Electrostatic Control of Photoisomerization in Channelrhodopsin 2.

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.