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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteins with a BLUF (sensor of blue light using flavin adenine dinucleotide) domain represent a newly recognized class of photoreceptors that is widely distributed in the genomes of photosynthetic bacteria, cyanobacteria, and Euglena. Recently, Okajima et al. [Okajima, K., Yoshihara, S., Geng, X., Katayama, M. and Ikeuchi, M. (2003) Plant Cell Physiol. 44 (Suppl), 162] purified BLUF protein Tll0078 encoded in the genome of thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by expressing the protein in Escherichia coli. We investigated the photocycle of Tll0078 by measuring the picosecond fluorescence kinetics, transient absorption changes, and the UV-visible absorption spectra at 10 to 330 K. The absorption spectrum of the FAD moiety of Tll0078 showed a 10-nm red shift upon illumination at 278-330 K. The quantum efficiency of the formation of the red-shifted form was 29%. Illumination at 10 K, on the other hand, caused only a 5-nm red shift in about one-half of the protein population. The 5-nm-shifted form was stable at 10 K. The 5-nm red-shifted form was converted into the 10-nm red-shifted form at 50-240 K upon warming in the dark. At room temperature, the 10-nm red-shifted final product appeared within 10 ns after laser flash excitation. The lifetime of the fluorescence of FAD was found to be 120 ps at room temperature. These results reveal a fast and efficient photoconversion process from the singlet-excited state to the final product at room temperature. A photocycle of BLUF protein is proposed that includes the 5-nm red-shifted intermediate form as the precursor for the 10-nm red-shifted final product. The temperature dependence of each step of the photocycle is also discussed.
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PMID:Primary intermediate in the photocycle of a blue-light sensory BLUF FAD-protein, Tll0078, of Thermosynechococcus elongatus BP-1. 1579 52

The sensor proteins for blue light using the FAD (BLUF) domain belong to the third family of the photoreceptor proteins using a flavin chromophore, where the other two families are phototropins and cryptochromes. As the first structure of this BLUF domain, we have determined the crystal structure of the Tll0078 protein from Thermosynechococcus elongatus BP-1, which contains a BLUF domain bound to FAD, at 2A resolution. Five Tll0078 monomers are located around the non-crystallographic 5-fold axis to form a pentamer, and two pentamers related by 2-fold non-crystallographic symmetry form a decameric assembly. The monomer consists of two domains, the BLUF domain at the N-terminal region and the C-terminal domain. The overall structure of the BLUF domain consists of a five-stranded mixed beta-sheet with two alpha-helices running parallel with it. The isoalloxazine ring of FAD is accommodated in a pocket formed by several highly conserved amino acid residues in the BLUF domain. Of these, the three apparent key residues (Asn31, Asn32 and Gln50) were substituted with Ala. Mutant proteins of N31A and N32A showed a nearly normal 10nm spectral shift of the flavin upon illumination, while the Q50A mutant did not exhibit such a shift at all. On the basis of the crystal structure, we discussed a possible role of Gln50, which is structurally and functionally linked with the critical Tyr8 (FAD-Gln50-Tyr8 network), with regard to the light-induced spectral shift of the BLUF proteins.
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PMID:Structure of a cyanobacterial BLUF protein, Tll0078, containing a novel FAD-binding blue light sensor domain. 1587 64

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.
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PMID:Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria. 1600 96

PixD (Tll0078, Slr1694) is a BLUF (sensor of blue light using FAD)-type blue light receptor protein of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 and the mesophilic cyanobacterium Synechocystis sp. PCC 6803. BLUF protein is known to show light-induced approximately 10 nm red shift of flavin absorption that is coupled with strengthening of the hydrogen bond between the O(4) of the isoalloxazine ring and a certain amino acid residue. According to the 3D structure of TePixD we determined, O(4) of the ring is linked to Gln50 and Asn32. A survey of flavin-interacting residues by site-directed mutagenesis showed that Gln50 but not Asn32 is essential for the normal red-shifting photoreaction. Here, we further studied the role of Gln50 and its close neighbor Tyr8. All the mutated proteins of Gln50 and Tyr8 (Q50A, Q50N, Y8A and Y8F) lost the normal red-shifting photoreaction. Y8A, Y8F and Q50N, instead, showed a light-induced flavin triplet state and a low yield of subsequent flavin reduction that is analogous to the photocycle of the LOV (light-oxygen-voltage-sensing) domain of phototropins, while Q50A did not. Fourier-transform infrared (FT-IR) analysis of N32A showed that O(4) of the ring is hydrogen-bonded to Asn32 both in the light and dark. These results, together with the 3D structure, indicate that the hydrogen bond network of Tyr8-Gln50-O(4)/N(5) (flavin) is critical for the light reaction of the BLUF domain. Based on the structural and functional similarities of the BLUF and the LOV domain of phototropins, we propose that the interaction between apoprotein and N(5) of flavin determines the photoreaction of the flavin-binding sensors.
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PMID:Fate determination of the flavin photoreceptions in the cyanobacterial blue light receptor TePixD (Tll0078). 1695 75

