Membrane protein structures by solution NMR

通过溶液 NMR 确定膜蛋白结构

• 批准号: 8140470
• 负责人: JAMES Jeiwen CHOU
• 金额: $ 229.72万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8140470
• 关键词: Membrane; protein; structures; solution; NMR

The use of solution NMR to determine MP structure is now in a rapid state of growth. Recently, this approach has been employed to solve a number of new MP structures of high biological impact, demonstrating its potential in MP structural genomics. However, there is not yet a specialized center that focuses on broad development of this important emerging area of structural biophysics. We propose a Protein Structure Initiative (PSI) Center that aims to develop an efficient solution NMR pipeline for solving MP structures. This Center consists of a team of investigators who are among the most productive in the world in solving MP structures by NMR, including James Chou, Gerhard Wagner, Charles Sanders, and Volker Dotsch. The Center also includes a synthetic core for providing the materials used in MP NMR, an NMR/computational core for developing faster methods of structure determination, and an administrative core for management and dissemination of technology. To drive technology development and to test the proposed pipeline, we have selected 10 MP targets for which the structures are not known, including membrane-embedded transporters, enzymes, and receptors. These targets are polytopic helical MPs with 3-7 transmembrane helices and with sizes from 18-43 kDa. The Center will develop technologies that will have immediate and practical impact on structure determination. They include (1) cell-free expression platforms for production of MPs and for screening for NMR-feasible MP targets; (2) new detergents, bicelles and MP refolding methods; (3) non-uniform sampled high resolution 4D NOESYs; (4) new strategies for selective isotope labeling of methyl groups for acquiring long-range NOEs; (5) novel reagent for site-directed paramagnetic tagging and universal DNA-nanotube alignment media for RDC measurements; and (6) RDC-based molecular fragment replacement and structure calculation protocols. Although the phrase "high-throughput" does not yet apply to MP in any technological context, we aim to establish a NMR tool package within the proposed funding period that has the capacity for systematic production of MP structures, while delivering the structures of the target MPs.

使用溶液核磁共振确定 MP 结构目前正处于快速增长状态。最近，这种方法已被用来解决许多具有高生物学影响的新 MP 结构，展示了其在 MP 结构基因组学中的潜力。然而，目前还没有一个专门的中心专注于结构生物物理学这一重要新兴领域的广泛发展。我们提出了一个蛋白质结构倡议（PSI）中心，旨在开发一种有效的解决核磁共振管道来解决 MP 结构。该中心由世界上在通过 NMR 解决 MP 结构方面最具生产力的研究人员组成，其中包括 James Chou、Gerhard Wagner、Charles Sanders 和 Volker Dotsch。该中心还包括一个用于提供 MP NMR 所用材料的合成核心、一个用于开发更快的结构测定方法的 NMR/计算核心，以及一个用于管理和传播技术的管理核心。为了推动技术开发并测试拟议的管道，我们选择了 10 个结构未知的 MP 靶点，包括膜嵌入转运蛋白、酶和受体。这些靶标是具有 3-7 个跨膜螺旋且大小为 18-43 kDa 的多面螺旋 MP。该中心将开发对结构测定产生直接和实际影响的技术。它们包括 (1) 用于生产 MP 和筛选 NMR 可行的 MP 靶标的无细胞表达平台； (2)新的去污剂、bicelles和MP复性方法； (3)非均匀采样高分辨率4D NOESY； (4) 甲基选择性同位素标记以获得长程 NOE 的新策略； (5) 用于定点顺磁标记的新型试剂和用于 RDC 测量的通用 DNA 纳米管对准介质； (6)基于RDC的分子片段替换和结构计算协议。尽管“高通量”一词在任何技术背景下尚不适用于 MP，但我们的目标是在拟议的资助期内建立一个 NMR 工具包，该工具包具有系统生产 MP 结构的能力，同时提供目标结构议员们。