Conformational Changes of Channelrhodopsin-2
Conformational Changes of Channelrhodopsin-2
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DOI：
10.1021/ja8084274
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发表时间：
2009-06-03
影响因子：
15
通讯作者：
Heberle, Joachim
中科院分区：
文献类型：
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作者：
Radu, Ionela;Bamann, Christian;Heberle, Joachim
通道视紫红质-2 （ChR2）是绿藻莱茵衣藻中作为趋光受体的一类新型光门控离子通道的成员。该蛋白被用于光遗传学，其中神经回路在高时空控制下受到光刺激。尽管ChR2在生理实验中的应用迅速增加，但其反应机制尚不清楚。在这里，我们应用振动光谱来追踪视网膜发色团光激发后ChR2的结构变化。不同光循环中间体的FT-IR差异光谱显示，在通道的导电状态（P-3）之前（Pi态）和之后（P-4）的光化学反应阶段与蛋白质主链的大构象变化有关，这表明酰胺I波段的强烈差异。检测了质子化羧基氨基酸侧链（D1 56, E90）的关键氢键变化，并对其作用机理进行了探讨。我们利用延长P-3寿命的C128T突变体，应用FT-IR和共振拉曼光谱研究了ChR2的导电P-3态。最后，提出了一个机制模型，将观察到的ChR2的结构变化与通道电导的变化联系起来。
Channelrhodopsin-2 (ChR2) is a member of the new class of light-gated ion channels which serve as phototaxis receptors in the green alga Chlamydomonas reinhardtii. The protein is employed in optogenetics where neural circuits are optically stimulated under high spatiotemporal control. Despite its rapidly growing use in physiological experiments, the reaction mechanism of ChR2 is poorly understood. Here, we applied vibrational spectroscopy to trace structural changes of ChR2 after light-excitation of the retinal chromophore. FT-IR difference spectra of the various photocycle intermediates revealed that stages of the photoreaction preceding (Pi state) and succeeding (P-4) the conductive state of the channel (P-3) are associated with large conformational changes of the protein backbone as indicate by strong differences in the amide I bands. Critical hydrogen-bonding changes of protonated carboxylic amino acid side chains (D1 56, E90) were detected and discussed with regard to the functional mechanism. We used the C128T mutant where the lifetime Of P-3 is prolonged and applied FT-IR and resonance Raman spectroscopy to study the conductive P-3 state of ChR2. Finally, a mechanistic model is proposed that links the observed structural changes of ChR2 to the changes in the channel's conductance.