摘要
细菌视紫红质(bacteriorhodopsin,bR)是嗜盐杆菌R1M1(Halobacterium salinarum R1M1)紫膜上以视黄醛(Retinal)为发色团的七次跨膜光敏受体蛋白,具有质子泵功能。位于视黄醛键合区胞内侧的色氨酸残基182(tryptophan 182,W182)在整个微生物视紫红质家族中极具保守性,通过与视黄醛多烯侧链的相互作用影响着b R光循环和质子泵浦能力。本研究采用定点突变技术,利用原位紫外-可见光吸收光谱、闪光动力学光谱和p H滴定等手段并结合分子动力学模拟,对W182F和W182A突变体的吸收光谱特性、光循环特征、质子泵的泵浦能力、关键基团的pKa值等改变的分子机制进行了深入探究,从而阐明W182在bR光循环中的功能。研究结果表明,W182F突变对视黄醛键合区造成的扰动较大,而W182A突变则对蛋白胞内半通道造成了较大影响;两种突变对胞外半通道的影响存在不同分子作用机制,致使M中间态的衰减呈现出较大的区别。
Bacteriorhodopsin(bR),a seven-transmembrane photoreceptor protein with retinal as the chromophore found in purple membrane of Halobacterium salinarum R1M1,which has proton pump function.Tryptophan 182(W182),one of the most conserved aromatic residues within the microbial rhodopsin family proteins,locates in the intracellular side of the retinal binding pocket and plays a very important role in affecting the photocycle and proton pumping activity of bR through the interaction with the retinal polyene chain.In this study,site-directed mutagenesis of W182F and W182A,in combination with in-situ UV-vis absorption spectroscopy,light-induced transient absorption change spectroscopy,pH titration and molecular dynamics simulations,were employed to elaborate the functional role of W182 in the bR photocycle.Our results showed that the mutation of W182F caused a great disturbance to the binding region of retinal,while the mutation of W182A had a great effect on the intracellular half channel of the protein.The two mutations had different molecular mechanisms on the effect of proton transport on the extracellular half channel,resulting in a significant difference in the decay of M intermediate state.
作者
王铭
陈思瑾
赵欣
Wang Ming;Chen Sijin;Zhao Xin(Shanghai Key Laboratory of Magnetic Resonance,School of Physics and Electronic Science,East China Normal University,Shanghai,200062)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第7期2685-2695,共11页
Genomics and Applied Biology
基金
国家自然科学基金项目(21475045,30970657)
上海市自然科学基金项目(19ZR1416300)共同资助
