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

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。膜蛋白占人类基因组的30%，因此，对现代结构生物学具有巨大的兴趣。然而，它们的X射线高分辨率结构通常难以获得，因为脂质的存在对结晶构成了挑战。从这个角度来看，固态核磁共振（SSNMR）光谱是一种独特的替代方法来研究膜蛋白在其天然脂质环境中，而不需要结晶。这是由于最近的进展，在样品制备技术使用磁性排列的双层，它提供了膜模拟和充分的水合作用。此外，SSNMR方法产生直接依赖于取向的观测值作为原子分辨率的结构测定的输入。该项目旨在通过从根本上改进当前的SSNMR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：（一）完成膜蛋白的三维结构测定;（二）建立研究膜蛋白分子间和分子内接触的新方法;（三）检测配体结合并阐明相关的构象变化;（四）根据依赖于角度的NMR限制计算蛋白质结构的有效算法;（v）对特定蛋白质及其配体的应用。后者将包括离子通道及其激动剂和拮抗剂;病毒蛋白;和较大的膜结合受体（肾上腺素能和嗅觉受体）。这些目标将通过开发新的SSNMR实验双标记的蛋白质样品的光谱分配，阐明分子内相互作用，并测量方向依赖的观测值，如偶极耦合和化学位移。最终的目标是分配光谱，并计算完整的三维蛋白质结构，从一个单一的统一标记的蛋白质样品仅基于SSNMR数据。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。本文所开发的方法预期可用于其它应用，包括溶液NMR和高度有序的无机化合物如沸石和其它纳米结构的NMR。这项研究的基本结果，在膜中的蛋白质结构测定的固态核磁共振方法将被包括作为课程的一部分，涵盖生物结构测定的现代方法的研究生课程的大分子结构。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.