SITE-DIRECTED MUTAGENESIS OF CARDIAC A-TROPOMYOSIN

α-原肌球蛋白是一个复杂的多基因家族成员 含有多种同种型。 表达的TM类型取决于 特定细胞类型的发育阶段和生理状态，或 组织，尽管未公布心肌的此类信息 在发育和肥大过程中。 Alpha Alpha-TM存在于成人中 小型哺乳动物的心脏和骨骼肌。 作为理解不同TM异构体的意义的一种方式， 正常和病理状态下，α TM的定点突变将 进行调查结构功能关系，在这方面 重要的调节蛋白。 Alpha-TM的全长cDNA克隆， 在建 该cDNA克隆将在E.杆菌 以产生可纯化用于体外研究的非融合蛋白。 定点突变将用于解决以下具体问题： 基于螺旋线圈模型的TM的功能是： 有离散的肌动蛋白结合位点吗 什么是真正的程度， 肌钙蛋白结合位点？ TM结构的哪些特征对于 与肌动蛋白和肌球蛋白的协同作用？ 保守和 已知TM序列的非保守区将被考虑到 选择诱变位点的考虑因素，以及 发育和肥大中的心脏亚型，正如它们所知。 寡核苷酸定向突变是改变单个突变的方法， 以已知的方式。 对于局部随机诱变，亚硫酸氢钠 胞嘧啶的催化脱氨基作用将与 寡核苷酸 突变蛋白质的分析将包括与肌动蛋白的结合 和肌钙蛋白以及肌动球蛋白ATP酶协同激活的测定 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.