REGULATION OF CONTRACTION IN MUSCLE AND NON-MUSCLE CELLS

实验室的长期目标是了解 调节肌肉和非肌肉细胞的收缩。 涉及肌钙蛋白的横纹肌细丝调节 原肌球蛋白是特别感兴趣的。 实验概述于 本发明建议使用定点突变来研究 肌钙蛋白C的结构-功能关系 肌钙蛋白C结合 通过与肌钙蛋白I相互作用调节收缩 和肌钙蛋白T 结构的晶体学分析 显示肌钙蛋白C，像钙调蛋白，有两个钙结合， 由长的中央螺旋连接的结构域。 结构性 蛋白质的功能，这是重要的，作为一个 钙结合调节蛋白与 关于调节中钙结合状态的信息 域的其他组成部分的细丝将是 研究了 已构建了编码禽肌钙蛋白C的cDNA， 寡核苷酸和蛋白质已表达和纯化 来自大肠杆菌 长螺旋连接的功能作用 肌钙蛋白C的两个结构域， 调节蛋白，将通过改变其结构进行评估， 各种方式：它的螺旋度和稳定性，它的长度和 域相对于彼此的取向。 寡核苷酸定向诱变将用于改变 单个残基并进行缺失和插入。 的 将分析突变体的钙结合、复合物形成 与其他肌钙蛋白组分，肌动球蛋白的调节 ATP酶，并最终取代皮肤纤维和X射线 结晶学 实验的另一个目的是 有助于我们对以下要求的一般理解 蛋白质中的α-螺旋结构。 第二个具体目标是确定地点， 肌钙蛋白I和肌钙蛋白T结合的结构要求 肌钙蛋白C位点。 我们将使用两种方法： 肌钙蛋白C-钙调蛋白嵌合体和 蛋白质的表面，不有助于 蛋白质的疏水核心。 嵌合体分析应 表明哪些残基对于肌钙蛋白C特异性 功能协调发展的 随后将对特定残基进行诱变。

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.