Membrane protein structures by solution NMR

利用溶液核磁共振测定MP结构的研究正处于快速发展阶段。最近，这种方法已被用来解决一些新的MP结构的高生物影响，显示其在MP结构基因组学的潜力。然而，目前还没有一个专门的中心，专注于广泛发展的结构生物物理学这一重要的新兴领域。我们提出了一个蛋白质结构倡议（PSI）中心，旨在开发一个有效的解决方案NMR管道解决MP结构。该中心由一组研究人员组成，他们是世界上通过NMR解决MP结构的最富有成效的研究人员之一，包括James Chou，Gerhard瓦格纳，Charles Sanders和Volker Dotsch。该中心还包括一个合成核心，用于提供MP NMR中使用的材料，一个NMR/计算核心，用于开发更快的结构测定方法，以及一个管理核心，用于管理和传播技术。为了推动技术开发和测试拟议的管道，我们选择了10个结构未知的MP目标，包括膜嵌入式转运蛋白，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的多位螺旋MP。该中心将开发对结构确定具有直接和实际影响的技术。它们包括（1）用于生产MP和用于筛选NMR可行的MP靶标的无细胞表达平台;（2）新的去污剂、双胞和MP重折叠方法;（3）非均匀采样的高分辨率4D NOESY;（4）用于获得长距离NOE的甲基的选择性同位素标记的新策略;（5）新的同位素标记方法。（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;和（6）基于RDC的分子片段置换和结构计算方案。虽然“高通量”一词在任何技术背景下都不适用于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MP的结构。

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.