Insights into the mechanisms of light‐oxygen‐voltage domain color tuning from a set of high‐resolution X‐ray structures

Abstract Light‐oxygen‐voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All the currently known LOV have absorption maxima range ~440 to ~450 nm, it is not clear whether they shifted significantly using mutations. Here, we generated a panel domain variants by mutating key chromophore‐proximal glutamine aminoacid thermostable flavin based fluorescent protein CagFbFP (Gln148) asparagine, aspartate, glutamate, histidine, lysine arginine. Absorption spectra all mutants blue‐shifted, with maximal shift 8 nm observed for Q148H variant. While its Q148N/D/E sensitive pH, Q148H/K/R reveal moderate red induced byacidic pH. To gain further insight, determined high resolution crystal structures studied at resolutions from 1.07 Å Q148D 1.63 Q148R. Whereas some variants, 148 remains vicinity flavin, Q148K, Q148R partially Q148D, C‐terminus unlatches side chain residue reoriented away chromophore. Our results explain absence color shifts replacing Gln148 charged aminoacids pave way rational design color‐shifted proteins.