Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study

Abstract

Aureochromes (AUREO) act as blue-light photoreceptors in algae. They consist of a light-, oxygen-, voltage-sensitive (LOV) domain and a DNA-binding basic region/leucine zipper. Illumination of the flavin cofactor in LOV leads to the formation of an adduct, followed by global structural changes. Here, we first applied UV/vis spectroscopy to characterize the photocycle of full-length aureochrome 1c ( Pt AUREO1c) from the diatom Phaeodactylum tricornutum . With a time constant of 850 s and a quantum yield of 23%, Pt AUREO1c reveals a faster recovery time and a much lower sensitivity toward light than Pt AUREO1a, pointing to its role as a high light sensor in vivo . UV/vis spectroscopy offers details on the local recovery of the flavin chromophore. However, kinetic information on the global structural recovery of full-length AUREO or any other multidomain LOV protein is missing. This information is essential not least for the photoreceptors' applications as optogenetic devices. Therefore, we established a procedure to apply small-angle X-ray scattering on Pt AUREO1c in a time-resolved manner employing an in-house setup. In combination with UV/vis spectroscopy under similar conditions, we revealed a discrepancy between the recovery of the global protein structure and the adduct lifetime. Accordingly, we propose to supplement the photocycle by an intermediate state (I447), which decays with a time constant of about 800 s and prolongs the lifetime of the signaling state.

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

“…Nevertheless, asynchronous dark relaxation of chromophore, backbone and native side-chains was previously reported for Slr1694, a related photoreceptor belonging to the BLUF (bluelight using flavin adenine dinucleotide) family, based on a hybrid UV/Vis/IR approach (63). Also in agreement with our observations, a delayed recovery of the protein structure with respect to the flavin adduct decay has been recently described for a dimeric two-domain LOV photoreceptor based on smallangle X-ray scattering and UV/Vis (64).…”

“…The escape from heterogeneous traps rather than the nucleation rate has been proposed to account for the residue-specific folding rates of α-helices ( 66 ). In other LOV sensors, dynamic interactions at protein/protein interfaces are considered additional rate-limiting factors for the conformational recovery ( 64 ). Our results highlight the A’α element, where both CNF31 and CNF35 reside, as a central hub in controlling the dark recovery rate of EL222.…”

Steric and Electronic Interactions at Gln154 in ZEITLUPE Induce Reorganization of the LOV Domain Dimer Interface

“…Light, oxygen, or voltage (LOV) domain-containing proteins are abundant in nearly all kingdoms of life, where they function as sensory proteins to regulate a diverse array of adaptive responses. , Structurally, LOV proteins consist of a central five-stranded β-sheet flanked on one side by four helical elements that cradle a photoactive flavin-based cofactor (FAD, FMN, and riboflavin) , (Figure ). Upon photoexcitation, a covalent bond is formed between a conserved cysteine residue and the C4a position of the flavin cofactor (C4a adduct). − The C4a adduct is reversible in the presence of UVA/violet light or will spontaneously decay in the dark with variable kinetics ranging from seconds to days. ,, The result is a tunable photochemical sensor that can modulate activity over a wide range of environmental light intensities. − …”

Sensing and Signalling in Diatom Responses to Abiotic Cues

“…Evidence from silenced and knockout lines of P. tricornutum further indicates that PtAureo1a and PtAureo1b might be involved in regulation of photoacclimation (Table 21.1;Schellenberger Costa et al 2013b;Mann et al 2017;Madhuri et al 2019). PtAureo1c might be a high light sensor in vivo because it recovers faster and is much less sensitive to light than PtAUREO1a (Bannister et al 2019). Recent studies indicate that aureochromes may have a large impact on the cells.…”

“…However, information about the spectral and functional properties of these and other diatom rhodopsins is still lacking, so their function as light sensors remains to be established. chromophore, absorption spectrum, and regulated processes are indicated for: cryptochrome photolyase 1 (PtCPF1) (Coesel et al 2009); plant-like Cry (PtCryP) (Juhas et al 2014); aureochromes PtAureo1a (Heintz and Schlichting 2016) and PtAureo1c (Bannister et al 2019) and diatom phytochromes (PtDPH and TpDPH) (Fortunato et al 2016). Domains involved in the light sensing are indicated in blue or red.…”