Blue Light Sensor Research Articles

3TA2-08 PELDOR測定による青色光センサータンパク質SyPixD重合体のタンパク間相互作用の解析(光生物-視覚・光受容,第47回日本生物物理学会年会)

3TA2-08 PELDOR測定による青色光センサータンパク質SyPixD重合体のタンパク間相互作用の解析(光生物-視覚・光受容,第47回日本生物物理学会年会)

1P-060 青色光センサータンパク質フォトトロピンの光反応に対するCrowding効果(蛋白質-機能(反応機構,生物活性など),第47回日本生物物理学会年会)

1P-060 青色光センサータンパク質フォトトロピンの光反応に対するCrowding効果(蛋白質-機能(反応機構,生物活性など),第47回日本生物物理学会年会)

In Vivo Generation of Flavoproteins with Modified Cofactors

To understand flavoprotein mechanisms and reactivity, biochemical and biophysical methods are usually employed, and differences between wild-type and mutated proteins with altered primary structures are placed under specific consideration. Alternatively, the cofactor can be modified, and modified flavoproteins can be studied accordingly. Here we present an efficient and general method for modifying the cofactor of flavoproteins in vivo. The modified cofactor is incorporated into apoprotein during protein biosynthesis in a riboflavin-auxotrophic Escherichia coli strain, which expresses a bacterial riboflavin transporter to import flavins from the medium. This system was used to introduce roseoflavin into the riboflavin-binding protein dodecin and into microbial blue-light photoreceptors of the BLUF (blue-light sensors using FAD) and LOV (light oxygen voltage) families. The modified photoreceptors showed absorption and fluorescence different from those of proteins carrying their natural cofactor or chromophores in solution, but did not show any photochemical reaction as implied by former physiological studies.

Formation of Interacting Spins on Flavosemiquinone and Tyrosine Radical in Photoreaction of a Blue Light Sensor BLUF Protein TePixD

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.

Bacterial bilin- and flavin-binding photoreceptors

The growing number of sequenced prokaryotic genomes reveals a wide distribution of open reading frames (ORFs) that putatively encode for red- and blue light sensing photoreceptors. They comprise the bilin-binding phytochromes and the flavin-binding cryptochromes, LOV and BLUF proteins, indicating that about 1/4 of bacteria do possess at least one of these photosensory proteins. The distribution of red- and blue-light sensors among different prokaryotic phyla and classes, and their functional activity as light-switched systems are the subject of this perspective. These photoreceptors were originally found in plants by following the associated physiological responses induced by the respective spectral irradiation. Genome-based approaches now require the assignment of a photochemical/physiological function to the heterologously expressed gene product. Database searches demonstrate in some cases several genes of one category in a certain prokaryot, indicating the presence of more than one type of red- or blue-light sensing properties, but also show a combination of proteins with both spectral sensitivities. Another interesting feature now "comes into light": according to their nature as biological sensors, these photoreceptors are equipped with signalling domains, initiating a cellular response, thereby constituting modular systems switchable by light. It is seen that many of these signalling domains, now found together with light-inducible sensing domains, were already described for other stimuli, e.g., osmo-regulation, oxygen, hydrogen, chemicals, or pH. In some cases, the same type of signalling domain can be found in a red- or a blue-light sensing photoreceptor. Following the characterization of their photochemistry, for several of these bacterial photoreceptors physiological functions are now assigned.

LOV-Like Flavin-Cys Adduct Formation by Introducing a Cys Residue in the BLUF Domain of TePixD

BLUF and LOV are blue-light sensor domains that possess flavin as a common chromophore but exhibit distinct photoreactions. Ile66 located in the BLUF domain of a cyanobacterial photosensor protein, TePixD, was replaced with Cys to mimic the LOV domain. Light-induced Fourier transform infrared spectra of the I66C TePixD showed that a flavin-Cys adduct, typical of the photoinduced intermediates of LOV domains, was formed in the I66C BLUF domain. This result demonstrates that different types of flavin photoreactions can be realized in the same domain if key amino acids are properly arranged near the flavin and the domain structure itself is not a crucial factor to determine the photoreaction type.

