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

使用稠合多环醚阐明膜蛋白活性状态的结构原理

基本信息

批准号：
16201044
负责人：
TACHIBANA Kazuo
金额：
$ 32.2万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16201044/
关键词：
marine toxin polycyclic ether membrane protein lipid bilayer Suzuki cross coupling Gambierol photo-affinity labeling helical structure 分子認識機構

项目摘要

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.

含有稠合多环醚的海洋毒素是研究膜蛋白功能的有前途的化学工具。我们的策略包括以下方案：（i）通过化学合成制备简化的稠合多环醚库； (ii) 获得聚醚-膜蛋白复合物模型结构信息的方法开发。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 光谱证实了它们识别出螺旋结构。为了开发膜蛋白模拟系统，我们使用 bicelle（DMPC/DHPC：磷脂聚集体）系统完善了模型膜系统。已经使用 31 P-NMR、DLS和DSC对其依赖于温度的形态变化进行了表征。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），新的碳-碳键被区域选择性地引入 Ras 蛋白的目标位置（位置 32 或 174）。大肠杆菌抑制因子 tRNA^<Phe> (tRNA^<Phe> cuA) 在高镁离子浓度下被大肠杆菌苯丙氨酰-tRNA 合成酶 (G294-PheRS) 的 A294G 突变体用 4-碘-L-苯丙氨酸错误酰化。将预酰化的 tRNA 添加到大肠杆菌无细胞系统中，合成在特定目标位置含有 4-碘-L-苯丙氨酸残基的 Ras 蛋白。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。通过蛋白质印迹和 LC-MS/MS 证实了 Ras 蛋白的位点特异性生物素化。