用于研究膜蛋白中柔性残基的固体NMR新方法

Structural analyses of membrane proteins (MPs) are hotspots and challenges for the researches of proteins nowadays. Solid-state NMR (ssNMR) has special advantages in structural analyses of MPs in native-like lipid-bilayer environments and its spectral line-width is not limited by the weight of MPs. As a consequence, ssNMR is regarded as a very prospective technique for elucidating the structures of MPs. However, ssNMR which is mainly based on dipolar couplings, is difficult to analyze the structures of flexible residues in MPs effectively, and thus restricts the understanding of its functions involving in signal conduction, molecular transport and ligand recognition. In this project, we will develop new ssNMR methods simultaneously based on J-couplings and dipolar couplings, in order to achieve the structural analysis of flexible residues and promote the studies of these function-related sites in MPs. The implement of this project will be able to enhance the ability of SSNMR to study MPs.

膜蛋白的结构分析是蛋白质研究的热点和难题。固体NMR可在接近天然环境的磷脂膜中研究膜蛋白，且分辨率不受膜蛋白分子量大小的限制，因此它被认为是研究膜蛋白结构的非常有前景的分析技术。但是，当前固体NMR方法主要是基于D-耦合作用，难以有效地对膜蛋白的柔性残基进行结构分析，进而阻碍了人们对柔性残基在膜蛋白中参与的信号传导、分子转运、配体识别等功能的研究。本项目拟设计同时基于J-耦合和D-耦合的固体NMR新方法，目的是实现膜蛋白中柔性残基的结构分析、促进人们对其在膜蛋白中相关功能的研究。本项目的顺利实施将提高固体NMR研究膜蛋白的能力。

固体NMR难以对膜蛋白的柔性残基进行有效地分析。主要原因是缺乏针对柔性残基特点的、稳定的偶极重耦方法。本项目利用模拟结合实验的方法，设计了两种全新的15N-13C异核偶极重耦方法。第一种方法是具有频率选择性的DBP，能够选择性地建立15N-13Cα相关或15N-13C'相关，建立蛋白质的信号归属；第二种方法是宽带DBP方法（Bro-DBP），比当前经典的TEDOR方法更灵活更高效，可用于膜蛋白的定量实验。新发展的两种DBP方法实现了快速重耦，有利于膜蛋白中核磁信号弛豫较快的柔性残基的研究；同时，DBP方法形式简单、性能稳定、设置简便，具有较好的实用性。