W86F/W86A突变对细菌视紫红质光循环影响的差异性分析

Differential Analysis of the Effects of W86A and W86F Mutations on the Bacteriorhodopsin Photocycle

细菌视紫红质(bacteriorhodopsin,bR)是一种光驱受体蛋白,每个活性单元由3个单体组成,每个单体由一分子视蛋白和一分子的视黄醛发色团共价结合而成,其功能为从胞内向胞外定向传输质子,利用形成的质子浓度梯度将光能转化为化学能。光照后视黄醛发色团构型发生all-trans向13-cis转变,蛋白的构象也随之发生了一系列具有稳定中间态的变化并驱动质子的定向传递。为探讨bR视黄醛键合区保守性氨基酸色氨酸86(Tryptophan 86,W86)对其光循环中间态和质子泵功能的影响,本研究采用定点突变技术将W86突变为侧链大小不同的F86和A86,通过原位紫外-可见光吸收光谱、闪光光解光谱、pH滴定、固体核磁共振等技术手段,探究W86F和W86A突变对bR光循环及质子泵功能影响差异性的分子机制。结果表明,W86F和W86A突变均造成了bR暗适应状态下视黄醛顺反异构平衡向顺式构型占优的方向移动,且W86F突变可造成反式构型的完全消失。此外,无论是W86F还是W86A突变都造成了蛋白光循环中间态的减弱且衰减延长,以及质子泵功能减弱,但影响机制各有所不同,这可能与这两个突变对视黄醛多烯链上电子云的分布以及周围残基造成的扰动程度不同有关。

Bacteriorhodopsin(bR)is a light-driven photoreceptor.Each active unit is made up of three monomers and each monomer contains one opsin molecule and one retinal(Ret)chromophore covalently bonded to each other,functioning as a proton pump to transport protons from the cytoplasmic side to the extracellular side directionally to convert light energy into chemical energy through formed proton concentration gradient.Photo-isomerization of the Ret chromophore from the all-trans to the 13-cis configuration triggers cyclic conformation changes that include a series of stable photo-intermediates to transfer a proton vectorial across the cell membrane.Tryptophan 86(W86),one of the key aromatic amino acid residues in the Ret binding pocket,is highly conservative in all microbial rhodopsin family proteins.To reveal the mechanism of the coupling of W86 with the bR photocycle,in this study,in situ solid-state NMR correlation experiments,combined with light-induced transient absorption change measurements,pH titration and site-specific mutation,were employed to elucidate the functional roles of W86 during the bR photocyc.In order to investigate the effect of Tryptophan 86(W86),a conserved amino acid in BR retinal bond region,on its photocyclic intermediate state and proton pump function,we mutated W86 into F86 and A86 with different side chain sizes by site mutation technique.In situ UV-Vis absorption spectroscopy,flash photolysis spectroscopy,pH titration,solid state nuclear magnetic resonance and other techniques were used to explore the molecular mechanism of the difference between W86F and W86A mutations on BR photocirculation and proton pump functions.The results showed that both W86F and W86A mutations shift the dark-adapted isomerization thermal equilibrium of Ret towards a cis dominant direction with the all-trans isomer completely disappearing in W86F.In addition,both W86F and W86A mutations lead to weakened and prolonged attenuation of the intermediate state of the protein photocycle,as well as weakened proton pump function,but the influencing mechanisms are different,which may be related to the difference in the distribution of electron clouds on the retinal-polyene chain and the degree of disturbance caused by the surrounding residues.