Investigation of membrane binding mechanism of peripheral membrane protein based on solid state NMR using magnetically aligned membrane bilayers at room temperature

磷脂酶C(PLC)与细胞膜上的磷脂酰肌醇4，5-二磷酸(PIP_2)通过Peckstrin同源(PH)结构域结合，并通过催化域催化PIP_2生成二酰甘油和1，4，5-三磷酸肌醇(IP_3)两种第二信使。本研究探讨了人PLC-δ-1PH结构域的脂结合机制。首先，用基于Native-PAGE的方法研究了α2-螺旋对IP_3结合活性和热稳定性的贡献。然后，用溶液核磁共振检测IP_3结合引起的HPH的局部结构变化。研究发现，HPH中的分子内相互作用网络控制其配体结合。此外，通过固体核磁共振分析还发现，HPH与PIP_2包埋的脂质双层表面结合后，产生了不均匀的结构，表明HPH最终不可逆地插入到脂质双层中。

Phospholipase C (PLC) binds to phosphatidylinositol 4,5-bisphosphate (PIP_2) in the cell membrane through the pleckstrin homology (PH) domain, and hydrolyzes PIP_2 to produce two second messengers, diacylglycerol and inositol 1,4,5-triphosphate (IP_3), by the catalytic domain. In this study, lipid binding mechanism of human PLC-δ1 PH domain (hPH) was explored. First, the contributions of the α2-helix to the IP_3 binding activity and thermal stability of the hPH domain were investigated by developed approach based on Native-PAGE methods. Then, linked local structural changes of hPH induced by the IP_3 binding were detected by solution NMR. It was found that intra molecular interaction network in hPH controls its ligand binding. Furthermore, through the analyses of solid state NMR, it was also found that hPH induces inhomogeneous structure after binding to a surface of PIP_2 embedded lipid bilayers and implied that hPH is finally inserted to lipid bilayers irreversibly.