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

Signal Transduction in Light-Oxygen-Voltage Receptors Lacking the Active-Site Glutamine

“…Our data however illustrate that even when these residues are replaced, LOV receptors can principally transduce light signals, although likely with reduced amplitude. These considerations transcend the optogenetic deployment of LOV receptors and also concern the widespread applications of cysteine-deficient (and often additionally glutamine-deficient 66 ) LOV modules as fluorescent proteins 67,68 and photosensitizers for molecular oxygen 53 .…”