Department of Chemistry and The PULSE Institute, Stanford University, Stanford, California 94305, United States.
SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, United States.
J Am Chem Soc. 2021 Apr 14;143(14):5425-5437. doi: 10.1021/jacs.1c00058. Epub 2021 Apr 1.
Channelrhodopsin 2 (ChR2) is the most commonly used tool in optogenetics. Because of its faster photocycle compared to wild-type (WT) ChR2, the E123T mutant of ChR2 is a useful optogenetic tool when fast neuronal stimulation is needed. Interestingly, in spite of its faster photocycle, the initial step of the photocycle in E123T (photoisomerization of retinal protonated Schiff base or RPSB) was found experimentally to be much slower than that of WT ChR2. The E123T mutant replaces the negatively charged E123 residue with a neutral T123 residue, perturbing the electric field around the RPSB. Understanding the RPSB photoisomerization mechanism in ChR2 mutants will provide molecular-level insights into how ChR2 photochemical reactivity can be controlled, which will lay the foundation for improving the design of optogenetic tools. In this work, we combine ab initio nonadiabatic dynamics simulation, excited state free energy calculation, and reaction path search to comprehensively characterize the RPSB photoisomerization mechanism in the E123T mutant of ChR2. Our simulation agrees with previous experiments in predicting a red-shifted absorption spectrum and significant slowdown of photoisomerization in the E123T mutant. Interestingly, our simulations predict similar photoisomerization quantum yields for the mutant and WT despite the differences in excited-state lifetime and absorption maximum. Upon mutation, the neutralization of the negative charge on the E123 residue increases the isomerization barrier, alters the reaction pathway, and changes the relative stability of two fluorescent states. Our findings provide new insight into the intricate role of the electrostatic environment on the RPSB photoisomerization mechanism in microbial rhodopsins.
通道视紫红质 2（ChR2）是光遗传学中最常用的工具。由于其光循环速度比野生型（WT）ChR2 更快，因此当需要快速神经元刺激时，ChR2 的 E123T 突变体是一种有用的光遗传学工具。有趣的是，尽管其光循环速度更快，但实验发现 E123T 中光循环的初始步骤（视黄醛质子化席夫碱或 RPSB 的光异构化）比 WT ChR2 慢得多。E123T 突变体用中性 T123 残基取代带负电荷的 E123 残基，扰乱了 RPSB 周围的电场。了解 ChR2 突变体中 RPSB 光异构化机制将为如何控制 ChR2 光化学反应性提供分子水平的见解，这将为改进光遗传学工具的设计奠定基础。在这项工作中，我们结合从头算非绝热动力学模拟、激发态自由能计算和反应路径搜索，全面表征了 ChR2 的 E123T 突变体中 RPSB 的光异构化机制。我们的模拟与之前的实验一致，预测 E123T 突变体的吸收光谱红移和光异构化显著减慢。有趣的是，尽管激发态寿命和吸收最大值存在差异，我们的模拟预测突变体和 WT 的光异构化量子产率相似。突变后，E123 残基上负电荷的中和增加了异构化势垒，改变了反应途径，并改变了两种荧光态的相对稳定性。我们的发现为微生物视紫红质中静电环境对 RPSB 光异构化机制的复杂作用提供了新的见解。
