Experimental Characterization of In Silico Red-Shift-Predicted iLOV(L470T/Q489K) and iLOV(V392K/F410V/A426S) Mutants

[Image: see text] 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 reduce phototoxicity and allow for better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOV(L470T/Q489K) and iLOV(V392K/F410V/A426S), which were previously computationally proposed by ( KhrenovaJ. Phys. Chem. B2017, 121 ( (43), ), pp 10018−1002528992704) to have red-shifted excitation and emission spectra. While iLOV(L470T/Q489K) is about 20% brighter compared to the WT in vitro, it exhibits a blue shift in contrast to quantum mechanics/molecular mechanics (QM/MM) predictions. Additional optical characterization of an iLOV(V392K) mutant revealed that V392 is essential for cofactor binding and, accordingly, variants with V392K mutation are unable to bind to FMN. iLOV(L470T/Q489K) and iLOV(V392K/F410V/A426S) are expressed at low levels and have no detectable fluorescence in living cells, preventing their utilization in imaging applications.