Light-induced radicals were detected by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) in the BLUF-domain protein TePixD of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The illumination of TePixD at 5-200 K derived an EPR signal with a separation of 85 G between the main peaks around g = 2, showing a typical Pake's pattern of magnetic dipole-dipole interaction between two nearby radicals. Longer illumination induced an EPR signal at g = 2.0045, which was assigned as a neutral flavosemiquinone FADH(*). The FADH(*) formation occurred in parallel with a decrease in Pake's doublet. The Pake's doublet was not detected in a mutant TePixD protein in which a tyrosine residue was replaced with phenylalanine (Y8F protein). A pulsed ENDOR study suggested that the Pake's doublet had arisen from the interaction between a neutral flavosemiquinone radical and a neutral tyrosine radical, i.e., the FADH(*)-Y8(*) state. An EPR simulation of the Pake's doublet showed that the distance between FAD and Y8 is 2.2 A shorter than that calculated from the X-ray crystallography structure in the dark-adapted state, suggesting the modification of the protein conformation in the photoinduced FADH(*)-Y8(*) state. The Pake's doublet signal was detected by 10 K illumination in the sample which was immediately frozen after 273 K illumination, corresponding to the red-shifted state F(490). On the other hand, the signal was not detected in the sample which was incubated for 10 min at 273 K in the dark after 273 K illumination, corresponding to the dark-adapted state D(471). In the sample annealed at 160 K for 10 min after 160 K illumination, corresponding to the partially red-shifted state J(11), the Pake's doublet signal was detected by the 10 K illumination. On the basis of these observations, we concluded that the interaction with the FADH(*)-Y8(*) state occurred after the second photoexcitation of the photoinduced red-shifted states in the photocycle of TePixD.
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PMID:Formation of interacting spins on flavosemiquinone and tyrosine radical in photoreaction of a blue light sensor BLUF protein TePixD. 1897 4

The photochemical reaction dynamics of a BLUF (sensors of blue light using FAD) protein, PixD, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) were studied by pulsed laser-induced transient grating method. After the formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as expansion of partial molar volume with a time constant of 40 micros, whereas the other reaction phase represented a change in the diffusion coefficient D [i.e., the diffusion-sensitive conformational change (DSCC)]. D decreased from 4.9 x 10(-11) to 4.4 x 10(-11) m2 s(-1) upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 x 10(-11) m2 s(-1) upon formation of the second intermediate. From a global analysis of signals at various grating wavenumbers, the time constant of D-change was determined to be 4 ms. Although the magnitude and rate constant of the faster volume change were independent of protein concentration, the amplitude of the signal that reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution: a reactive species exhibiting the DSCC, and a second species that is nonreactive. The fraction of these species was found to be dependent on the concentration. The difference in reactivity was attributed to the different oligomeric states of TePixD (i.e., pentamer and decamer). The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in the signal transduction of this photosensory protein.
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PMID:Oligomeric-state-dependent conformational change of the BLUF protein TePixD (Tll0078). 1945 99

To reveal macromolecular crowding effects on a chemical reaction of a BLUF (sensors of blue light using FAD) protein (PixD from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 [TePixD, Tll0078]), the photoreaction was studied at various concentrations of the macromolecule Ficoll-70 by UV/Vis absorption spectroscopy and the pulsed laser-induced transient grating (TG) method. The absorption spectrum did not change with varying concentration of Ficoll-70. The crowding did not affect the quantum yield of the spectral red shift reaction, recovery rate of the product, rate constant of the volume change reaction and the magnitude of the volume change. However, the magnitude of the TG signal representing the diffusion-sensitive conformation change significantly increased on addition of Ficoll-70. This dependence was attributed to the crowding effect on the TePixD decamer-pentamer equilibrium in the solution. This result indicates that the TePixD reaction is more efficient in cellular than in in vitro conditions.
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PMID:Macromolecular crowding effects on reactions of TePixD (Tll0078). 2111 71