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

红膜蛋白和纤维蛋白占所有蛋白质的三分之一以上，具有许多重要的生物学功能。然而，由于它们的非晶态和不溶性，它们不能用单晶X射线衍射和溶液状态核磁共振(核磁共振)来表征。固体核磁共振是研究这些分子三维结构的一种强有力的探针，但到目前为止，它对蛋白质结构的测定效率很低，因为通常只有少量的自旋标记(如13C和15N)可以被结合到蛋白质中来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，并将其与多维固体核磁共振相结合，通过实验获得许多结构参数，从而更有效地阐明蛋白质的结构。这一标记计划将利用氨基酸的生物合成途径来实现对13C的选择性但广泛的浓缩。这将产生许多用于核间距离测量的自旋对，同时减少统一标记13C的蛋白质中存在的复杂的多自旋相互作用。这种标记方法将在一种具有良好特性和稳定的蛋白质泛素上进行测试。基于这一标记方案，将探索两种类型的固体核磁共振实验。第一种类型的实验通过测量骨架扭转角f和y来探索蛋白质的二级结构。第二种类型的实验从核间距离，特别是不同残基侧链上15N和13C之间的长距离来限制蛋白质的三级结构。在整个职业生涯中，洪博士一直在核磁共振领域接受培训，她的兴趣一直是了解小肽的结构。在POWRE的支持下，她打算进入大多肽领域，利用蛋白质生物合成和同位素标记的分子生物学技术。这些支持将为她提出的研究提供所需的启动，并使她能够将教学扩展到高级核磁共振和大分子生物物理学领域，供麻省理工大学的本科生和研究生使用。在此支持下，洪博士还打算组织一系列关于生物分子结构确定的入门核磁共振和二维核磁共振的研讨会。

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.