Ritter Eglof, Stehfest Katja, Berndt Andre, Hegemann Peter, Bartl Franz J
Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
J Biol Chem. 2008 Dec 12;283(50):35033-41. doi: 10.1074/jbc.M806353200. Epub 2008 Oct 16.
Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of tau = 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu(90) is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family.
通道视紫红质-2（ChR2）是一种微生物型视紫红质，也是一种光门控阳离子通道，可控制衣藻的趋光性。我们在COS细胞中表达ChR2，对其进行纯化，随后通过闪光光解、紫外可见光谱和傅里叶变换红外差示光谱对这种不同寻常的光感受器进行研究。我们鉴定出野生型ChR2的几种瞬态光产物，并将它们的动力学和分子特性与ChR2突变体E90Q的动力学和分子特性进行比较。基于光谱数据，我们建立了一个包含六个可区分中间体的光循环模型。这个光循环与团藻中与ChR2相关的通道视紫红质的光循环有相似之处，但也存在显著差异。我们发现分子变化包括视黄醛异构化、羧酸氢键的变化以及蛋白质主链结构的大幅改变。这些改变比在细菌视紫红质等其他微生物视紫红质的光循环中观察到的改变更强，并且与动物视紫红质中发生的改变有关。紫外可见光谱和傅里叶变换红外差示光谱揭示了在暗态恢复过程中存在的两个具有不同时间常数（τ = 6和40秒）的晚期中间体。Glu(90)的羧基侧链参与了缓慢转变。我们结合视紫红质家族的其他成员讨论了ChR2光循环过程中的分子变化。
