SITE-DIRECTED MUTAGENESIS OF CARDIAC A-TROPOMYOSIN

α原肌球蛋白(Alpha TM)是一个复杂的多基因家族的成员 含有多种异构体的。所表达的TM的类型取决于 特定细胞类型的发育阶段和生理状态 组织，尽管这样的信息没有发表在心肌上 在发育和肥大期间。阿尔法阿尔法-TM在成人中存在 小型哺乳动物的心肌和骨骼肌。 作为一种理解不同TM亚型的意义的方法 Alpha TM Will的正常和病理状态、定点突变 来研究这种结构与功能的关系 重要的调节蛋白。Alpha-TM全长cDNA克隆为 正在建设中。完整的cdna克隆将在大肠杆菌中表达。 生产一种非融合蛋白，可以提纯用于体外研究。 定点突变将被用于解决特定的问题 基于盘绕线圈模型的TM的结构功能： 有离散的肌动蛋白结合位点吗？真正的影响范围是什么？ 肌钙蛋白结合部位？TM结构的哪些特征对 肌动蛋白和肌球蛋白的协同作用？保守的和 已知TM序列的非保守区将被纳入 在选择诱变地点时应考虑的问题，以及 发育和肥大中的心脏异构体，如所知的。 寡核苷酸定向诱变是一种改变单一 以一种已知的方式建立基地。对于局部随机诱变，亚硫酸氢钠 胞嘧啶的催化脱氨将与 寡核苷酸。突变蛋白的检测将包括与肌动蛋白的结合。 肌钙蛋白与肌动球蛋白ATPase协同激活的测定 在肌钙蛋白存在和不存在的情况下。

Alpha-Tropomyosin (Alpha TM) is a member of a complex multigene family containing multiple isoforms. The type of TM expressed depends on the developmental stage and physiological state of a particular cell type or tissue, although such information is not published for cardiac muscle during development and hypertrophy. Alpha Alpha-TM is present in adult cardiac and skeletal muscle in small mammals. As a way to understand the significance of the different TM isoforms in normal and pathological states, site-directed mutagenesis of Alpha TM will be carried out to investigate structure-function relationships in this important regulatory protein. A full length cDNA clone of Alpha-TM is under construction. The complete cDNA clone will be expressed in E. coli to produce a non-fusion protein that can be purified for studies in vitro. Site-directed mutagenesis will be used to address specific questions about the function of TM based on the coiled-coil model for its structure: are there discrete actin binding sites? What is the true extent of the troponin binding site? What features of TM structure are important for cooperative interaction with actin and myosin? The conserved and non-conserved regions of known TM sequences will be taken into consideration in selecting sites for mutagenesis, as well as changes in cardiac isoforms in development and hypertrophy, as they become known. Oligonucleotide directed mutagenesis is the method for changing single bases in a known fashion. For local random mutagenesis, sodium bisulfite catalyzed deamination of cytosine will be used in conjunction with oligonucleotides. Assays for mutant proteins will include binding to actin and troponin and assay of cooperative activation of the actomyosin ATPase in the presence and absence of troponin.