A dark and constitutively active mutant of the tiger salamander UV pigment.
A dark and constitutively active mutant of the tiger salamander UV pigment.
复制标题
虎蝾螈紫外线色素的深色且具有组成活性的突变体。
DOI：
10.1021/bi047898f
复制
发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Oprian,DanielD
中科院分区：
文献类型：
--
作者：
Kono,Masahiro;Crouch,RosalieK;Oprian,DanielD
虎斑UV视锥色素的三个突变体(F86L/T93P/S118T；牛视紫质编号)似乎被捕获在类似于变紫红质-II的开放构象中。该色素能够在黑暗中激活转导蛋白，而无配体的脱辅基蛋白也能够结构性地激活转导蛋白。该色素允许质子和氯离子从溶液中进入活性部位，因为它显示出与pH和氯化钠有关的吸收光谱，而野生型色素没有观察到。然而，当三个突变体与11-顺式-9-去甲基视黄醇重组后，野生型的光依赖活性和pH无关的吸收光谱被恢复。这些结果表明，结合天然发色团不能使蛋白质失活，因为蛋白质(可能是118位残基)与发色团的9-甲基之间存在空间相互作用。此外，9-去甲基视网膜再生色素的吸收光谱比相同视网膜类似物产生的人蓝或火蜥蜴UV野生色素的吸收光谱更宽，在吸收最大处的消光率更低。广谱似乎由两个或更多物种组成，并可以通过两种野生型色素的比例光谱之和很好地拟合。结合发色团似乎将色素捕获在两种或两种以上的构象中。这里报道的三个突变体代表了第一个暗活性锥体色素和结构性活性锥体视蛋白的例子。
A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.