Membrane protein structures by solution NMR

描述(申请人提供)：使用溶液核磁共振来确定MP结构目前正处于快速发展的状态。最近，这种方法已经被用于解决一些具有高生物影响的新的MP结构，展示了它在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这一重要的新兴结构生物物理学领域的广泛发展。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方案核磁共振流水线来解决MP结构。这个中心由一组研究人员组成，他们是世界上用核磁共振技术解决MP结构的最有成效的人之一，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核磁共振所用材料的合成核心，一个用于开发更快结构确定方法的核磁共振/计算核心，以及一个用于管理和传播技术的行政核心。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靶标，包括膜嵌入的转运体、酶和受体。这些靶点是多面体螺旋MPS，具有3-7个跨膜螺旋，大小为18-43 kDa。该中心将开发将对结构确定产生直接和实际影响的技术。它们包括(1)用于生产的无细胞表达平台 这些研究包括：(1)MPS和用于筛选核磁共振可行的MP靶标的新方法；(2)新的洗涤剂、双组分和MP复性方法；(3)非均匀采样的高分辨率4D NOESYs；(4)用于获得远程NOE的甲基选择性同位素标记的新策略；(5)用于RDC测量的新型顺磁标记试剂和通用DNA-纳米管排列介质；以及(6)基于RDC的分子片段替换和结构计算方法。尽管“高通量”一词在任何技术背景下还不适用于MP，但我们的目标是在拟议的供资期限内建立一个核磁共振工具包，该工具包有能力系统地生产MP结构，同时交付目标MP的结构。 与公共卫生相关：MPS约占药物靶标的50%，但由于解决MPS结构涉及的技术挑战，通常无法获得用于合理药物设计的MPS结构。这项赠款中提议的核磁共振技术将构成一条系统地生产MP结构的强大管道。这条流水线将很好地补充现有的基于结晶学的中心在MP结构空间的覆盖范围。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: MPs constitute about 50% of drug targets, but their structures for rational drug design are generally not available due to the technical challenges involved in solving MP structures. The NMR technologies proposed in this grant would constitute a robust pipeline for systematic production of MP structures. This pipeline would nicely complement the existing crystallography-based Centers in the coverage of MP structure space.