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 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 aminoacid 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 byacidic 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 aminoacid 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 aminoacids and pave the way for rational design of color‐shifted flavin based fluorescent proteins.

The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins

“…Therefore, we conclude that irrespective of the buffer and post-translational modification, the C terminus of the iLOV-Q489K and CagFbFP-Q148K variants is unlatched, the LOV core domain is opened up to allow solvent ingress, and the introduced lysine occupies an extended conformation facing away from the FMN chromophore. The related variant CagFbFP-Q148R, which was recently reported, also shows blue-shifted spectral properties, featuring an unlatched C terminus (26). We thus suggest these structural changes as the characteristic features for a blue-shifted absorption and emission spectrum, that is, when compared with the parental FbFPs.…”

Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species

“…In prolinesubstituted CagFbFP variants, the temperature of both transitions is raised by up to ~ 2°C [31]. In the variants generated to achieve spectral tuning, both transitions are shifted to lower temperatures [32,33]. Finally, two or even three different transitions were observed in variants with extended flexible loops of different lengths, which were designed for identification of the best potential split sites during the process of generating a split flavin-based fluorescent reported based on CagFbFP [34].…”

Experimental Characterization of In Silico Red-Shift-Predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S Mutants

“…Several studies recently also provided a molecular characterization of the spectral effects of mutations on iLOV to gain a mechanistic understanding. − Röllen and colleagues have shown that iLOV V392T/Q489K fluorescence has a slight red-shift with an emission spectrum maximum of 502 nm . Similar mutations on iLOV homolog protein CagFbFP I52T/Q148K with the emission maximum of 504 nm and another blue-shifted variant CagFbFP Q148K with the emission maximum of 491 nm were used in fluorescence microscopy experiments and were successfully spectrally separated .…”