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.