Complex active site structures influence absorption spectrum of Chrimson wild type and mutants

The red light-activated channelrhodopsin Chrimson is widely used in optogenetic applications, including vision and hearing restoration. Despite structural insights from X-ray crystallography and their identification of another red-shifted mutant (S169A), the molecular determinants of its large spectral shift remain incompletely understood. In this study, we present a computational analysis of wild type Chrimson and several mutants within a QM/MM approach. A key finding is the pronounced flexibility of the active site, where multiple conformations interconvert on the nano-second scale. We also highlight the role of residue S169, whose hydrogen bonding influences the torsional flexibility of the nearby counterion E165, thus affecting the prevalence of distinct structural motifs at the active site. The comparison of experimental and QM/MM MD-sampled absorption spectra supports the validity of our computational models. A direct hydrogen bond between counterions is identified as one of the key factors contributing to the red-shifted absorption spectrum, with increased occurrence observed in the red-shifted S169A mutant.