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

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• 作者：Mehdi D. Davari ; Benita Kopka ; Marcus Wingen ; Marco Bocola ; Thomas Drepper ; Karl-Erich Jaeger ; Ulrich Schwaneberg ; Ulrich Krauss
• 刊名：Journal of Physical Chemistry B
• 出版年：2016
• 出版时间：April 7, 2016
• 年：2016
• 卷：120
• 期：13
• 页码：3344-3352
• 全文大小：428K
• ISSN：1520-5207

文摘

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 K489_in forming a hydrogen bond with the O4 atom of FMN chromophore and (ii) a most populated conformer K489_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.