Experimental Characterization of Red-Shift-Predicted iLOV and iLOV Mutants.

iLOV is a flavin mononucleotide-binding fluorescent protein used for cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to reduce phototoxicity and allow for better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOV and iLOV, which were previously computationally proposed by (KhrenovaJ. Phys. Chem. B2017, 121 ( (43), ), pp 10018-10025) to have red-shifted excitation and emission spectra. While iLOV is about 20% brighter compared to the WT , it exhibits a blue shift in contrast to quantum mechanics/molecular mechanics (QM/MM) predictions. Additional optical characterization of an iLOV mutant revealed that V392 is essential for cofactor binding and, accordingly, variants with V392K mutation are unable to bind to FMN. iLOV and iLOV are expressed at low levels and have no detectable fluorescence in living cells, preventing their utilization in imaging applications.