SITE-DIRECTED MUTAGENESIS OF TROPOMYOSIN

原肌球蛋白的定点诱变

• 批准号: 2217902
• 负责人: Sarah Ellen Hitchcock-DeGregori
• 金额: $ 24.31万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2217902
• 关键词: Escherichia coli; acetylation; actins; binding proteins; chemical structure function; complementary DNA; gene expression; genetic manipulation; molecular genetics; muscle contraction; mutant; myosins; posttranslational modifications; protein sequence; protein structure; site directed mutagenesis; striated muscles; tissue mosaicism; tropomyosin; troponin

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.

研究的长期目标是了解 原肌球蛋白在细丝相关调节中的功能。 Tropomyosin 与肌钙蛋白沿着细丝组装，它们主要 负责横纹肌收缩的钙依赖性调节 肌肉. 原肌球蛋白使细肌丝作为一种合作的 单位 该建议侧重于一套假设的基本结构 原肌球蛋白功能所需的特征。 从更广泛的角度来看， 原肌球蛋白是卷曲螺旋纤维蛋白质的最佳研究实例。 分子遗传学提供了一个前所未有的机会， 原肌球蛋白结构与功能关系 总的来说是线圈。 方法学方法将是定点诱变， 原肌球蛋白cDNA和突变型和野生型cDNA在E. 大肠杆菌用于生产待用生物化学分析的蛋白质， 生物物理和细胞方法。 具体目标是： 1. 为了获得一种功能完整的重组原肌球蛋白， 肌动蛋白结合和调节特性的原肌球蛋白分离自 肌肉;翻译后乙酰化在原肌球蛋白功能中的作用。 2. 为了确定保守的氨基末端在功能中的作用 是肌动蛋白结合位点吗 3. 为了确定原肌球蛋白的内部周期性重复，它们的 长度和序列对于肌动蛋白结合和调节是重要的。 4. 探讨原肌球蛋白外显子结构的功能意义。 5. 为了评估七肽重复序列的参数， 短距离和长距离通信的决定因素，稳定性和 原肌球蛋白的协同性。