Photoreceptors containing the light‐oxygen‐voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark‐state recovery are not well understood. 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. 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. Time‐resolved multi‐probe UV/visible and IR spectroscopy experiments of the lit‐to‐dark transition revealed four dynamical events. 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. 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.

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm−1, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200–2200 cm−1) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm−1. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins.

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at forty-five positions of the bacterial transcription factor EL222. 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. Time-resolved multi-probe UV/Visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. 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. 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.SIGNIFICANCEThe kinetics of fold switching between non-illuminated and blue-light-irradiated states in the transcription factor EL222 is important for understanding the signal transduction mechanism of LOV photoreceptors. Here we combine two native probes, the FMN chromophore (absorption bands in the UV/Visible region) and the protein backbone (amide bands in the infrared region), with genetically encoded cyano (C≡ N)-containing phenylalanine residues as infrared reporters of protein microenvironments. EL222 structural dynamics is more complex than expected if using a single type of probe. Local changes around residues 31 and 35 precede FMN-protein adduct rupture, which in turn precedes the global protein conformational relaxation. Our findings point the way forward to obtaining comprehensive descriptions of kinetic transitions in LOV and other photosensors.TEASERFold switching kinetics from lit to dark state in a photoreceptor is revealed by infrared-active non-canonical amino acids.