POWRE: New Approaches for Efficient Determination of Protein Structures by Solid-State NMR and Isotopic Labeling

POWRE：通过固态核磁共振和同位素标记有效测定蛋白质结构的新方法

• 批准号: 9870373
• 负责人: Mei Hong
• 金额: $ 7.5万
• 依托单位: University of Massachusetts Amherst
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-15 至 1999-08-17
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9870373&HistoricalAwards=false
• 关键词: POWRE; New; Approaches; Efficient; Determination; POWRE; New; Approaches; Efficient; Determination

9870373HongMembrane proteins and fibrous proteins constitute more than a third of all proteins and carry out many important biological functions. Yet they cannot be characterized by single-crystal X-ray diffraction and solution-state nuclear magnetic resonance (NMR) due to their noncrystalline and insoluble nature. Solid-state NMR is a powerful probe of the three-dimensional structure of these molecules, but to date it is inefficient for protein structure determination, since normally only a small number of spin labels (e.g. 13C and 15N) can be incorporated into the protein to measure a few molecular structural parameters at a time. The goal of this research is to develop a novel isotopic labeling approach and combine it with multi-dimensional solid-state NMR to obtain many structural parameters per experiment, thus elucidating protein structure more efficiently. This labeling scheme will exploit the biosynthetic pathways of amino acids to achieve selective but extensive enrichment of 13C. This will create many spin pairs for internuclear distance measurements while reducing the complex multi-spin interactions present in uniformly 13C-labeled proteins. This labeling approach will be tested on a well-characterized and stable protein, ubiquitin. Based on this labeling scheme, two types of solid-state NMR experiments will be explored. The first type of experiment probes the protein secondary structure by measuring the backbone torsion angles f and y. The second type of experiment constrains the protein tertiary structure from internuclear distances, especially long-range distances between the 15N and 13C on the side chains of different residues.While trained in the area of NMR throughout her professional life, Dr. Hong's interest has been understanding the structure of small peptides. With POWRE support, she intends to move into the arena of large polypeptides, employing molecular biological techniques of protein biosynthesis and isotopic labeling. The support will provide her the needed start-up for the proposed study, and allow her to expand her teaching into areas of advanced NMR and biophysics of macromolecules to both undergraduate and gradute students at U Mass. With the support, Dr. Hong also intends to organize a number of workshops on introductory NMR and two-dimensional NMR for biomolecular structure determination.

9870373Hongmmbrane蛋白和纤维蛋白构成了所有蛋白质的三分之一以上，并执行许多重要的生物学功能。然而，由于非结晶性和不溶性性质，它们的特征是单晶X射线衍射和溶液状态核磁共振（NMR）。固态NMR是这些分子三维结构的强大探针，但迄今为止，蛋白质结构的测定效率低下，因为通常只能将少量的自旋标记（例如13C和15N）掺入蛋白质中以测量少数分子结构参数。这项研究的目的是开发一种新型的同位素标记方法，并将其与多维固态NMR结合使用，以每个实验获得许多结构参数，从而更有效地阐明蛋白质结构。这种标记方案将利用氨基酸的生物合成途径，以实现选择性但广泛的富集13c。这将创建许多用于临时距离测量值的自旋对，同时还原以均匀13C标记的蛋白质存在的复杂多旋转相互作用。这种标记方法将在特征良好且稳定的蛋白质泛素上进行测试。基于此标签方案，将探讨两种类型的固态NMR实验。第一种实验通过测量骨架扭转角F和y来探测蛋白质二级结构。第二种实验限制了蛋白质的三级结构，尤其是在不同残基的侧链的15n和13c之间的远距离距离。洪博士在整个职业生涯中接受了NMR的训练，但洪博士的兴趣一直在理解小肽的结构。 在Powre的支持下，她打算采用蛋白质生物合成和同位素标记的分子生物学技术进入大多肽领域。 支持将为她提供拟议的研究所需的初创企业，并让她将教学扩展到美国马萨诸塞州大学的本科生和毕业生的高级NMR和生物物理学领域。在支持的情况下，Hong博士还打算组织许多介绍性NMR的研讨会，以实现NMR的入门和二维Nmr结构，以确定生物质量的二维结构。