的信息 域到其他成分的细丝将被 调查过了。 鸡肌钙蛋白C编码基因的构建 寡核苷酸及其蛋白的表达和纯化 来自E.Coli。长螺旋连接细胞的功能作用 肌钙蛋白C的两个结构域--钙的保守结构 调节蛋白，将通过改变其结构在一个 多种途径：其螺旋度和稳定性，其长度和 域之间的相对方向。 将使用寡核苷酸定向突变来改变 单个残基，并进行删除和插入。这个 将对突变株进行钙结合、络合物形成等分析 对于其他肌钙蛋白成分，肌动肌球蛋白的调节 ATPase，并最终取代皮肤纤维和X射线 结晶学。这些实验的另一个目的是 有助于我们对以下各项要求的总体理解 蛋白质中的α-螺旋结构。 第二个具体目标是确定地点和 肌钙蛋白I和肌钙蛋白T结合的结构要求 我们将使用两种方法：构建 肌钙蛋白C-钙调蛋白嵌合体及其残基的诱变 蛋白质的表面不会对 蛋白质的疏水核心。对嵌合体的分析应该 指出哪些残基对肌钙蛋白C的特异性至关重要 功能。随后将对特定残留物进行诱变。

The long term goal of the laboratory is to understand mechanisms of regulation of contraction in muscle and non-muscle cells. Striated muscle thin filament regulation involving troponin and tropomyosin is of particular interest. Experiments are outlined in the present proposal to use site-directed mutagenesis for study of structure-function relationships in troponin C. Troponin C binds calcium and regulates contraction via interaction with troponin I and troponin T. Crystallographic analysis of the structure has shown that troponin C, like calmodulin, has two calcium binding domains connected by a long central helix. The structural features of the protein that are important for its function as a calcium binding regulatory protein and transmission of information about the state of calcium binding in the regulatory domain to other components of the thin filament will be investigated. A cDNA encoding avian troponin C has been constructed from oligonucleotides and the protein has been expressed and purified from E. coli. The functional role of the long helix connecting the two domains of troponin C, a conserved structure in calcium regulatory proteins, will be evaluated by altering its structure in a variety of ways: its helicity and stability, its length and orientation of the domains relative to each other. Oligonucleotide directed mutagenesis will be used to change individual residues and to make deletions and insertions. The mutants will be analyzed for calcium binding, complex formation with other troponin components, regulation of the actomyosin ATPase, and ultimately substitution in skinned fibers and X-ray crystallography. An additional aim of the experiments is to contribute to our general understanding of the requirements for alpha-helical structure in proteins. The second specific aim is to determine the location and structural requirements of the troponin I and troponin T binding sites on-troponin C. We will use two approaches: construction of troponin C-calmodulin chimeras and mutagenesis of residues on the surface of the protein that do not contribute to the hydrophobic core of the protein. Analysis of chimeras should indicate which residues are critical for troponin C specific functions. Mutagenesis of specific residues will follow.