SEGMENTAL LABELING FOR PROTEIN STRUCTURE BY NMR

描述(逐字摘自申请者摘要)：《缪尔和考伯恩》 实验室建议最终检验这一假说 利用表达蛋白连接的同位素标记可以显著延长 一系列可用核磁共振进行结构研究的目标。在演示之后 这种方法的实用性最近，有人建议将 允许选择性标记蛋白质内部片段的程序，以 显著提高了方法的效率和实用性 打算使它们成为例行化的，并用一系列 重要的靶系统，包括src同源适配器、crk、 限制性内切酶，TaqI，GTP酶效应域，DBL同源性 与顺序结合的Pleckstrin同源结构域结合，以及 MAP激酶通路中的支架/接头。 这些技术改进将包括选择更好的方法来 制备含有所需化学成分的重组多肽片段 表达蛋白连接的反应性基团以及一种 固相表达蛋白连接策略将允许多个 多肽片段要高效、快速地拼接在一起。这个 节段性同位素标记方法的发展将具有重要意义 拓宽表达蛋白连接的应用范围，特别是通过 关于核磁共振，这样溶液中的结构研究就可以与 核磁共振中的其他技术发展，适用于更重要的范围 与健康相关的生物学中的分子结构问题。直接举例说明 与该项目的联系可能会导致重组DNA技术的改进 通过增加对限制性内切酶的了解，更深入地了解 涉及正常发育和内稳态的信号转导机制， 以及与感染、癌症和衰老相关的疾病。

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.