Flavocytochrome c sulfide dehydrogenase (FCC) is an important enzyme of sulfur metabolism in sulfur-oxidizing bacteria, and its catalytic properties have been extensively studied. However, the ultrafast dynamics of FCC is not well understood. We present ultrafast transient absorption and fluorescence spectroscopy measurements to unravel the early events upon excitation of the heme and flavin chromophores embedded in the flavocytochrome c (FccAB) from the bacterium Thiocapsa roseopersicina. The fluorescence kinetics of FccAB suggests that the majority of the photoexcited species decay nonradiatively within the first few picoseconds. Transient absorption spectroscopy supports these findings by suggesting two major dynamic processes in FccAB, internal conversion occurring in about 400 fs and the vibrational cooling occurring in about 4 ps, mostly affecting the heme moiety.

• Publication type
• Journal Article MeSH

The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

• MeSH
• Bacterial Proteins * chemistry   metabolism   genetics   MeSH
• DNA-Binding Proteins * chemistry   metabolism   MeSH
• DNA * metabolism   chemistry   MeSH
• Flavin Mononucleotide * chemistry   metabolism   MeSH
• Flavins * chemistry   metabolism   MeSH
• Photoreceptors, Microbial * chemistry   metabolism   MeSH
• Kinetics MeSH
• Protein Conformation radiation effects   MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Oxidation-Reduction MeSH
• Molecular Dynamics Simulation MeSH
• Light * MeSH
• Transcription Factors * chemistry   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• DNA-Binding Proteins * MeSH
• DNA * MeSH
• Flavin Mononucleotide * MeSH
• Flavins * MeSH
• Photoreceptors, Microbial * MeSH
• Transcription Factors * MeSH

Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.

• Keywords
• cytokines, genetically encoded non-canonical amino acids (ncAA), interleukin-24, optobinders, ortho-nitrobenzyltyrosine (NBY), photocaged proteins, photoxenoprotein engineering, protein-protein interactions (PPI),
• Publication type
• Journal Article MeSH

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.

• Keywords
• femtosecond-stimulated Raman spectroscopy (FSRS), flavins, kinetic isotope effect (KIE), lifetime distribution analysis (LDA), light-oxygen-voltage (LOV) photosensors, maximum entropy method, photobiology, photochemistry, protein structural dynamics, time-resolved vibrational spectroscopy, transient visible absorption (visTA) spectroscopy,
• MeSH
• Spectrum Analysis, Raman * methods   MeSH
• Spectrophotometry, Infrared MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH