Contribution of glutamic acid in the conserved E/DRY triad to the functional properties of rhodopsin.
Contribution of glutamic acid in the conserved E/DRY triad to the functional properties of rhodopsin.
复制标题
保守的 E/DRY 三联体中谷氨酸对视紫红质功能特性的贡献。
作者：
Sato;K.;Yamashita;T.;and Shichida;Y.
视紫红质是一种专门用于光接受的G蛋白偶联受体，含有一个吸收光的发色团视网膜，通过质子化的希夫碱键与视蛋白的赖氨酸残基结合。光将视紫红质转化为活性态后视紫红质II （MII）及其前体后视紫红质I （MI）的平衡混合物，它们分别具有去质子化和质子化的希夫碱发色团。这种平衡被认为不是依赖于希夫碱发色团的pKaof，而是依赖于螺旋III中高度保守的E/DRY三联体中的谷氨酸E134。我们对E134及其附近残基进行了突变分析，以检验这种平衡是否真的依赖于E134的pKaof，并获得E134对视紫红质G蛋白激活特性的贡献线索。除E134D外，134位的所有单突变体都失去了典型的ph依赖平衡，表明E134的羧基负责平衡。有趣的是，134位突变对MII或MII光谱及MII的G蛋白激活效率影响不大，但会引起MI - MII平衡的改变。在134位含有疏水或含酰胺残基的突变体形成有利于MII的平衡，导致光诱导G蛋白激活效率提高。另一方面，野生型的视蛋白活性低于突变体，表现出合理的光依赖活性。这些结果强烈提示E134的进化意义不是G蛋白活性的增加，而是抑制视蛋白活性。
Rhodopsin is a G protein-coupled receptor specialized for photoreception and contains a light-absorbing chromophore retinal that binds to the lysine residue of opsin through a protonated Schiff base linkage. Light converts rhodopsin to an equilibrium mixture of the active state metarhodopsin II (MII) and its precursor, metarhodopsin I (MI), which have deprotonated and protonated Schiff base chromophores, respectively. This equilibrium was thought to depend on the pKaof not the Schiff base chromophore but glutamic acid E134 in the highly conserved E/DRY triad in helix III. We performed mutational analyses of E134 and nearby residues to examine whether the equilibrium is really dependent on the pKaof E134 and to obtain clues about the contribution of E134 to the G protein activation characteristics of rhodopsin. All the single mutants at position 134 except for E134D lost the characteristic pH-dependent equilibrium, indicating that the carboxyl group of E134 is responsible for the equilibrium. Interestingly, mutation at position 134 caused little change in the MI or MII spectra or G protein activation efficiency of MII, while it caused a shift of the MI–MII equilibrium. The mutants containing hydrophobic or amide-containing residues at position 134 formed an equilibrium in favor of MII, resulting in an increase in light-induced G protein activation efficiency. On the other hand, the wild type exhibited an opsin activity lower than those of the mutants, which exhibited reasonable light-dependent activities. These results strongly suggest that the evolutionary significance of E134 is not an increase in G protein activity but rather suppression of the opsin activity.