SITE DIRECTED MUTAGENESIS OF TROPOMYOSIN

这项研究的长期目标是了解其发病机制 原肌球蛋白在细丝连锁调节中的作用。原肌球蛋白 与肌钙蛋白沿着细丝聚集在一起，它们主要是 负责纹状体收缩的钙依赖性调节 肌肉。原肌球蛋白使细丝发挥协同作用 单位。 该提案集中于一系列关于基本结构的假设 功能所需的原肌球蛋白的特性。从更广泛的角度来看， 原肌球蛋白是研究得最好的卷曲状纤维蛋白。 分子遗传学为研究提供了前所未有的机会 原肌球蛋白的结构-功能关系 大体上是线圈。 方法论方法将是定点突变。 原肌球蛋白基因及突变型和野生型基因在E. 大肠杆菌，用于生产生化分析的蛋白质， 生物物理和细胞方法。 具体目标是： 1.获得具有完整功能的重组原肌球蛋白 原肌球蛋白的肌动蛋白结合和调节特性 肌肉；翻译后乙酰化在原肌球蛋白功能中的作用。 2.确定保守的氨基末端在功能中的作用 肌肉原肌球蛋白；它是肌动蛋白结合部位吗？ 3.为了确定原肌球蛋白的内部周期性重复序列是否，他们的 长度和序列对肌动蛋白的结合和调控很重要。 4.确定原肌球蛋白外显子结构的功能意义。 5.评估七肽重复序列的参数为 短距离和远程通信的决定因素、稳定性和 原肌球蛋白的协同作用。

The long term objective of the research is to understand the mechanism of tropomyosin function in thin filament linked regulation. Tropomyosin assembles with troponin along filaments and together they are primarily responsible for calcium dependent regulation of contraction in striated muscles. Tropomyosin allows the thin filament to function as a cooperative unit. The proposal focuses on a set of hypotheses concerning the basic structural features of tropomyosin required for function. From a broader perspective, tropomyosin is the best studied example of a coiled-coil fibrous protein. Molecular genetics offers an unprecedented opportunity to investigate structure-function relationships in tropomyosin in particular and coiled coils in general. The methodological approach will be site-directed mutagenesis of tropomyosin cDNAs and overexpression of mutant and wildtype cDNAs in E. coli for production of protein to be analyzed using biochemical, biophysical and cellular methods. The Specific Aims are: 1. To obtain a fully functional recombinant tropomyosin that retains the both actin binding and regulatory properties of tropomyosin isolated from muscle; the role of post-translational acetylation in tropomyosin function. 2. To determine the role of the conserved amino terminus in the function of muscle tropomyosin; is it an actin binding site? 3. To determine if the internal periodic repeats of tropomyosin, their length and sequence are important for actin binding and regulation. 4. To determine the functional significance of tropomyosin exon structure. 5. To evaluate the parameters of the heptapeptide repeat that are determinants of short range and long range communication, stability and cooperativity in tropomyosin.