Fig. 2: Mutagenesis of FAST and absorption/emission spectra of brightest mutants.

a Residues, rationally selected for mutagenesis in the current study as the closest to the pyridine moiety of N871b (shown with letters and numbers). b Residues that were mutated, because their substitutions proved favorable for other fluorogens in the literature. c Electrostatic potential distribution over the contact surface of N871b according to DFT calculations. Red zones correspond to the negative potential, blue - to the positive one. d Absorption and emission spectra of various fluorogens in complexes with FAST protein and its brightest mutants. Absorption spectra are normalized to the protein/ligand complex concentration and represented in the molar absorptivity coefficient scale. Emission spectra are normalized to the FQY.