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

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 ofin silicored-shift predicted iLOVL470T/Q489Kand iLOVV392K/F410V/A426Smutants

“…The mechanistic understanding of the spectral effects of the tested iLOV mutants requires more detailed molecular characterization efforts like recently applied by Goncharov et al 24 , Remeeva et al 25 and Röllen et al 26 . In the latter, authors have shown that iLOV V392T/Q489K has a slight red shift with emission spectrum maxima of 502 nm.…”

“…Several studies recently also provided a molecular characterization of the spectral effects of mutations on iLOV to gain a mechanistic understanding [27][28][29] . Röllen et al 29 (and its homolog CagFbFP I52T/Q148K ) was mutated at the same sites as in our study, the V392T mutation does not reduce the affinity of iLOV for FMN as much as V392K does (FMN ΔΔG for V392T 0.67 kcal mol -1 vs for V392K 1.05 kcal mol -1 predicted by PremPLI 26 ). For the rational design of iLOV variants, using in silico predictors could guide researchers to filter out mutations that would disrupt protein stability and/or reduce FMN binding.…”

“…Several studies recently also provided a molecular characterization of the spectral effects of mutations on iLOV to gain a mechanistic understanding 27–29 . Röllen et al 29 have shown that iLOV V392T/Q489K fluorescence has a slight red-shift with an emission spectrum maximum of 502 nm.…”

“…Several studies recently also provided a molecular characterization of the spectral effects of mutations on iLOV to gain a mechanistic understanding 27–29 . Röllen et al 29 have shown that iLOV V392T/Q489K fluorescence has a slight red-shift with an emission spectrum maximum of 502 nm. Its homolog mutant CagFbFP I52T/Q148K (emission maximum 504 nm) and another blue-shifted variant CagFbFP Q148K (emission maximum 491 nm) were used in fluorescence microscopy experiments and were successfully spectrally separated.…”