Structural Requirements for Key Residues and Auxiliary Portions of a BLUF Domain

BlrB in Rhodobacter sphaeroides is a single domain, flavin-based blue light sensor protein in the BLUF family of photoreceptors. Consistent with other members of this family, blue light excitation induces a putative signaling state characterized by a 10 nm red shift in the UV-visible absorbance spectrum. Structural and spectroscopic characterization of truncated BlrB constructs establishes that the C-terminal 50 amino acids of this protein are essential to its structural integrity despite not being part of the canonical BLUF domain architecture. Mutagenesis studies support the critical roles of Tyr9, Asn33, and Gln51 for flavin binding and the integrity of the BLUF domain fold. Comparison of solution NMR spectra of BlrB acquired under dark and light conditions indicates very limited light-dependent conformational changes except for a few interesting residues: Trp92, Met94, and Ile127. Notably, the Ile127 side chain experiences significant chemical shift changes despite the fact that it is far ( approximately 15 A) from the flavin chromophore in the C-terminal extension. These data suggest that the light-induced signal is propagated from the flavin through the beta sheet to the last two alpha helices in the C-terminal extension, potentially providing a mechanism to transmit this change to initiate a cellular response to blue light.

2P-258 青色光受容BLUFドメインへのシステイン導入によるLOV様Cys-フラビン結合の形成(光生物・視覚,光受容(2),第46回日本生物物理学会年会)

2P-258 青色光受容BLUFドメインへのシステイン導入によるLOV様Cys-フラビン結合の形成(光生物・視覚,光受容(2),第46回日本生物物理学会年会)

Role of Phe1010 in Light-Induced Structural Changes of the neo1-LOV2 Domain of Adiantum

Phototropin (phot) is a blue-light sensor protein that elicits several photo responses in plants. Phototropin has two flavin mononucleotide (FMN)-binding domains, LOV1 and LOV2, in its N-terminal half. The C-terminal half is a blue-light-regulated Ser/Thr kinase. Various functional studies have reported that only LOV2 is responsible for the kinase activity, whereas the X-ray crystallographic structures of the LOV1 and LOV2 domains are almost identical. How does such a functional difference emerge? Our previous FTIR study of the LOV domains of Adiantum neochrome1 (neo1) showed that light-induced protein structural changes are small and temperature independent for neo1-LOV1, whereas the structural changes are large and highly temperature dependent for neo1-LOV2, which involve loops, alpha-helices, and beta-sheets. These observations successfully explained the different functions in terms of protein structural changes. They also suggested the presence of some crucial amino acids responsible for greater protein structural changes in the LOV2 domain. Here, we focused on phenylalanine-1010 (Phe1010) in neo1-LOV2, where FMN is sandwiched between Phe1010 and the reactive cysteine. Phenylalanine at this position is conserved for LOV2 domains, while the corresponding amino acid is leucine for LOV1 domains in almost all plant phototropins. We observed that unlike wild-type LOV2, the FTIR spectra of F1010L LOV2 exhibited no temperature dependence in the alpha-helical and beta-sheet regions and that spectral changes in amide-I of these regions were significantly reduced, which was similar to LOV1. Thus, the replacement of phenylalanine with leucine converts neo1-LOV2 into neo1-LOV1 in terms of protein structural changes that must be related to the different functions. We will discuss the roles of phenylalanine and leucine in the LOV2 and LOV1 domains, respectively.

