ASSEMBLY OF ISOFORMS OF THE MYOSIN ROD

肌球蛋白杆异构体的组装

• 批准号: 3056716
• 负责人: STEVEN H GROSSMAN
• 金额: $ 3.53万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-31 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3056716
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; chickens; circular dichroism; fluorescence spectrometry; intermolecular interaction; molecular cloning; myogenesis; myosins; protein sequence; sedimentation; site directed mutagenesis

The embryonic and neonatal homodimeric isoforms of myosin heavy chain are coexpressed during muscle development but do not form a hybrid. The proposed study seeks to ascertain if thermodynamic or biological factors determine subunit association. Isolated rod segments will be denatured, renatured and assayed for formation of the heterodimer. The high degree of sequence homology between the rod subunit isoforms necessitates application of new methods to detect the hybrid. These include use of a thiol- blocking/disulfide-forming method coupled with non-reducing SDS-PAGE, a resonance energy transfer procedure, and an immobilized subunit hybridization technique. If hybrids are not detected, the rod isoforms will be expressed in E. coli and subjected to site specific mutagenesis in regions where the sequences are known to differ. The assay will be used to determine the effects of each mutation in the homodimer on heterodimer formation. If hybrids are detected, similar mutagenesis studies will be done to determine those features of the sequence upon which association depends. Understanding these structural features of myosin dimerization is important in describing how myofibrils assemble and function in the contractile process.

肌球蛋白重链的胚胎和新生儿同型二聚体亚型是 在肌肉发育过程中共表达，但不形成杂种。 的 拟议的研究旨在确定热力学或生物学因素是否 确定亚基关联。 分离的杆段将变性， 复性并测定异二聚体的形成。 的高度 视杆细胞亚基同种型之间的序列同源性需要应用 新的方法来检测混合体。 这些包括使用硫醇- 封闭/二硫键形成法结合非还原SDS-PAGE， 共振能量转移过程，以及固定化亚基 杂交技术 如果未检测到杂交体， 将以E表示。大肠杆菌，并进行位点特异性诱变， 已知序列不同的区域。 该试验将用于 确定同二聚体中的每个突变对异二聚体的影响 阵 如果检测到杂交体，将进行类似的诱变研究。 这样做是为了确定这些特征的序列上， 看情况了 了解肌球蛋白二聚化的这些结构特征， 重要的是描述肌原纤维如何组装和功能， 收缩过程