Experimental characterization of in silico red-shift predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S mutants

Abstract

iLOV is a flavin mononucleotide-binding fluorescent protein used for in vivo cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to have reduced phototoxicity and better tissue penetration. In this 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 mutants bearing the V392K mutation lost the ability to bind FMN. While iLOVL470T/Q489K is about 20% brighter compared to WT iLOV in vitro, it exhibits a blue shift in contrast to QM/MM predictions. Additionally, both mutants are expressed at low levels and have undetectable fluorescence in living cells, which prevents their utilization in imaging applications. Our results suggest that rational fluorescent protein design efforts can benefit from in silico protein stability and ligand affinity calculations.