Photoreactions of Tyr8- and Gln50-Mutated BLUF Domains of the PixD Protein of Thermosynechococcus elongatus BP-1: Photoconversion at Low Temperature without Tyr8

We studied the photoreaction of a blue-light sensor PixD protein of Thermosynechococcus elongatus that has the blue-light-using flavin (BLUF) domain. The Tyr8 and Gln50 residues of the protein were modified to phenylalanine, alanine, or asparagine (Y8F, Y8A, Q50N, and Q50A) by site-directed mutagenesis. The following results were obtained. (1) At room temperature, blue-light illumination induced the red shift of the absorption bands of flavin in the wild-type (WT) protein but not in the Y8F, Y8A, Q50A, and Q50N mutant proteins, as reported [Okajima, K., et al. (2006) J. Mol. Biol. 363, 10-18]. (2) At 80 K, neither the Q50N nor the Q50A mutant protein accumulated the red-shifted form. (3) At 80 K, the Y8F protein photoaccumulated the red-shifted forms to an extent that was half that in the WT protein at a 43-fold slower rate, and the Y8A protein to the one-fourth the extent at a 137-fold slower rate. (4) The red-shifted form in the Y8F protein was stable below 240 K and became unstable above 240 K in the dark. (5) The illumination of the Y8F protein at 150 K accumulated the red-shifted form at the beginning, and the prolonged illumination accumulated the flavin anions by the secondary photoreaction. (6) The results indicate that Tyr8 is not indispensable for the accumulation of the red-shifted form at least at 80 K. (7) Photoconversion mechanisms in the WT and Tyr8-mutated proteins are discussed in relation to the schemes with and without the electron transfer between Tyr8 and flavin in the first step of the photoconversion.

Blue light diminishes interaction of PAS/LOV proteins, putative blue light receptors in Arabidopsis thaliana, with their interacting partners

The light, oxygen, or voltage (LOV) domain that belongs to the Per-ARNT-Sim (PAS) domain superfamily is a blue light sensory module. The Arabidopsis thaliana PAS/LOV PROTEIN (PLP) gene encodes three putative blue light receptor proteins, PLPA, PLPB, and PLPC, because of its mRNA splicing variation. PLPA and PLPB each contain one PAS domain at the N-terminal region and one LOV domain at the C-terminal region, while the LOV domain is truncated in PLPC. RNA gel blot analysis showed that PLP mRNA was markedly expressed after exposure to salt or dehydration stress. Yeast two-hybrid screening led to the isolation of VITAMIN C DEFECTIVE 2 (VTC2), VTC2-LIKE (VTC2L), and BEL1-LIKE HOMEODOMAIN 10 proteins (BLH10A and BLH10B) as PLP-interacting proteins. The molecular interaction of PLPA with VTC2L, BLH10A or BLH10B, and that of PLPB with VTC2L were diminished when yeasts were grown under blue light illumination. Furthermore, the possible binding of flavin chromophore to PLPA and PLPB was demonstrated. These results imply that the LOV domain of PLPA and PLPB functions as a blue light sensor, and suggest the applicability of these interactions to blue light-dependent switching in transcriptional regulation in yeast or other organisms.

Heterogeneous Environment of the S−H Group of Cys966 near the Flavin Chromophore in the LOV2 Domain of Adiantum Neochrome1

The primary photochemistry of the blue-light sensor protein, phototropin, is adduct formation between the C4a atom of the flavin mononucleotide (FMN) chromophore and a nearby, reactive cysteine (Cys966), following decay of the triplet excited state of FMN. The distance between the C4a position of FMN and the sulfur atom of Cys966 is 4.2 A in the LOV2 domain of Adiantum neochrome 1 (neo1-LOV2), a fusion protein of phototropin containing the phytochrome chromophoric domain. We previously reported the presence of an unreactive fraction in neo1-LOV2 at low temperatures, which presumably originated from the heterogeneous environment of Cys966 [Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191]. The present study showed that (i) 28% forms an adduct at 77 K (state I), (ii) 50% forms an adduct at 150 K but not at 77 K (state II), and (iii) 22% does not form an adduct at 150 K (state III). By Fourier transform infrared (FTIR) spectroscopy, we observed the S-H stretching frequencies at 2570 and 2562 cm-1 for state I and at 2563 cm-1 for state II, suggesting that the microenvironment of the S-H group of Cys966 determines the reactivity at low temperatures. Adduct formation is more efficient for state I than for states II and III. Molecular dynamics simulation strongly suggests that the observed multiple structures originate from the isomeric forms of Cys966. We thus concluded that there are multiple local structures of FMN and cysteine in neo1-LOV2, each of which is thermally converted by protein fluctuation at physiological temperatures.

