Mutational analyses identify a single amino acid critical for colour tuning in proteorhodopsins.

Microbial rhodopsins are light-activated proteins that contain seven transmembrane alpha-helices. Spectral tuning in microbial rhodopsins is a useful optogenetic tool. In this study, we report a new site that controls spectral tuning. In the proteorhodopsins ISR34 and ISR36, a single amino-acid substitution at Cys189 caused an absorption maximum shift of 44 nm, indicating spectral tuning at a specific site. Comparison of single amino acid substitutions was conducted using photochemical and photobiological approaches. The maximum absorption for red-shift was measured for mutations at positions 189 and 105 in ISR34, both residues being equally important. Structural changes resulting from amino acid substitutions are related to pK a values, pumping activity and spectral tuning.