Ockenfels Andreas, Schapiro Igor, Gärtner Wolfgang
Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, D-45470 Mülheim, Germany.
Photochem Photobiol Sci. 2016 Feb;15(2):297-308. doi: 10.1039/c5pp00322a. Epub 2016 Feb 1.
A series of vitamin-A aldehydes (retinals) with modified alkyl group substituents (9-demethyl-, 9-ethyl-, 9-isopropyl-, 10-methyl, 10-methyl-13-demethyl-, and 13-demethyl retinal) was synthesized and their 11-cis isomers were used as chromophores to reconstitute the visual pigment rhodopsin. Structural changes were selectively introduced around the photoisomerizing C11=C12 bond. The effect of these structural changes on rhodopsin formation and bleaching was determined. Global fit of assembly kinetics yielded lifetimes and spectral features of the assembly intermediates. Rhodopsin formation proceeds stepwise with prolonged lifetimes especially for 9-demethyl retinal (longest lifetime τ3 = 7500 s, cf., 3500 s for retinal), and for 10-methyl retinal (τ3 = 7850 s). These slowed-down processes are interpreted as either a loss of fixation (9dm) or an increased steric hindrance (10me) during the conformational adjustment within the protein. Combined quantum mechanics and molecular mechanics (QM/MM) simulations provided structural insight into the retinal analogues-assembled, full-length rhodopsins. Extinction coefficients, quantum yields and kinetics of the bleaching process (μs-to-ms time range) were determined. Global fit analysis yielded lifetimes and spectral features of bleaching intermediates, revealing remarkably altered kinetics: whereas the slowest process of wild-type rhodopsin and of bleached and 11-cis retinal assembled rhodopsin takes place with lifetimes of 7 and 3.8 s, respectively, this process for 10-methyl-13-demethyl retinal was nearly 10 h (34670 s), coming to completion only after ca. 50 h. The structural changes in retinal derivatives clearly identify the precise interactions between chromophore and protein during the light-induced changes that yield the outstanding efficiency of rhodopsin.
合成了一系列具有修饰烷基取代基的维生素 A 醛（视黄醛）（9-去甲基、9-乙基、9-异丙基、10-甲基、10-甲基-13-去甲基和 13-去甲基视黄醛），并将它们的 11-顺式异构体用作发色团来重构视觉色素视紫红质。在光异构化的 C11=C12 键周围选择性地引入了结构变化。确定了这些结构变化对视紫红质形成和漂白的影响。组装动力学的全局拟合得出了组装中间体的寿命和光谱特征。视紫红质的形成是逐步进行的，寿命延长，特别是对于 9-去甲基视黄醛（最长寿命 τ3 = 7500 s，相比之下，视黄醛的 τ3 = 3500 s）和 10-甲基视黄醛（τ3 = 7850 s）。这些减缓的过程被解释为在蛋白质构象调整过程中固定作用的丧失（9dm）或空间位阻的增加（10me）。量子力学和分子力学（QM/MM）相结合的模拟提供了对视黄醛类似物组装的全长视紫红质的结构洞察。测定了漂白过程（微秒到毫秒时间范围）的消光系数、量子产率和动力学。全局拟合分析得出了漂白中间体的寿命和光谱特征，揭示了显著改变的动力学：野生型视紫红质以及漂白的和 11-顺式视黄醛组装的视紫红质最慢的过程分别发生在 7 秒和 3.8 秒的寿命，而对于 10-甲基-13-去甲基视黄醛，这个过程接近 10 小时（34670 s），仅在约 50 小时后才完成。视黄醛衍生物中的结构变化清楚地确定了在光诱导变化期间发色团与蛋白质之间的精确相互作用，这些变化产生了视紫红质的卓越效率。
