Membrane protein structures by solution NMR

利用溶液核磁共振测定MP结构的方法目前正处于快速发展的状态。最近，该方法已被用于解决一些具有高生物学影响的新MP结构，显示了其在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，旨在开发一个有效的解决核磁共振管道来解决MP结构。该中心由一组研究人员组成，他们是世界上最富有成效的核磁共振解MP结构，包括James Chou， Gerhard Wagner， 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，一个核磁共振/计算核心，用于开发更快的结构确定方法，以及一个管理和传播技术的行政核心。为了推动技术发展和测试拟议的管道，我们选择了10个结构未知的MP靶点，包括膜嵌入转运体，酶和受体。这些靶标是具有3-7个跨膜螺旋的多面体螺旋MPs，大小在18-43 kDa之间。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)无细胞表达平台，用于生产MPs和筛选核磁共振可行的MPs靶点；(2)新型清洗剂、单胞剂和MP再折叠方法；(3)非均匀采样高分辨率四维NOESYs；(4)甲基选择性同位素标记获取远程NOEs的新策略；(5)定位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的分子片段置换和结构计算方案。尽管“高通量”一词尚未适用于任何技术背景下的MP，但我们的目标是在拟议的资助期内建立一个核磁共振工具包，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.