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）通过pleckstrin同源结构域（PH）与细胞膜上的磷脂酰肌醇4，5-二磷酸（PIP_2）结合，并通过催化结构域水解PIP_2产生第二信使甘油二酯和肌醇1，4，5-三磷酸（IP_3）。本研究探讨了人PLC-δ1 PH结构域（hPH）的脂质结合机制.首先，利用改进的Native-PAGE方法研究了α2-螺旋对hPH结构域的IP_3结合活性和热稳定性的贡献。然后，通过溶液NMR检测到IP_3结合引起的hPH的连接局部结构变化。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在与包埋有PIP_2的脂双层表面结合后，导致了不均匀的结构，暗示hPH最终不可逆地插入脂双层。

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.