Abstract
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.
Highlights
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 rotation of the glutamine was found to be sensitive to the LOV domain topology and, in particular, to the stabilisation provided by residues of the adjacent b-sheet
Infrared spectra of four possible isomers were collected along a tentative reaction coordinate up to the adduct, and key marker bands were followed, such as the carbonyl stretching vibrations of flavin mononucleotide (FMN), the side chain glutamine, and the asparagine residues
Summary
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. EL222 of Erythrobacter litoralis has its effector helix-turn-helix (HTH) domain bound to the photoactive LOV domain in the dark.[21] Illumination exposes the HTH domain and allows the protein to dimerise, bind to DNA and activate gene transcription.[22] RsLOV the LOV protein of Rhodobacter sphaeroides, has still an unclear physiological function, but it is known to self-associate as dimer in the dark.[23] RsLOV forms the FMN–cysteine adduct in adequate yields, and dissociates to a monomeric state under illumination, as evidenced by fluorescence experiments.[24] AsLOV2 is the most photosensitive of the two LOV domains present in phototropin 1 of Avena sativa, and is reported to assist the autophosphorylation of the kinase domain, promoting plant growth.[25] It is postulated that a vital role in the AsLOV2 ability to propagate signals is played by the unfolding of its Ja helix.[26] The metastable cysteine–FMN covalent bond is cleaved in the dark on timescales ranging from seconds to hours depending on the particular combination of LOV domain and the associated effector modules.[27]
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