Non-irradiated LOV proteins absorb maximally at ~450 nm while their irradiated counterparts peak at ~390 nm (Figure 1b left panel). UV/vis spectroscopy has been instrumental in monitoring the photoconversion of FMN (Figures 1 and S1). Although high-resolution structural information of the lit state(s) of EL222 is missing, it is known that blue-light excitation of the flavin mononucleotide (FMN) chromophore embedded in the LOV domain leads to uncaging of the associated HTH module, oligomerization and subsequent DNA binding and gene expression (Nash et al. In the photocontrolled transcription factor EL222 from Erythrobacter litoralis, the well-studied dark state is characterized by a monomeric “closed” conformation featuring extensive interactions between the light-oxygen-voltage (LOV) and helix-turn-helix (HTH) domains (Nash et al. The conformation and dynamics of photoreceptor proteins are orchestrated by light, which regulates changes at the secondary, tertiary and sometimes quaternary levels of structure (Kottke et al. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222.
The mechanism and kinetics of dark-state recovery are not well understood. Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light.
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