UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

这项提案的主要目标是开发和应用方法来 阐明蛋白质在溶液中的动态结构。现在是时候了 普遍认识到蛋白质没有静态结构，而且 X射线数据必须补充关于侧链的运动信息和 主链原子如果对蛋白质的性质有更全面的了解 互动是要实现的。 我们建议合成多种13C和15N标记的氨基酸和 将它们结合到葡萄球菌核酸酶中，去肽胰岛素酰胺 和大肠杆菌信号转导蛋白IIIGlc。这些分子将 然后用高场(200-600 MHz)核磁共振进行研究 (核磁共振)光谱学。我们对研究侧链特别感兴趣 在这些蛋白质中运动，因此我们的主要目的是合成 每隔一条侧链碳上用C-13标记的氨基酸。然后用 现场特定的T1、T2和远程NOE数据在手，我们希望能够 描绘一幅结构和动态的详细图景 这些标有标签的地点。 为了实施这些项目，我请求支持三个项目 四年本科生。他们将承担初选 负责所有标记氨基酸、多肽的合成 和胰岛素衍生物，他们会例行公事地纯化和分析他们的 产物经层析和核磁共振分析。约克公司的200 MHz宽带FT-核磁共振 (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能够充分参与准备， 对其C-13和N-15标记产物进行表征和分析 本科生一般不能获得前沿技术的帮助。

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.