Elucidation of structural principal in active state of membrane proteins using fused polycyclic ethers

含稠环醚的海洋毒素是研究膜蛋白功能的重要化学工具。我们的策略包括以下方案：（i）通过化学合成制备简化的稠合多环醚库;（ii）开发获得聚醚-膜蛋白复合物模型结构信息的方法。我们成功地合成了基于B-烷基Suzuki-Miyaura交叉偶联的简化聚醚，并使用CD光谱确认了它们的螺旋结构。为了建立膜蛋白模拟系统，我们用双分子膜（DMPC/DHPC：磷脂聚集体）系统对模型膜系统进行了修饰。已经使用1HP-NMR、DLS和DSC进行了它们的温度依赖性形态变化的表征<31>。此外，我们开发了几种蛋白质标记技术。通过两种类型的有机钯反应（Mizoroki-Heck和Sonogashira反应），一个新的碳-碳键已经区域选择性地引入Ras蛋白的靶位置（位置32或174）。大肠杆菌A294 G突变株可使大肠杆菌抑制基因tRNA^<Phe>（tRNA^<Phe>cuA）与4-碘-L-苯丙氨酸发生错酰化反应。大肠杆菌苯丙氨酰-tRNA合成酶（G294-PheRS）在高镁离子浓度下的活性。将预酰化的tRNA加入E.大肠杆菌无细胞系统中合成了一种在特定靶位含有4-碘-L-苯丙氨酸残基的Ras蛋白。将含有4-碘-L-苯丙氨酸残基的iF-Ras蛋白与乙烯基化或炔丙基化生物素进行有机钯反应。通过Western印迹和LC-MS/MS证实Ras蛋白的位点特异性生物素化。

Marine toxins which contain fused polycyclic ethers are promising chemical tools for investigation on membrane protein functions. Our strategy is consisted with following scheme: (i) preparation of simplified fused polycyclic ether library by chemical synthesis; (ii) methodogy development for obtaining structural information of polyether-membrane protein complex model. We succeeded in synthesis of simplified poly ethers based on B-alkyl Suzuki-Miyaura cross coupling and confirmed them recognized a helical structure using CD spectroscopy. For development of membrane protein mimic system we polished up model membrane system using bicelle (DMPC/DHPC: phospholipid aggreregates) system. Characterization of their temperature dependent morphological changes has been done using^<31>P-NMR, DLS, and DSC. Furthermore we developed several protein labeling techniques. A new carbon-carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki-Heck and Sonogashira reactions). An Escherichia coli suppressor tRNA^<Phe> (tRNA^<Phe> cuA) was mis-acylated with 4-iodo-L-phenylalanine by the A294G mutant of E. coli phenylalanyl-tRNA synthetase (G294-PheRS) at a high magnesium ion concentration. The pre-acylated tRNA was added to an E. coli cell-free system to synthesize a Ras protein containing 4-iodo-L-phenylalanine residue at specific target position. The iF-Ras proteins containing 4-iodo-L-phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS.