Heterologous expression and spectroscopic characterization of halo-adapted LOV photoreceptors

CORONEL YONATHAN JAVIER; DIAZ, JOAQUÍN AUGUSTO; ZANNIER, FEDERICO; ESSEN, LARS OLIVER; FARIAS, MARIA EUGENIA; ABATEDAGA, INES; VALLE, LORENA

Córdoba

Congreso; SAB 2023-LI Reunión Anual de la Sociedad Argentina de Biofísica; 2023

Sociedad Argentina de Biofísica

The aim of this work was to study the photophysics of Light-Oxygen-Voltage (LOV) photoreceptors containing flavin mononucleotide (FMN) as a cofactor. The selected proteins belong to a microorganism of the Archaea domain, specifically Halorubrum sp DM2, in collaboration with Dr. Albarracín's group. The DM2LOV1 and DM2LOV2 domains were studied in the Laboratory of Molecular Photobiology (INBIONATEC). The recombinant proteins were produced in a heterologous expression system and purified using affinity chromatography. The DM2LOV2 domain was expressed by binding the FMN cofactor, while DM2LOV1 was expressed as an apoprotein. In vitro, spectroscopic characterization of the DM2LOV2 domain was performed using PBS buffer with 0.5 M NaCl. Subsequently, the effect of adding KCl at different concentrations (0.5 M, 1.0 M, and 1.5 M) was studied.Preliminary results using absorption spectroscopy and stationary fluorescence suggest that DM2LOV2 behaves differentially in the presence or absence of KCl. The protein responds to blue light forming the characteristic adduct, but the irradiation power needed is 10 times higher than that used for similar domains studied in our group. The emission spectra show a split in the emission band observed with NaCl. The kinetic study shows differences in the formation times of the light-adapted state, DM2LOV2390, and in the thermal recovery times of the dark-adapted state, DM2LOV2449. Stationary anisotropy reveals a greater restriction in cofactor mobility with KCl aggregate compared to protein only in NaCl.