Absorption spectra of opsin-based pigments are tuned from the UV to the red regions by interactions of the chromophore with surrounding amino acid residues. Both vertebrates and invertebrates possess long-wavelength-sensitive (LWS) opsins, which underlie color vision involving "red" sensing. The LWS opsins have independently evolved in each lineage, which suggests the existence of diverse mechanisms in spectral tuning. In vertebrate LWS opsins, the mechanisms underlying spectral tuning have been well characterized by spectroscopic analyses with recombinant pigments of wild type (WT) and mutant opsins. However in invertebrate LWS opsins including insect ones, the mechanisms are largely unknown due to the difficulty in obtaining recombinant pigments. Here we have overcome the problem by analyzing heterologous action spectra based on light-dependent changes in the second messenger in opsin-expressing cultured cells. We found that WTs of two LWS opsins of the butterfly, Papilio xuthus, PxRh3 and PxRh1 have the wavelengths of the absorption maxima at around 570 nm and 540 nm, respectively. Analysis of a series of chimeric mutants showed that helix III is crucial to generating a difference of about 15 nm in the wavelength of absorption maxima of these LWS opsins. Further site-directed mutations in helix III revealed that amino acid residues at position 116 and 120 (bovine rhodopsin numbering system) are involved in the spectral tuning of PxRh1 and PxRh3, suggesting a different spectral tuning mechanism from that of primate LWS opsins.

中文翻译：

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在异源作用光谱学揭示的蝴蝶长波长敏感视觉视蛋白中由螺旋III介导的光谱调谐。

通过生色团与周围氨基酸残基的相互作用，将视蛋白基颜料的吸收光谱从UV调整为红色区域。脊椎动物和无脊椎动物都具有长波长敏感（LWS）视蛋白，它们是涉及“红色”感测的彩色视觉的基础。LWS视蛋白在每个谱系中都独立进化，这表明在光谱调谐中存在多种机制。在脊椎动物的LWS视蛋白中，通过使用野生型（WT）和突变视蛋白的重组色素进行光谱分析，已经很好地表征了潜在的光谱调节机制。但是，在包括昆虫在内的无脊椎动物LWS视蛋白中，由于很难获得重组色素，其机理在很大程度上尚不清楚。在这里，我们通过分析表达视蛋白的培养细胞中第二信使中依赖于光的变化来分析异源作用谱，从而克服了这个问题。我们发现蝴蝶的两个LWS视蛋白WTapis xuthus，PxRh3和PxRh1的WT的最大吸收波长分别在570 nm和540 nm左右。对一系列嵌合突变体的分析表明，螺旋线III对于在这些LWS视蛋白的最大吸收波长上产生约15 nm的差异至关重要。螺旋III中的进一步定点突变表明，位置116和120（牛视紫红质编号系统）的氨基酸残基参与了PxRh1和PxRh3的光谱调谐，这表明与灵长类LWS视蛋白的光谱调谐机制不同。