氮氧自由基自旋探针R1在特定空间位点上对bacteriorhodopsin产生颜色调谐效应的机理分析

Color Tuning in Bacteriorhodopsin Induced by Nitroxide Free Radical Spin Probe R1 at Specific Site

蛋白质结构研究中的氮氧自由基自旋探针R1,在特定的空间位置上会对bacteriorhodopsin的结构与功能产生影响。该文作者发现:将bacteriorhodopsin中远离视黄醛的第192位Gly替换为自旋探针R1后,产生了显著的颜色调谐,最大吸收峰有28nm的蓝移。但用R1替换Gly192邻近的Gly195和Ala196,未产生颜色调谐效应;替换螺旋E末端的Val130和Tyr131,以及螺旋B末端的Leu62,均无颜色调谐。进一步表征bacteriorhodopsin光循环中间体O和M的衰减过程发现:R1替换前,Gly192Cys(+)的O态衰减寿命约为847.7ms;但替换后,其衰减显著加快至308.2ms。M态的衰减寿命在R1替换后反而显著延长,尤其是慢组分Ms从24.8ms延长至83.6ms。Gly192R1的顺磁共振信号揭示:该位置上的R1处于低运动性状态,表明R1可能与局部环境的结构组分发生了直接作用。晶体结构分析发现:R1可能直接与质子传递通道中的"三体水分子"作用。由于"三体水分子"通过长程的氢键网络与视黄醛活性中心相连,并且参与质子在bacteriorhodopsin中的传递,因此自旋探针R1在膜表面Gly192位置处产生的颜色调谐,可能是一种分子内的长程作用结果。

Nitroxide Spin probe R1 widely used in protein structural research may affect structure and function of bacteriorhodopsin at specific site. This work reported a significant color tuning, about 28 nm blue shift of maximum absorption, happened at Gly192 substituted with a spin probe R1. However, no color tuning at Gly195 and Ala196, which are close to Gly192, was observed. Color tuning at Val130 and Tyr131 on helix E and Leu62 on helix B was not observed either. Analysis of intermediate O in bacteriorhodopsin photocycle showed that the lifetimes of O was about 847.7ms in Gly192Cys（＋） and decreased to 308.2 ms in Gly192R1. The decay of intermediate M was significantly prolonged after R1 substitution. Especially the slow decay component Ms had a lifetime of 24.8 ms in Gly192Cys （＋） and a lifetime of 83.6ms in Gly192R1. The electron paramagnetic resonance signal of Gly192R1 showed that R1 at this site was in an immobile state, and might have strong tertiary contacts with nearby structural elements. Structural analysis suggested that the spin probe R1 at Gly192 might have a direct interaction with the ＂water trimer＂ in bacteriorhodopsin proton transport channel. Since the ＂water trimer＂ is connected with the retinal active site through long-distance hydrogen bonded network and participates in proton translocation, the authors suggest that color tuning induced by spin probe R1 at Gly192 may be caused by a long-range interaction within bacteriorhodopsin.