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

通过固态核磁共振研究膜蛋白的高分辨率结构和配体诱导的构象变化：方法开发和应用

• 批准号: 0843520
• 负责人: Alexander Nevzorov
• 金额: $ 77.69万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843520&HistoricalAwards=false
• 关键词: Resolution; Structures; Ligand; Induced; Conformational

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.

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，由于脂质的存在给结晶带来了挑战，它们的 X 射线高分辨率结构通常很难获得。从这个角度来看，固态核磁共振（SSNMR）光谱代表了一种独特的替代方法，可以在天然脂质环境中研究膜蛋白，而无需结晶。这是由于使用磁性排列双层的样品制备技术的最新进展，该技术提供了膜模拟和完全水合。此外，SSNMR 方法可直接产生方向相关的可观测值，作为原子分辨率结构测定的输入。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：（i）完成膜蛋白的三维结构测定（ii）创建研究膜蛋白分子间和分子内接触的新方法； (iii) 检测配体结合并阐明相关的构象变化； (iv) 根据角度相关的 NMR 约束计算蛋白质结构的有效算法； (v)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（肾上腺素能受体和嗅觉受体）。这些目标将通过在双标记蛋白质样品上开发新的 SSNMR 实验来实现，这些实验用于光谱分配、分子内相互作用的阐明以及方向依赖的可观察量（例如偶极耦合和化学位移）的测量。最终目标是仅根据 SSNMR 数据从单个统一标记的蛋白质样品中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。本文开发的方法预计可用于其他应用，包括溶液 NMR 和高度有序无机化合物（如沸石和其他纳米结构）的 NMR。这项关于膜中蛋白质结构测定的固态核磁共振方法的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。