UNIQUELY LABELED AMINO ACIDS FOR PROTEIN NMR

用于蛋白质 NMR 的独特标记氨基酸

• 批准号: 6107219
• 负责人: PAUL E YOUNG
• 金额: $ 20.71万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107219
• 关键词: aminoacid; chemical structure function; chromatography; conformation; fibronectins; hormone analog; insulin; method development; methyl group; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein structure

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.

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。 它是新 一般认为蛋白质没有静态结构， X射线数据必须补充有关侧链的运动信息 和骨架原子，如果一个更完整的理解的性质， 蛋白质的相互作用。 我们建议合成各种13 C-和2 H-标记的氨基酸， 将它们整合到HIV-1蛋白酶，去五肽胰岛素酰胺中， 纤连蛋白的第9和第10 III型结构域。 这些分子将被 研究了高场（200 - 600 MHz）核磁共振（NMR） 谱 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 在每隔一个侧链碳上用13 C标记的酸。 然后，与网站- 具体的T1，T2和长期NOE数据在手，我们希望能够 描绘出这些区域及其周围的结构和动力学的详细画面， 标签网站 最近的研究表明，在许多蛋白质中， 在确定酶活性方面起着至关重要的作用。 因此，举例来说， 在蛋白质如HIV-1蛋白酶中，构象上的灵活性 移动的“翼片”区域可以控制基板的开-关访问， 对这一运动的理解可能会提供对理性 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 学生四年。 他们将承担主要责任， 合成所有标记的氨基酸、肽和胰岛素衍生物 他们将通过色谱法对产品进行常规纯化和分析 和NMR。 约克的200 MHz宽带FT-nmr，Sun Sparc 5工作站联网 使用Biosym NMR处理/建模软件的SGI，我们的固相 肽合成器将使这些学生充分参与 制备、表征和分析它们的13 C-和2 H-标记的 借助前沿技术生产的产品， 本科生