Chrimson is a naturally occurring channelrhodopsin with one of the most red-shifted absorption maxima (λmax = 590 nm). This unique absorption makes it an ideal candidate for noninvasive optogenetic applications. The origin of the absorption maximum is thought to stem from a single deprotonated counterion near the Schiff base. However, it remains unclear which of the two potential counterions, E165 and D295, is protonated. In addition, a third titratable residue (E132) near the chromophore may also play a role in the spectral tuning mechanism. A single mutation from serine to alanine (S169A) further red-shifts the absorption maximum to 608 nm. Here we elucidate the mechanism behind the red-shifted absorption maximum using classical molecular dynamics simulations in tandem with hybrid QM/MM simulations. We found that protonation of both E165 and E132 leads to the correct experimental absorption trend for the wild type. This was further validated for the S169A mutant. The derived understanding of the spectral shift will guide the experimental design of red-shifted microbial rhodopsins.
Church et al. (Wed,) studied this question.