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E。大肠杆菌为基础的无细胞（CF）表达系统，这些策略，然后将用于解决几个结构的人整合膜蛋白（hIMP）。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的结构测定效果不佳。跨膜（TM）螺旋束的内部流动性导致NMR光谱中的信号的强烈加宽，并产生信号分配、光谱分析和长程相互作用的检测的问题，这是建立TM 1-螺旋结构域的结构所必需的。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和稳健（目标1）;掺入可以用顺磁性标记修饰的非天然氨基酸，以测量弛豫增强和顺磁性化学位移效应（目标2）;在CF系统中实施13 C-甲基和19 F-标记，以获得用于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的（目标4）hIMP，以确定10个或更多个目标的高分辨率NMR结构（目标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).