Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

• MeSH
• cystein chemie   MeSH
• flavinmononukleotid chemie   MeSH
• fotochemické procesy MeSH
• fototropiny chemie   MeSH
• glutamin chemie   MeSH
• isomerie MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• normální rozdělení MeSH
• oves chemie   MeSH
• sekvence aminokyselin MeSH
• spektrofotometrie infračervená MeSH
• Sphingomonadaceae chemie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• flavinmononukleotid MeSH
• fototropiny MeSH
• glutamin 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.

• Klíčová slova
• 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
• Ramanova spektroskopie * metody   MeSH
• spektrofotometrie infračervená MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH