类胡萝卜素发色团对光驱质子泵aR4关键残基F146功能的影响

Effect of Carotenoid Chromophore on the Function of the Key Residue F146 in the Light-driven Proton Pump Archaerhodopsin-4

古紫质-4(archaerhodopsin-4,aR4)是一种类似于细菌视紫红质(bacterirhodopsin,bR)的光驱质子泵蛋白,同样具有三聚结构。其差异性在于,每个单体除了由一分子视蛋白和一分子视黄醛发色团共价结合而成外,单体间还镶嵌有一个类胡萝卜素发色团菌红素(bacterioruberin),同时二者具有不同的质子传输时序。苯丙氨酸-146(phenylalanine-146,F146)和甲硫氨酸-145(methionine-145,M145)是aR4和bR视黄醛键合区处在相同位置且唯一不同的关键残基。已有研究结果表明,bR的M145F突变可影响其暗适应态下视黄醛的顺反异构平衡,而且对其光循环有着重要的影响。然而,F146在aR4中的作用尚不清楚,特别是菌红素对质子泵视黄醛键合区关键残基的作用是否存在调控尚未见报道。为此,本研究采用定点突变技术、原位紫外-可见光吸收光谱、闪光动力学光谱、酸碱滴定和固体核磁共振等技术手段,对比分析F146M和M145F单点突变对aR4和bR视黄醛键合区和光循环影响的共性和差异性。研究结果表明,F146和M145在aR4和bR中均起到维持视黄醛顺反异构平衡的作用,突变可造成键合区装配紧密程度和胞外侧氢键网络的改变,从而参与调控质子释放至胞外的过程。此外,本研究发现菌红素不仅具有稳定aR4三聚结构的作用,更重要的是对键合区关键残基F146存在调控作用,菌红素的缺失可造成F146作用的弱化。

Archaerhodopsin-4(aR4),a bacteriorhodopsin(bR)like light-driven proton pump,possesses the similar trimeric structure.aR4 contains not only an opsin molecule and a covalently bound retinal chromophore,but also a carotenoid chromophore bacterioruberin that is packed in the crevices between the adjacent trimer subunits of the protein trimer.In addition,aR4 displays an opposite temporal order of proton release and uptake to that of bR.Phenylalanine-146(F146)and methionine-145(M145)are the key residues that locate within the same position in the retinal binding pocket of aR4 and bR,respectively,and the only different residue of the two binding pockets.It was reported that M145 F mutation can affect the cis-trans thermal equilibrium of retinal in the dark-adapted bR,and has an important effect on its photocycle.However,nothing is clear about the function of F146 in the proton pump aR4,especially any modulation of the carotenoid chromophore bacterioruberin to the function of the key residue in the retinal binding pocket.To address this research gap,site-specific mutagenesis,combined with in situ UV-Vis absorption spectroscopy,solid-state NMR,flash light-induced transient absorption change measurements,and pH titration were used to study the function commonality and difference of F146M and M145F mutation in aR4 and bR.Our results showed that both F146 and M145 played a role in maintaining the cis-trans thermal equilibrium of retinal in the dark-adapted proteins.Both F146M and M145F mutations could cause changes to the retinal binding pocket and the hydrogen bond network with the proton translocation channel,thus participating in the regulation of proton transfer.In addition,it was identified that bacterioruberin not only can stabilize the trimeric structure,but also can modulate the function of F146,a key residue of the retinal binding pocket,removal of bacterioruberin will weaken the effect of F146 in aR4.