Hydration and Temperature Similarly Affect Light-Induced Protein Structural Changes in the Chromophoric Domain of Phototropin

Phototropin is a blue-light sensor protein in plants, and LOV domain binds a flavin mononucleotide (FMN) as a chromophore. A photointermediate state, S390, is formed by light-induced adduct formation between FMN and an S-H group of nearby cysteine, which triggers protein structural changes for kinase activation in phototropin. We previously studied the low-temperature Fourier transform infrared (FTIR) spectra between the S390 and unphotolyzed states for a LOV2 domain of a phototropin from Adiantum (neo1-LOV2), and found that the protein structures of the S390 intermediate are highly temperature dependent (Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191). At physiological temperature, amide-I vibration at 1640-1620 cm-1 is significantly changed, implying structural alteration of beta-sheet region. Such changes are largely suppressed at low temperatures, though S390 is formed. This observation suggested the presence of progressive protein structural changes in the unique active state (S390). Here we report that the hydration dependence of the amide-I vibrational bands in neo1-LOV2 is similar to the temperature dependence. As hydration of the sample is lowered, amide-I vibration at 1640-1620 cm-1 is significantly reduced. Instead, amide-I vibration at 1694 cm-1 newly emerged at low hydration as well as at low temperature, which shows a weakened hydrogen bond in the loop region. Spectral coincidence between low hydrations and temperatures strongly suggested that protein structural changes are similarly restricted under such conditions. It is likely that protein fluctuations are prerequisite for formation of the active state of neo1-LOV2.

2P360 シロイヌナズナ青色光受容体Phototropin1のLOV2-kinase領域の光誘起構造変化(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)

2P360 シロイヌナズナ青色光受容体Phototropin1のLOV2-kinase領域の光誘起構造変化(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)

3P243 青色光受容タンパク質TePixDにおける、ESRによるフラボセミキノンと近傍アミノ酸間の光誘起電子スピン間相互作用の観測(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)

3P243 青色光受容タンパク質TePixDにおける、ESRによるフラボセミキノンと近傍アミノ酸間の光誘起電子スピン間相互作用の観測(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)

Three Putative Photosensory Light, Oxygen or Voltage (LOV) Domains with Distinct Biochemical Properties from the Filamentous Cyanobacterium Anabaena sp. PCC 7120

Light, oxygen or voltage (LOV) domains function as blue-light sensors in the phototropin family of photoreceptors found in plants, algae and bacteria. We detected putative LOV domains (Alr3170-LOV, A112875-LOV and A1r1229-LOV) in the genome of a filamentous cyanobacterium, Anabaena sp. PCC 7120. These cyanobacterial LOV domains are closely clustered with the known LOV domains. Alr3170-LOV and A112875-LOV carry the conserved cysteine residue unique to the photoactive LOV, whereas A1r1229-LOV does not. We expressed these three LOV domains in Escherichia coli and purified them. In fact, Alr3170-LOV and A112875-LOV that are conserved in Nostoc punctiforme, a related species, bound flavin mononuclcotide and showed spectral changes unique to known LOV domains on illumination with blue light. A1r3170-LOV was completely photoreduced and dark reversion was slow, whereas A112875-LOV was slowly photoreduced and dark reversion was rapid. For comparison, AvA112875-LOV in a closely related A. variabilis was also studied as a homolog of A112875-LOV. Finally, we observed that A1r1229-LOV that is not conserved in N. punctiforme showed no flavin binding.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.

