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。这是新的 普遍认识到蛋白质没有静态结构，而且 X射线数据必须补充有关侧链的运动信息 和主干原子，如果能更全面地理解 蛋白质的相互作用是要实现的。 我们建议合成多种13C和2H标记的氨基酸和 将它们结合到HIV-1蛋白酶中，去掉多肽胰岛素酰胺，然后 纤维连接蛋白的第9和第10类III域。然后这些分子将被 高场(200~600 MHz)核磁共振研究 光谱学。我们对研究侧链运动特别感兴趣。 因此我们的主要目的是合成氨基 每隔一个侧链标记的酸--碳链上有13C。然后用SITE- 具体的T1、T2和远程NOE数据在手，我们希望能够 描绘一幅这些建筑及其周围的结构和动态的详细图景 已标记的站点。最近的研究表明，在许多蛋白质中，灵活性可能 在确定酶活性方面起着至关重要的作用。因此，例如， 在HIV-1蛋白酶等蛋白质中，构象上的灵活性 可移动的“襟翼”区域可以控制衬底和一个 对这一动议的理解可能为理性的人提供至关重要的信息 蛋白酶抑制剂的设计。 为了实施这些项目，我请求支持两名本科生 四年的学生。他们将承担主要责任 所有标记氨基酸、多肽和胰岛素衍生物的合成 他们会常规地通过层析来提纯和分析他们的产品 和核磁共振。约克的200 MHz宽带FT-核磁共振，Sun Sparc5工作站联网 使用Biosym核磁共振处理/建模软件的SGI，以及我们的固相 多肽合成器将使这些学生充分参与到 ~(13)C和~2H标记化合物的制备、表征和分析 借助前沿技术的产品通常不适用于 本科生。

The major objective of this proposal is to develop and apply methods to elucidate the dynamic structure of proteins in solution. It is new generally recognized that proteins do not have static structures and that X-ray data must be supplemented by motional information about side-chain 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 2H-labeled amino acids and incorporate them into HIV-1 protease, despentapeptideinsulin amide, and the 9th and 10th type III domains of fibronectin. These molecules will then be studied by high field (200 600 MHz) nuclear magnetic resonance (Nmr) spectroscopy. We are especially interested in studying side-chain motion in these proteins and therefore our primary purpose is to synthesize amino acids labeled at every other side-chain carbon with 13C. 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. Recent work suggests that in many proteins flexibility may play a crucial role in determining enzymatic activity. Thus, for example, in proteins such as HIV-1 Protease, flexibility in the conformationally mobile "flap" region may control on-off access of the substrate and an understanding of this motion may provide information vital ta the rational design of protease inhibitors. To implement these projects, I am requesting support for two 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, Sun Sparc5 workstation networked to an SGI with Biosym nmr processing/modeling software, and our solid phase peptide synthesizer will enable these students to participate fully in preparing, characterizing, and analyzing their 13C- and 2H- labeled products with the aid of forefront technology not generally available to undergraduates.