The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.
Background: Aureochromes in stramenopiles are thought to function as light-regulated transcription factors, although the molecular mechanism is unknown. Results: Monomeric AUREO1 is present in reduced conditions and undergoes dimerization upon illumination. Conclusion: Blue light-induced dimerization enhances the affinity for the target sequence. Significance: AUREO1 is useful for understanding the blue light responses of stramenopiles, and for optogenetics and biophysical analyses.
Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at ؊40°C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995) Biochemistry 34, 12669 -12672) were observed at 1482, 1437, and 1163 cm ؊1 for PYP. However, the bands corresponding to these modes were not observed for PYP M , the blue-shifted intermediate, but the 1175 cm ؊1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYP M . A 1736 cm ؊1 band was observed for PYP, but the corresponding band for PYP M was not. Because it disappeared in the Glu-46 3 Gln mutant of PYP, this band was assigned to the C؍O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYP M .Photoactive yellow protein (PYP) 1 ( max ϭ 446 nm) (1) is proposed to be a photoreceptor protein for the negative phototaxis observed in the purple phototrophic bacterium, Ectothiorhodospira halophila (2). PYP belongs to the novel group of photoreceptor proteins (3, 4) whose structures are quite different from those of the other photoreceptor proteins studied so far. Namely, the protein moiety of PYP has an ␣/ fold structure (5) composed of 125 amino acids (6, 7). The chromophore is a p-coumaric acid (7-9) bound to a cysteine residue via a thioester bond.PYP absorbs a photon and enters the photocycle. We have analyzed the photocycle of PYP in detail by low temperature spectroscopy and identified several intermediates (10). Irradiation of PYP at Ϫ190°C yields PYP B ( max ϭ 489 nm) and PYP H ( max ϭ 442 nm), which are thermally converted to PYP L ( max ϭ 456 nm) through PYP BL ( max ϭ 400 nm) and PYP HL ( max ϭ 447 nm), respectively. The two pathways beginning with PYP B and PYP H join at PYP L and revert to PYP. Flash photolysis at ambient temperature identified two intermediates, pR ( max ϭ 465 nm) and pB ( max ϭ 355 nm) (11,12). pR is formed within 10 ns after flash excitation. It decays to pB over a submillisecond time scale and reverts to PYP within 1 s. It has been demonstrated that pR is the same species as PYP L (10) (In this paper, pR and pB are called PYP L and PYP M , respectively, to avoid confusion) and that PYP L is accumulated by irradiation of PYP at Ϫ80°C (10, 13). However, the precursors of PYP L have not been discovered by flash photolysis at room temperature.Recent studies have clarified some details of the events that take place during t...
Aureochrome-1 (AUREO1) is a blue light (BL) receptor responsible for the BL-induced blanching of a stramenopile alga, Vaucheria frigida. The AUREO1 protein contains a central basic region/leucine zipper (bZIP) domain, and a C-terminal light-oxygen-voltage-sensing (LOV) domain. BL induces the dimerization of monomeric AUREO1, which subsequently increases the affinity of this transcription factor for its target DNA [Hisatomi, O., et al. (2014) J. Biol. Chem. 289, 17379-17391]. We constructed a synthetic gene encoding N-terminally truncated monomeric AUREO1 (designated Photozipper) to elucidate the molecular mechanism of this BL-regulated transcription factor and to develop it as an optogenetic tool. In this study, four different Photozipper (PZ) protein constructs were prepared comprising different N-terminal truncations. The monomer-dimer equilibria of the PZ constructs were investigated in the dark and light states. Dynamic light scattering and size-exclusion chromatography analyses revealed that the apparent dissociation constants of PZ dimers with and without the ZIP region were ~100 and 30 μM, respectively, indicating that the ZIP region stabilized the monomeric form in the dark state. In the light state, fluorescence resonance energy transfer analyses demonstrated that deletion of the ZIP region increased the dissociation constant from ~0.15 to 0.6 μM, suggesting that intermolecular LOV-LOV and ZIP-ZIP interactions stabilized the dimeric forms. Our results suggest that synergistic interactions between the LOV and bZIP domains stabilize the monomeric form in the dark state and the dimeric form in the light state, which possibly contributes to the function of PZ as a BL-regulated molecular switch.
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