High-speed NMR methods for membrane protein analysis

用于膜蛋白分析的高速 NMR 方法

• 批准号: 8316193
• 负责人: SENYON CHOE
• 金额: $ 36.77万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8316193
• 关键词: Address; Amino Acids; Cell-Free System; Cells; Chemicals; Cysteine; Dependency; Detection; Detergents; Development; Drug Design; Escherichia coli; Human; In Vitro; Integral Membrane Protein; Isotope Labeling; Label; Measures; Membrane Proteins; Methods; Mutation; Process; Property; Protein Analysis; Relaxation; Research; Resolution; Sampling; Signal Transduction; Site; Spectrum Analysis; Speed; Structure; System; base; combinatorial; high throughput screening; innovation; novel; protein structure; research study; tool

DESCRIPTION (provided by applicant): The purpose of the proposed research is to develop a set of NMR strategies for membrane protein structure determination. Combined with our existing E. coli-based cell-free (CF) expression system, these strategies will then be used to solve several structures of human integral membrane proteins (hIMPs). The existing NMR structure determination methods are not effective for membrane proteins because of their intrinsic structural and dynamical properties. The internal mobility of transmembrane (TM) helical bundles causes strong broadening of the signals in NMR spectra and creates problems with signal assignment, spectra analysis, and detection of long-range interactions, which are necessary to build up the structure of the TM 1-helical domain. Therefore, we propose a set of NMR strategies utilizing the CF expression system, which will significantly speed up structure determination of membrane proteins. In particular, we will further develop the combinatorial dual-isotope labeling strategy in order to make the resonance assignment process fast and robust (Aim 1); incorporate unnatural amino acids that can be modified with a paramagnetic label to measure relaxation enhancement and paramagnetic chemical shift effects (Aim 2); implement 13C-methyl and 19F-labeling in the CF system to obtain additional long-range distance constraints for structure determination (Aim 3). These methods will be applied to preselected (Aim4) hIMPs to determine the high resolution NMR structures of 10 or more targets (Aim 5).

描述（由申请人提供）：本研究的目的是开发一套用于膜蛋白结构测定的 NMR 策略。结合我们现有的基于大肠杆菌的无细胞 (CF) 表达系统，这些策略将用于解析人类整合膜蛋白 (hIMP) 的几种结构。由于膜蛋白固有的结构和动力学特性，现有的核磁共振结构测定方法对膜蛋白并不有效。跨膜 (TM) 螺旋束的内部迁移率导致 NMR 谱中信号的强烈展宽，并产生信号分配、谱分析和长程相互作用检测的问题，而这些对于构建 TM 1- 结构是必需的螺旋域。因此，我们提出了一套利用CF表达系统的NMR策略，这将显着加快膜蛋白的结构测定。特别是，我们将进一步开发组合双同位素标记策略，以使共振分配过程快速且稳健（目标1）；纳入可以用顺磁标记修饰的非天然氨基酸，以测量弛豫增强和顺磁化学位移效应（目标 2）；在 CF 系统中实施 13C-甲基和 19F-标记，以获得结构测定的额外长程距离约束（目标 3）。这些方法将应用于预选 (Aim4) hIMP，以确定 10 个或更多目标的高分辨率 NMR 结构（目标 5）。