title: Photophysics of the LOV-Based Fluorescent Protein Variant iLOV-Q489K Determined by Simulation and Experiment.
abstract: 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 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 O4 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 K489in forming a hydrogen bond with the O4 atom of FMN chromophore and (ii) a most populated conformer K489out 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 K489out 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 K498in to K489out accounts for the change in the direction of the spectral shift from red to blue, thus reconciling theory and experiment.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University , 52056 Aachen, Germany.
authors.forename: Mehdi D
authors.initials: MD
authors.lastname: Davari
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich , 52426 Jülich, Germany.
authors.forename: Benita
authors.initials: B
authors.lastname: Kopka
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich , 52426 Jülich, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Wingen
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University , 52056 Aachen, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich , 52426 Jülich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Drepper
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich , 52426 Jülich, Germany.|Institut für Bio- und Geowissenschaften, IBG-1, Biotechnologie, Forschungszentrum Jülich , 52426 Jülich, Germany.
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University , 52056 Aachen, Germany.|DWI-Leibniz Institute for Interactive Materials , Forckenbeckstraße 50, 52056, Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich , 52426 Jülich, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Krauss
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D015870:Gene Expression; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D013050:Spectrometry, Fluorescence
chemical_list: D029681:Arabidopsis Proteins; D005420:Flavoproteins; D008164:Luminescent Proteins