High-speed NMR methods for membrane protein analysis

描述（由申请人提供）：拟议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，这些策略将用于解决人类整体膜蛋白（hIMPs）的几种结构。由于膜蛋白固有的结构和动力学性质，现有的核磁共振结构测定方法对膜蛋白并不有效。跨膜（TM）螺旋束的内部迁移导致了核磁共振光谱信号的强烈展宽，并给信号分配、光谱分析和远程相互作用的检测带来了问题，而这是构建TM - 1螺旋结构域所必需的。因此，我们提出了一套利用CF表达系统的核磁共振策略，这将大大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和稳健（Aim 1）；加入可以用顺磁标签修饰的非天然氨基酸，以测量松弛增强和顺磁化学位移效应（目标2）；在CF体系中实施13c -甲基和19f标记，以获得用于结构确定的额外远程距离约束（目标3）。这些方法将应用于预选（Aim4） hIMPs，以确定10个或更多目标的高分辨率NMR结构（Aim 5）。

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).