UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

本提案的主要目标是制定和应用各种方法， 阐明蛋白质在溶液中的动态结构。 现在 一般认为蛋白质没有静态结构， X射线数据必须由关于侧链的运动信息补充， 如果对蛋白质的性质有更全面的了解， 互动是要实现的。 我们建议合成各种13 C-和15 N-标记的氨基酸， 将它们整合到葡萄球菌核酸酶中，去五肽胰岛素酰胺 以及大肠coli信号转导蛋白IIIGlc. 这些分子将 然后通过高场（200-600 MHz）核磁共振进行研究 (nmr)谱 我们对研究侧链特别感兴趣 因此我们的主要目的是合成 在每隔一个侧链碳上用C-13标记的氨基酸。 然后用 我们拥有特定地点的T1、T2和远程NOE数据，希望能够 来描绘一幅详细的结构和动力学的图片 这些标记的网站。 为了实施这些项目，我请求支持三个项目， 本科生四年。 他们将承担主要 负责合成所有标记的氨基酸、肽 和胰岛素衍生物，他们将定期纯化和分析他们的 通过色谱法和NMR测定产物。 约克200 MHz宽带FT-NMR （在MBRS补充仪器奖的帮助下获得）和 新的UNIX工作站和我们最近购买的固相肽 合成器将使这些学生充分参与准备， 表征和分析其C-13和N-15标记的产物， 大学生一般得不到的前沿技术的帮助。

The major objective of this proposal is to develop and apply methods to elucidate the dynamic structure of proteins in solution. It is now generally recognized that proteins do not have static structures and that X-ray data must be supplemented by motional information about sidechain and backbone atoms if a more complete understanding of the nature of protein interactions is to be achieved. We propose to synthesize a variety of 13C- and 15N-labeled amino acids and incorporate them into staphylococcal nuclease, despentapeptideinsulin amide and the E. coli signal transducing protein, IIIGlc. These molecules will then be studied by high field (200-600 MHz) nuclear magnetic resonance (nmr) spectroscopy. We are especially interested in studying sidechain motion in these proteins and therefore our primary purpose is to synthesize amino acids labeled at every other sidechain carbon with C-13. Then with site-specific T1, T2, and long-range NOE data in hand, we hope to be able to paint a detailed picture of the structure and dynamics at and around these labeled sites. To implement these projects, I am requesting support for three undergraduate students for four years. They will bear primary responsibility for the synthesis of all the labeled amino acids, peptides and insulin derivatives and they will routinely purify and analyze their products by chromatography and nmr. York's 200 MHz broadband FT-nmr (acquired with the aid of an MBRS supplemental instrumentation award) and new UNIX workstation and our recently purchased solid phase peptide synthesizer will enable these students to participate fully in preparing, characterizing, and analyzing their C-13 and N-15 labeled products with the aid of forefront technology not generally available to undergraduates.