Ligand-induced population shift leads to the activation of rhodopsin, a G-protein coupled receptor
Ligand-induced population shift leads to the activation of rhodopsin, a G-protein coupled receptor
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配体诱导的群体转移导致视紫红质（一种 G 蛋白偶联受体）的激活
DOI：
10.1016/j.bpj.2014.01.020
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Y
中科院分区：
文献类型：
--
作者：
Maeda;R.;Hiroshima;M.;Yamashita;T.;Wada;A.;Nishimura;S.;Sako;Y.;Shichida;Y.;and Imamoto;Y
视紫红质是一种G蛋白偶联受体，视紫红质中的发色团根据其构型和质子化状态而起反激动或激动作用。视紫红质的光刺激导致活性状态（Meta-II）和其非活性前体（Meta-I）之间的pH依赖性平衡。在这里，我们监测构象变化的视紫红质使用荧光探针Alexa 594在细胞质表面，这表明荧光增加后产生的活性状态，通过单分子测量。单个光敏视紫红质分子的荧光强度在两种状态之间交替。有趣的是，对于无配体的视紫红质（视蛋白）也观察到了这种荧光交替，但对于暗态视紫红质则没有观察到。此外，通过荧光测量估计的Meta-I/Meta-II平衡的pH依赖性显著偏离基于吸收光谱的估计，表明Meta-I和Meta-II是两种构象异构体的混合物。我们的观察结果表明，视紫红质分子本质上采取积极和消极的构象，和配体视网膜移动构象平衡。这些发现提供了动态的见解G蛋白偶联受体的激活机制。
Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.