Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate
Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate
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DOI：
10.1371/journal.pone.0209506
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发表时间：
2018-12
期刊：
影响因子：
3.7
通讯作者：
Gaoxiang Mei;N. Mamaeva;S. Ganapathy;Peng Wang;W. J. DeGrip;K. Rothschild
中科院分区：
文献类型：
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作者：
Gaoxiang Mei;N. Mamaeva;S. Ganapathy;Peng Wang;W. J. DeGrip;K. Rothschild
微生物视紫红质已成为光遗传学领域的重要工具。然而，由于生物组织对可见光的强烈吸收和散射，在许多情况下，有效的体内光遗传学受到严重限制。最近，视蛋白位点定向诱变和模拟视网膜替代的结合产生了在近红外（NIR）中吸收最大的变形视紫红质变体。本研究利用紫外-可见-近红外吸收光谱和共振拉曼光谱研究了MMAR再生的绿色吸收蛋白紫红质（GPR）的双突变体D212N/F234S，这是一种含有甲胺修饰的β-离子环的视网膜类似物。在560,620,710和780 nm附近检测到4个不同的子组分吸收谱带，其中近红外谱带在pH <7.3处占主导地位，可见谱带在pH < 9.5处占主导地位。基于λmax和γC = c之间扩展的线性相关关系，1064 nm激发下的FT-Raman光谱在1482和1498 cm-1处显示了两个强乙烯带，对应于近红外子成分吸收带。该光谱在指纹区和HOOP模式区显示了O640光中间体在适应光的细菌紫红质光循环中的高度特征。相比之下，532 nm的激发增强了560 nm的成分，其表现出与适应光的细菌视紫红质和/或酸紫色形式的细菌视紫红质非常相似的能带。原生GPR及其突变体D97N在MMAR再生后也表现出相似的吸收和拉曼光谱，但近红外吸收组分的贡献较弱。基于这些结果，我们提出含有MMAR的GPR-D212N/F234S的近红外吸收来自于o型发色团，其中希夫碱反离子D97被质子化，MMAR由于共轭多烯段的扭曲而采用非平面的全反式结构。这种结构的特点是广泛的电荷离域，很可能涉及MMAR发色团中的氮原子。
Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm-1 corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λmax and γC = C. This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O640 photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all-trans configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore.