WHY ARE BLUE VISUAL PIGMENTS BLUE - A RESONANCE RAMAN MICROPROBE STUDY
WHY ARE BLUE VISUAL PIGMENTS BLUE - A RESONANCE RAMAN MICROPROBE STUDY
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DOI：
10.1073/pnas.86.5.1515
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发表时间：
1989-03-01
影响因子：
11.1
通讯作者：
MATHIES, RA
中科院分区：
文献类型：
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作者：
LOPPNOW, GR;BARRY, BA;MATHIES, RA
共振拉曼显微镜已被开发用于研究单个感光细胞视觉色素中视网膜假体组的结构。通过将探测激光聚焦在冷冻在 77 K 冷台上的完整光感受器上来获得拉曼振动光谱。为了阐明蓝色视色素的波长调节机制，我们使用该装置研究了蟾蜍（Bufo marinus）“绿杆”中发现的 440 nm 吸收色素的发色团结构。绿棒颜料的 9-顺式异视紫红质形式表现出 1662-cm-1 C.dbd.NH+ 希夫碱拉伸模式，在氘取代的水中转变为 1636-cm-1。这表明与蛋白质的希夫碱键被质子化。希夫碱的质子化足以解释这种色素的 4440 nm 吸收最大值，而无需引起任何额外的蛋白质-发色团相互作用。烯键带以及对扰动敏感的 C-10-C-11 和 C-14-C-15 拉伸模式与溶液中 9-顺式质子化视网膜希夫碱具有相同频率的观察结果支持了不存在额外扰动。我们证明蓝色视色素含有未受扰动的质子化席夫碱，这提供了实验证据，表明导致 500 nm 吸收色素中视蛋白位移的蛋白质电荷扰动在蓝色色素的视蛋白中被去除，如序列数据所示。
A resonance Raman microscope has been developed to study the structure of the retinal prosthetic group in the visual pigments of individual photoreceptor cells. Raman vibrational spectra are obtained by focusing the probe laser on intact photoreceptors frozen on a 77 K cold stage. To elucidate the mechanism of wavelength regulation in blue visual pigments, we have used this apparatus to study the structure of the chromophore in the 440-nm absorbing pigment found in "green rods" of the toad (Bufo marinus). The 9-cis isorhodopsin form of the green rod pigment exhibits a 1662-cm-1 C.dbd.NH+ Schiff base stretching mode that shifts to 1636-cm-1 in deuterium-substituted H2O. This demonstrates that the Schiff base linkage to the protein is protonated. Protonation of the Schiff base is sufficient to explain the 4440-nm absorption maximum of this pigment without invoking any additional protein-chromophore interactions. The absence of additional perturbations is supported by the observation that the ethylenic band and the perturbation-sensitive C-10-C-11 and C-14-C-15 stretching modes have the same frequency as those of the 9-cis protonated retinal Schiff base in solution. Our demonstration that a blue visual pigment contains an unperturbed protonated Schiff base provides experimental evidence that the protein charge perturbation responsible for the opsin shift in the 500-nm absorbing pigments is removed in the opsins of blue pigments, as suggested by the sequence data.