Structure and mechanism of a eukaryotic seven-transmembrane protein investigated by solid state NMR

• 作 者: Jing Liu Chang Liu Shenlin Wang
• 机 构: College of Chemistry and Molecular Engineering,Peking University,China;Beijing NMR center,Peking Uni
• 出 处: 2016年分析化学前沿国际研讨会及中美分析化学研讨会
• 发表日期: 2016年3期
• 关键词: Solid-state NMR Seven-transmembrane proteins Protein topology Sparse 13C labelin

　　Solid-state NMR(SSNMR)is emerging as a powerful tool for investigating membrane proteins.Its main advantage is that it allows for studies of membrane proteins in their native-like,lipid-embedded environments.However,low sensitivity,poor resolution and spectral complexity are still the major obstacles for the applications of SSNMR for large membrane proteins.In my presentation I will introduce our recent progress in SSNMR methodologies towards structure determination of eukaryotic membrane proteins,using an example of a rhodopsin from L.maculans(LR).Firstly,I will discuss the SSNMR sample requirements,focusing on sparse 13C labeling approaches,optimized for eukaryotic methyltrophic yeast P.pastrois expression systems,which were achieved by using mixed carbon sources with different 13C labeling patterns.These schemes improve spectral resolution by 1.5 fold,produce site-specific labeling patterns and facilitate in obtaining long-range distance restraints.Secondly,I will discuss the joint application of the paramagnetic relaxation enhancements and the non-uniform sampling approaches towards speeding up the 3D SSNMR experiments.With improved sampling schemes,the S/N per time unit was enhanced by 4.0 times.Using the improved SSNMR methods,we obtained backbone and side-chain assignments of LR,revealing the presence of seven transmembrane helices.LR is retinal-binding light-driven proton pump,and is also known as a promising optogenetic tool.The active center includes two Asp residues and a protonated Schiff-base formed by retinal and NH3 group of a Lys residue.Selective Asp carboxyl side-chain experiments indicated that both the two critical Asp residues form strong internal H-bond or salt bridge.On the other hand,the water-edited SSNMR experiments and SSNMR based H/D exchange experiments suggested the rapid exchange between solvent water and proton of Schiff base.This indicated the participation of water molecule in the pathway of proton pump of LR.