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）：拟议研究的目的是为确定膜蛋白结构的确定制定一组NMR策略。结合我们现有的基于大肠杆菌的细胞（CF）表达系统，这些策略将用于解决人类整合膜蛋白（HISP）的几种结构。现有的NMR结构确定方法对于膜蛋白而言无效，因为它们具有内在的结构和动力学特性。跨膜（TM）螺旋束的内部迁移率会导致NMR光谱中信号的强大扩展，并在信号分配，光谱分析和检测长期相互作用的情况下引起了问题，这对于建立TM 1螺旋域的结构是必不可少的。因此，我们提出了使用CF表达系统的一组NMR策略，该策略将大大加快膜蛋白的结构测定。特别是，我们将进一步制定组合双异位标记策略，以使共振分配过程快速，稳健（AIM 1）；掺入不自然的氨基酸，可以用顺磁性标签修饰，以测量弛豫增强和顺磁化学移位效应（AIM 2）；在CF系统中实现13C-甲基和19F标记，以获得结构确定的其他远程距离约束（AIM 3）。这些方法将应用于预选的（AIM4）HIMP，以确定10个或更多目标的高分辨率NMR结构（AIM 5）。