Experimental Characterization of In Silico Red-Shift-Predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S Mutants

Pierre Wehler; Daniel Armbruster; Andreas Günter; Erik Schleicher; Barbara Di Ventura; Mehmet Ali Öztürk

doi:10.1021/acsomega.2c01283

Experimental Characterization of In Silico Red-Shift-Predicted iLOV^L470T/Q489K and iLOV^{V392K/F410V/A426S} Mutants

ACS Omega. 2022 Jun 1;7(23):19555-19560. doi: 10.1021/acsomega.2c01283. eCollection 2022 Jun 14.

Authors

Pierre Wehler^{1

2}, Daniel Armbruster^{1

2}, Andreas Günter³, Erik Schleicher³, Barbara Di Ventura^{1

2}, Mehmet Ali Öztürk^{1

2}

Affiliations

¹ Institute of Biology II, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
² Centers for Biological Signalling Studies BIOSS and CIBSS, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
³ Institute of Physical Chemistry, University of Freiburg, Albertstr. 21, 79104 Freiburg, Germany.

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 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 10018-10025) 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.