SEGMENTAL LABELING FOR PROTEIN STRUCTURE BY NMR

通过 NMR 对蛋白质结构进行分段标记

• 批准号: 6386609
• 负责人: Tom Muir
• 金额: $ 25.88万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386609
• 关键词: bioimaging /biomedical imaging; esterification; nuclear magnetic resonance spectroscopy; protein structure; recombinant proteins; stable isotope double label; structural biology; technology /technique development; thioester

DESCRIPTION (Verbatim from the applicant's abstract): The Muir and Cowburn laboratories propose to test definitively the hypothesis that segmental isotopic labeling using expressed protein ligation can extend significantly the range of targets amenable to structural studies using NMR. Having demonstrated the practicality of this approach recently, it is proposed to extend the procedure to permit selective labeling of internal segments of a protein, to improve significantly the efficiency and practicality of the methods with a view to making them routine, and to developing these approaches with a range of significant target systems, including the src homology adaptor, Crk, the restriction endonuclease, TaqI, the GTPase effector domain, Dbl homology in conjunction with the sequentially associated Pleckstrin homology domain, and scaffold/adaptors in the MAP kinase pathways. These technological improvements will include the selection of better ways to prepare recombinant polypeptide segments containing the necessary chemically reactive groups for expressed protein ligation, as well as the development of a solid-phase expressed protein ligation strategy which will allow multiple polypeptide segments to be efficiently and quickly pieced together. The development of the segmental isotopic labeling methods will significantly broaden the applications of expressed protein ligation, specifically with regard to NMR, so that structural studies in solution can, in combination with other technological developments in NMR, apply to a much more significant range of molecular structural problems in health related biology. Examples directly connected to the project may lead to improvements in recombinant DNA technology by increased understanding of restriction enzymes, deeper insight into mechanisms of signal transduction involving normal development and homeostasis, as well as disorders associated with infection, cancer, and aging.

描述（逐字从申请人的摘要中）：Muir和Cowburn 实验室建议确定测试一个分段的假设 使用表达蛋白结扎的同位素标记可以显着扩展 使用NMR适合结构研究的目标范围。演示 最近，这种方法的实用性，提议扩展 允许选择性标记蛋白质内部段的程序， 显着提高方法的效率和实用性 查看使它们常规，并以一系列的范围开发这些方法 重要的目标系统，包括SRC同源性适配器，CRK， 限制性核酸内切酶，TAQI，GTPase效应子域，DBL同源性 与依次关联的pleckstrin同源域的结合，并 MAP激酶途径中的脚手架/适配器。 这些技术的改进将包括选择更好的方法 准备重组多肽片段，并在化学上进行必要的化学 用于表达蛋白质连接的反应性组，以及 固相表示蛋白质连接策略，该策略将允许多个 多肽片段将有效，快速拼凑在一起。这 分段同位素标记方法的开发将显着 扩大表达蛋白质连接的应用，特别是 关于NMR，因此解决方案中的结构研究可以与 NMR中的其他技术发展，适用于更重要的范围 健康相关生物学中的分子结构问题。示例直接 连接到项目可能会改善重组DNA技术 通过增加对限制酶的理解，对 信号转导的机制涉及正常发育和稳态， 以及与感染，癌症和衰老有关的疾病。