Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Institute for Integrative Neurobiology and Department of Biology, Faculty of Science and Engineering, Konan University, Kobe 658-8501, Japan.
Proc Natl Acad Sci U S A. 2017 Jun 6;114(23):6028-6033. doi: 10.1073/pnas.1701088114. Epub 2017 May 22.
Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.
Ci-opsin1 是一种存在于无脊椎动物幼体眼点中的可见光敏感视蛋白。这种无脊椎动物视蛋白在视蛋白系统发育树中属于脊椎动物视觉和非视觉视蛋白群。Ci-opsin1 在位置 113 和 181 处含有候选抗衡离子（谷氨酸残基）；前者是脊椎动物视觉视蛋白谱系中新获得的位置，而后者是广泛存在于无脊椎动物视蛋白中的古老位置。在这里，我们表明 Ci-opsin1 中的 Glu113 和 Glu181 协同作用作为抗衡离子，赋予 Ci-opsin1 分子特性处于脊椎动物和无脊椎动物型视蛋白之间。Ci-opsin1 中的抗衡离子协同作用通过 E113Q 和 E181Q 突变体表现出 pH 依赖性光谱位移来证明，而只有脊椎动物视紫红质中的 E113Q 突变才会产生这种光谱位移。在吸收光时，Ci-opsin1 在质子化和去质子化的 Schiff 碱的两种中间体之间形成平衡，分别是 MI 样和 MII 样中间体。添加 G 蛋白会导致平衡向 MI 样中间体移动，表明 Ci-opsin1 在其活性状态下具有质子化的 Schiff 碱，类似于无脊椎动物型视蛋白。Ci-opsin1 的 G 蛋白激活效率介于脊椎动物和无脊椎动物型视蛋白之间。有趣的是，E113Y 和 E181S 突变分别将 Ci-opsin1 的分子特性改变为类似于无脊椎动物型或双稳态视蛋白以及脊椎动物远古/脊椎动物远古长或单稳态视蛋白的特性。这些结果强烈表明，抗衡离子 Glu113 的获得是脊椎动物/被囊动物共同祖先中视觉视蛋白分子特性变化的关键步骤，是脊椎动物视觉视蛋白进化的关键步骤。
