Verhoeven Michiel A, Bovee-Geurts Petra H M, de Groot Huub J M, Lugtenburg Johan, DeGrip Willem J
Leiden Institute of Chemistry, Leiden University, The Netherlands.
J Mol Biol. 2006 Oct 13;363(1):98-113. doi: 10.1016/j.jmb.2006.07.039. Epub 2006 Jul 28.
The C-11=C-12 double bond of the retinylidene chromophore of rhodopsin holds a central position in its light-induced photoisomerization and hence the photosensory function of this visual pigment. To probe the local environment of the HC-11=C-12H element we have prepared the 11-methyl and 12-methyl derivatives of 11-Z retinal and incorporated these into opsin to generate the rhodopsin analogs 11-methyl and 12-methyl rhodopsin. These analog pigments form with much slower kinetics and lower efficiency than the native pigment. The initial photochemistry and the signaling activity of the analog pigments were investigated by UV-vis and FTIR spectroscopy, and by a G protein activation assay. Our data indicate that the ultrafast formation of the first photointermediate is strongly perturbed by the presence of an 11-methyl substituent, but much less by a 12-methyl substituent. These results support the current concept of the mechanism of the primary photoisomerization event in rhodopsin. An important stronghold of this concept is an out-of-plane movement of the C-12H element, which is facilitated by torsion as well as extended positive charge delocalization into the C-10-C-13 segment of the chromophore. We argue that this mechanism is maintained principally with a methyl substituent at C-12. In addition, we show that both an 11-methyl and a 12-methyl substitutent perturb the photointermediate cascade and finally yield a low-activity state of the receptor. The 11-methyl pigment retains about 30% of the G protein activation rate of native rhodopsin, while the 12-methyl chromophore behaves like an inverse agonist up to at least 20 degrees C, trapping the protein in a perturbed Meta-I-like conformation. We conclude that the isomerization region of the chromophore and the spatial structure of the binding site are finely tuned, in order to achieve a high photosensory potential with an efficient pathway to a high-activity state.
视紫红质视黄醛发色团中的C-11=C-12双键在其光诱导的光异构化以及这种视觉色素的光感功能中占据核心地位。为了探究HC-11=C-12H基团的局部环境，我们制备了11-Z-视黄醛的11-甲基和12-甲基衍生物，并将它们掺入视蛋白中以生成视紫红质类似物11-甲基视紫红质和12-甲基视紫红质。这些类似色素形成的动力学比天然色素慢得多，效率也更低。通过紫外-可见光谱和傅里叶变换红外光谱以及G蛋白激活测定法研究了类似色素的初始光化学和信号传导活性。我们的数据表明，11-甲基取代基的存在强烈干扰了第一个光中间体的超快形成，但12-甲基取代基的干扰要小得多。这些结果支持了当前视紫红质初级光异构化事件机制的概念。这一概念的一个重要依据是C-12H基团的平面外移动，这通过扭转以及发色团C-10-C-13片段中的扩展正电荷离域来促进。我们认为，该机制主要在C-12处有甲基取代基时得以维持。此外，我们表明，11-甲基和12-甲基取代基都会干扰光中间体级联反应，并最终产生受体的低活性状态。11-甲基色素保留了天然视紫红质约30%的G蛋白激活率，而12-甲基发色团在至少20摄氏度时表现得像一种反向激动剂，将蛋白质捕获在一种受干扰的类似Meta-I的构象中。我们得出结论，发色团的异构化区域和结合位点的空间结构经过了精细调节，以便通过一条通向高活性状态的有效途径实现高的光感潜力。
