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

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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.