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

海洋毒素含有融合的多环醚，是研究膜蛋白功能的很有前途的化学工具。我们的策略包括以下方案：(I)通过化学合成制备简化的融合多环醚文库；(Ii)建立获取聚醚-膜蛋白复合体模型结构信息的方法学。我们成功地合成了基于B-烷基Suzuki-Miyaura交叉偶联的简化聚醚，并用圆二色谱确证了它们的螺旋结构。为了开发膜蛋白模拟系统，我们使用双柱(DMPC/DHPC：磷脂凝胶)体系对模型膜系统进行了改进。用核磁共振、差示扫描量热法和差示扫描量热法表征了它们随温度变化的形态变化。此外，我们还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反应和Sonogashira反应)，新的碳-碳键被区域选择性地引入到RAS蛋白的靶位(32位或174位)。大肠杆菌苯丙氨酰-tRNA合成酶(G294-phers)的A294G突变体在高镁离子浓度下错酰化了一种大肠杆菌抑制子tRNA^&lt；Phe&gt；(tRNA^&lt；Phe&gt；CUA)与4-碘-L-苯丙氨酸。将预酰化的tRNA加入到大肠杆菌无细胞体系中，合成靶位含有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蛋白与乙烯化或丙烯化生物素进行有机钯反应。用Western印迹和LC-MS/MS证实了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.