High-Resolution Structures and Ligand-Induced Conformational Changes of Membrane Proteins by Solid-State NMR: Methodology Development and Applications

该奖项由2009年美国复苏和再投资法案(公法111-5)资助。膜蛋白占人类基因组的30%，因此对现代结构生物学具有极大的兴趣。然而，由于脂类的存在带来了结晶方面的挑战，它们的X射线高分辨结构往往很难获得。从这个角度来看，固态核磁共振波谱是研究天然脂质环境中的膜蛋白的一种独特的替代方法，而不需要结晶。这是由于使用磁性排列双层膜的样品制备技术的最新进展，这种双层膜提供了膜模拟和完全水合。此外，SS核磁共振方法产生与取向直接相关的可观测数据，作为原子分辨率结构确定的输入。该项目旨在通过从根本上改进当前的SS核磁共振技术来回答重要的生物学和生物物理问题。该项目的具体目标是：(I)完整的膜蛋白三维结构测定；(Ii)建立研究膜蛋白分子间和分子内接触的新方法；(Iii)配体结合的检测和相关构象变化的阐明；(Iv)根据角度相关的核磁共振约束计算蛋白质结构的有效算法；(V)在特定蛋白质及其配体上的应用。后者将包括离子通道及其激动剂和拮抗剂；病毒蛋白；以及更大的膜结合受体(肾上腺素能和嗅觉受体)。这些目标将通过在双标记蛋白质样品上进行新的SS核磁共振实验来实现，包括光谱分配、阐明分子内相互作用以及测量与方向有关的观测数据，如偶极耦合和化学位移。最终目标是仅根据SS核磁共振数据从单个均匀标记的蛋白质样本分配光谱并计算完整的三维蛋白质结构。该项目将涉及不同种族和地理背景的博士后、研究生和本科生。这里将要开发的方法有望在其他应用中找到它们的用途，包括溶液核磁共振和高有序无机化合物的核磁共振，例如沸石和其他纳米结构。关于膜中蛋白质结构测定的固体核磁共振方法的这项研究的基本结果将作为涵盖现代生物结构测定方法的大分子结构研究生课程的一部分。计算算法和模拟程序将提供给感兴趣的研究小组。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Membrane proteins constitute 30% of the human genome and, therefore, are of tremendous interest for modern structural biology. However, their X-ray high-resolution structures are often difficult to obtain due to the challenges in crystallization posed by the presence of lipids. From this view, solid-state Nuclear Magnetic Resonance (SSNMR) spectroscopy represents a unique alternative method to study membrane proteins in their native lipid environment without the need for crystallization. This is due to the recent advances in sample preparation techniques using magnetically aligned bilayers, which provide both membrane mimetics and full hydration. In addition, SSNMR methods yield directly orientationally dependent observables as input for structure determination at atomic resolution. This project is aimed at answering important biological and biophysical questions by radically improving current SSNMR techniques. The specific aims of this project are: (i) complete three-dimensional structure determination of membrane proteins (ii) creation of new methods for the studies of inter- and intramolecular contacts in membrane proteins; (iii) detection of ligand binding and elucidation of the associated conformational changes; (iv) efficient algorithms for calculating protein structures from angular-dependent NMR restraints; (v) applications to specific proteins and their ligands. The latter will include ion channels and their agonists and antagonists; viral proteins; and larger membrane-bound receptors (adrenergic and olfactory receptors). These goals will be accomplished through the development of new SSNMR experiments on doubly labeled protein samples for both spectral assignment, elucidation of intramolecular interactions, and measurement of orientationally dependent observables such as dipolar couplings and chemical shifts. The final goal is to assign the spectrum and calculate complete three-dimensional protein structure from a single uniformly labeled protein sample solely based on SSNMR data. This project will involve postdocs, graduate and undergraduate students with different ethnic and geographical backgrounds. The methods to be developed herein are expected to find their use in other applications including solution NMR and NMR of highly ordered inorganic compounds such as zeolites and other nanostructures. The basic results of this research on solid-state NMR methods of protein structure determination in membranes will be included as part of the curriculum for a graduate course on Macromolecular Structure covering modern methods of biological structure determination. The computational algorithms and simulations programs will be made available to interested research groups.