Excitation and emission spectra of in silico red-shift predicted iLOV mutants

Abstract

AbstractiLOV is a flavin mononucleotide (FMN)-binding fluorescent protein (FbFP) used for in vivo cellular imaging similar to the green fluorescent protein (GFP). Contrary to GFP, iLOV fluoresces independently of molecular oxygen, making its usage in low-oxygen conditions possible. Moreover, iLOV is smaller than GFP, increasing the likelihood of retaining full functionality when creating fusions with proteins of interest. Nonetheless, GFP remains to date the most widely used FP in molecular biology, also due to the availability of multiple spectrally tuned variants allowing multi-color imaging experiments. To expand the range of applications of iLOV, spectrally tuned red-shift variants are desirable to have reduced phototoxicity and better tissue penetration. In this short report, we experimentally tested two iLOV mutants, iLOVL470T/Q489K and iLOVV392K/F410V/A426S, which were previously computationally proposed by Khrenova et al. (DOI: 10.1021/acs.jpcb.7b07533) to have red-shifted excitation and emission spectra. We found that only the triple mutant has moderately red-shifted excitation and emission spectra. Both mutants exhibit strongly decreased in vivo fluorescence intensity, which impedes their employment in live cell imaging. Finally, we show that the single iLOVV392K mutant suffices to red-shift the emission spectrum.