膜环境对膜蛋白DAGK结构与功能的影响

The structures of membrane proteins are profoundly influenced by membrane–protein interactions. To date, more than 90% structures of membrane protein in the PDB have been determined in membrane mimetic environments-detergent micelles. However, it is very difficult to obtain membrane mimetic environments that support the native structure and function of a membrane protein. Escherichia coli diacylglycerol kinase (DAGK) is a very important transmembrane protein of which three dimensional structure has been determined in detergent micelles or lipid cubic phase by solution-state NMR or X-Ray crystallography. Due to the different membrane mimetic environments, the two structures differ obviously. Solid-state NMR is a powerful tool for studying structures of membrane proteins in the native-like membrane environments. This project aims to use Solid-state NMR to determin the sturcture of DAGK in Escherichia coli total lipids bilayer. Then, we will expain the influence of membrane environments on membrane protein structure and function by comparation the structures of DAGK active domain in different membrane environments.

膜蛋白的结构与功能深受所处环境的影响。目前，在PDB数据库中收录的膜蛋白结构90%以上是在膜模拟环境—去垢剂胶束中解析的。然而，在去垢剂的环境中，膜蛋白的真实结构能否被维持是备受结构生物学家关注的问题。大肠杆菌甘油二脂激酶(DAGK)是一个很重要的三跨膜蛋白，其三维结构已经被液体核磁共振和X-Ray晶体衍射的方法在去垢剂胶束或脂质立方相中所解析，然而由于环境的不同，得到的结构差别很大，并且这两种结构都不是在DAGK的天然膜环境中进行解析的。固体核磁共振技术可以在接近膜蛋白天然环境的磷脂双层膜中解析其三维结构，是测定与证实膜蛋白结构的有力工具。本项目拟采用固体核磁共振技术在大肠杆菌磷脂膜的环境下对DAGK的三维结构进行解析，并分析不同环境下DAGK活性区域的结构变化，阐释膜蛋白结构与功能对膜环境的依赖性。

膜蛋白的结构与功能和其所处的膜模拟环境关系密切。膜蛋白大肠杆菌甘油二脂激酶(DAGK)的三维结构已有两种报道，分别是在去垢剂胶束中或脂质立方向中解析出来的，这两种结构之间存在明显的差异，更为重要的是，DAGK在天然膜环境中的结构尚未可知。本项目中，我们制备了DAGK在天然环境大肠杆菌磷脂双分子层中的脂质体，利用固体核磁共振技术解析了其高分辨率的三聚体结构；利用滴定的方法，分别研究了底物与DAGK相互作用的关键残基。我们通过比较磷脂环境与去垢剂、脂质立方向环境中结构与活性位点的差异性，分析了膜环境对膜蛋白DAGK结构与功能的影响。