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

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.

9870373红膜蛋白和纤维蛋白占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，由于它们的非结晶和不溶性，它们不能通过单晶X射线衍射和溶液态核磁共振（NMR）来表征。固态NMR是这些分子的三维结构的强有力的探针，但迄今为止，它对于蛋白质结构测定是低效的，因为通常只有少量的自旋标记（例如13C和15N）可以掺入蛋白质中以一次测量几个分子结构参数。本研究的目标是开发一种新的同位素标记方法，并将其与多维固态NMR相结合，以获得每个实验的许多结构参数，从而更有效地阐明蛋白质结构。该标记方案将利用氨基酸的生物合成途径来实现13 C的选择性但广泛的富集。这将为核间距离测量创建许多自旋对，同时减少均匀13C标记蛋白质中存在的复杂多自旋相互作用。这种标记方法将在充分表征和稳定的蛋白质泛素上进行测试。基于这种标记方案，将探索两种类型的固态NMR实验。第一类实验通过测量主链扭转角f和y来探测蛋白质二级结构。第二类实验是从核间距离，特别是不同残基侧链上15 N和13 C之间的长程距离来限制蛋白质三级结构。洪博士在整个职业生涯中接受过NMR领域的培训，她的兴趣一直是了解小肽的结构。 在POWRE的支持下，她打算进入大型多肽的竞技场，采用蛋白质生物合成和同位素标记的分子生物学技术。 该支持将为她提供所需的启动拟议的研究，并允许她扩大她的教学到先进的核磁共振和生物物理学的大分子领域的本科生和研究生在马萨诸塞州大学。 在此支持下，洪博士还打算组织一些关于核磁共振和二维核磁共振生物分子结构测定的研讨会。