Role of the C9 methyl group in rhodopsin activation:: Characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal
Role of the C9 methyl group in rhodopsin activation:: Characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal
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DOI：
10.1021/bi972060w
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发表时间：
1998-01-13
期刊：
影响因子：
2.9
通讯作者：
Sakmar, TP
中科院分区：
文献类型：
--
作者：
Han, M;Groesbeek, M;Sakmar, TP
视色素视紫红质的激活涉及视色素结合位点内的发色团和视蛋白之间的空间和静电相互作用。去除11-顺式-视黄醛的C-9甲基基团抑制G蛋白转导素的光依赖性活化，表明直接的空间接触。最近，我们已经表明，当跨膜螺旋3中间的Gly（121）被更大的疏水残基取代时，空间相互作用导致受体活化。为了更详细地了解C-9甲基在视紫红质结构和功能中的作用，我们首先研究了重组9-dm-Rho（用11-cis-9-demethylretinal重构的视蛋白）的性质。9-dm-Rho色素显示蓝移λ（max），增加羟胺反应性，并且降低激活转导素的能力。这些性质与C-9甲基是关键的结构锚或发色团在其结合位点的正确对接的假设一致。接下来，我们研究了视蛋白的Gly（121）和视黄醛的C-9甲基之间可能的相互作用，通过表征通过将突变体视蛋白（G121 A、-V、-I、-L和-W）与11-顺式-9-去甲基视黄醛组合产生的重组色素。突变体视蛋白G121 I、-L和-W不能结合发色团。然而，双突变体G121 L/F261 A结合11-cis-9-去甲基retinal以形成λ（max）为451 nm的稳定色素。当在膜中测定活性时，由于发色团上缺少C-9甲基而导致的9-dm-Rho对转导蛋白活化的减少可以通过用大的残基（亮氨酸、异亮氨酸或色氨酸）取代Gly（121）而部分恢复。这些结果支持受体活化的模型，该模型涉及发色团的C-9甲基与跨膜螺旋3上Gly（121）附近的视蛋白之间的空间相互作用。
Activation of the visual pigment rhodopsin involves both steric and electrostatic interactions between the chromophore and opsin within the retinal-binding site. Removal of the C-9 methyl group of 11-cis-retinal inhibits light-dependent activation of the G protein, transducin, suggesting a direct steric contact. More recently, we have shown that steric interactions lead to receptor activation when Gly(121) in the middle of transmembrane helix 3 is replaced by larger hydrophobic residues. In order to understand in more detail the role of the C-9 methyl group of retinal in the structure and function of rhodopsin, we first studied the properties of recombinant 9-dm-Rho (opsin reconstituted with 11-cis-9-demethylretinal). The 9-dm-Rho pigment displayed a blue-shifted lambda(max), increased hydroxylamine reactivity, and decreased ability to activate transducin. These properties are consistent with the hypothesis that the C-9 methyl group is a crucial structural anchor or the correct docking of the chromophore in its binding site. Next, we investigated the possible interaction between Gly(121) of opsin and the C-9 methyl group of retinal by characterizing recombinant pigments produced by combining mutant opsins (G121A, -V, -I, -L, and -W) with 11-cis-9-demethylretinal. Mutant opsins G121I, -L, and -W failed to bind the chromophore. However, the double mutant G121L/F261A bound 11-cis-9-demethylretinal to form a stable pigment with a lambda(max) of 451 nm. When activity was assayed in membranes, the reduction in transducin activation by 9-dm-Rho caused by the lack of a C-9 methyl group on the chromophore could be partially restored by replacing Gly(121) with a bulky residue (leucine, isoleucine, or tryptophan). These results support a model of receptor activation that involves steric interaction between the C-9 methyl group of the chromophore and the opsin in the vicinity of Gly(121) on transmembrane helix 3.