Photophysics of the LOV-Based Fluorescent Protein Variant iLOV-Q489K Determined by Simulation and Experiment

Light, oxygen, voltage (LOV) based fluorescent proteins (FPs) represent a promising alternative to fluorescent reporters of the green fluorescent protein family. For certain applications like multicolor imaging or the design of FRET-based biosensors, the generation of spectrally shifted LOV-based FPs would be required. In a recent theoretical study (Khrenova et al. J. Phys. Chem. B 2015, 119 (16), pp 5176-5183), the photophysical properties of a variant of the LOV-based fluorescent protein iLOV were predicted using quantum mechanics/molecular mechanics (QM/MM) approaches. The variant contained a lysine residue at the position of a highly conserved glutamine residue (Q489K), which directly interacts with the 04 and N5 atom of the flavin mononucleotide (FMN) chromophore. On the basis of QM/MM calculations, iLOV-Q489K was suggested to possess substantially red-shifted absorption and fluorescence-emission maxima with respect to parental iLOV. Here, we describe the experimental characterization of this variant, which, surprisingly contrary to the theoretical prediction, shows blue-shifted absorption and fluorescence-emission maxima. Using molecular dynamics (MD) simulations and QM/MM calculations, the molecular basis for the contradictory theoretical and experimental results is presented. Essentially, our computational analysis suggests that, in the Q489K variant, two possible side chain conformers exist: (i) a least populated conformer K489(in), forming a hydrogen bond with the O4 atom of FMN chromophore and (ii) a most populated conformer K489h(out) with the side-chain amino group flipped away from the FMN chromophore forming a new hydrogen bond with the backbone oxygen of G487. QM/MM calculated spectra of the K489(out) conformer are blue-shifted compared to the calculated spectra of parental iLOV, which is in accordance with experimental data. This suggests that the change in the conformation of K489 from K498(in), to K489(out) accounts for the change in the direction of the spectral shift from red to blue, thus reconciling theory and experiment.