Genome-Wide Analysis of Light Sensing inProchlorococcus

Prochlorococcus MED4 has, with a total of only 1,716 annotated protein-coding genes, the most compact genome of a free-living photoautotroph. Although light quality and quantity play an important role in regulating the growth rate of this organism in its natural habitat, the majority of known light-sensing proteins are absent from its genome. To explore the potential for light sensing in this phototroph, we measured its global gene expression pattern in response to different light qualities and quantities by using high-density Affymetrix microarrays. Though seven different conditions were tested, only blue light elicited a strong response. In addition, hierarchical clustering revealed that the responses to high white light and blue light were very similar and different from that of the lower-intensity white light, suggesting that the actual sensing of high light is mediated via a blue-light receptor. Bacterial cryptochromes seem to be good candidates for the blue-light sensors. The existence of a signaling pathway for the redox state of the photosynthetic electron transport chain was suggested by the presence of genes that responded similarly to red and blue light as well as genes that responded to the addition of DCMU [3-(3,4-dichlorophenyl)-1,1-N-N'-dimethylurea], a specific inhibitor of photosystem II-mediated electron transport.

The Blue-Light Receptor YtvA Acts in the Environmental Stress Signaling Pathway of Bacillus subtilis

The general stress response of the bacterium Bacillus subtilis is regulated by a partner-switching mechanism in which serine and threonine phosphorylation controls protein interactions in the stress-signaling pathway. The environmental branch of this pathway contains a family of five paralogous proteins that function as negative regulators. Here we present genetic evidence that a sixth paralog, YtvA, acts as a positive regulator in the same environmental signaling branch. We also present biochemical evidence that YtvA and at least three of the negative regulators can be isolated from cell extracts in a large environmental signaling complex. YtvA differs from these associated negative regulators by its flavin mononucleotide (FMN)-containing light-oxygen-voltage domain. Others have shown that this domain has the photochemistry expected for a blue-light sensor, with the covalent linkage of the FMN chromophore to cysteine 62 composing a critical part of the photocycle. Consistent with the view that light intensity modifies the output of the environmental signaling pathway, we found that cysteine 62 is required for YtvA to exert its positive regulatory role in the absence of other stress. Transcriptional analysis of the ytvA structural gene indicated that it provides the entry point for at least one additional environmental input, mediated by the Spx global regulator of disulfide stress. These results support a model in which the large signaling complex serves to integrate multiple environmental signals in order to modulate the general stress response.

Photochemistry in Phototropin, a Blue Light Sensor Protein in Plants

AbstractPhototropin (phot) is a blue‐light receptor in plants. The protein molecule has two FMN (Flavin MonoNucleotide) binding domains named LOV (Light‐, Oxygen‐, and Voltage‐sensing), which is a subset of the PAS (Per‐Arnt‐Sim) superfamily. Illumination of the phot‐LOV domains produces a covalent C(4a) flavin‐cysteinyl adduct (S390 intermediate) via a triplet‐excited state, which reverts to the original state in the dark. Chemical structure of the S‐H group of the reactive cysteine is important for the adduct formation, and several models have been proposed for efficient reaction. By means of Fourier‐transform infrared (FTIR) spectroscopy of the LOV2 domain of Adiantum phytochrome3 (phy3‐LOV2), a fusion protein of phot containing the phytochrome chromophoric domain, we have studied protonation state of the reactive cysteine (Cys966). Although it was proposed that the cysteine is deprotonated in LOV domains, we observed an S‐H stretch at ˜2570 cm−1. Since phy3‐LOV2 possesses only one cysteine at position 966, this observation clearly indicates that Cys966 is protonated. While transfer reaction of proton, hydrogen, or electron has been proposed from the cysteine in the triplet‐excited state, we also detected the S‐H stretch at 2537 cm−1 in the triplet‐excited state of phy3‐LOV2. This excludes the possibility of proton and hydrogen transfer reactions. These frequencies imply that the S‐H group is free from hydrogen bond in the unphotolyzed state, while it forms a strong hydrogen bond in the triplet‐excited state. We infer the hydrogen‐bonding acceptor to be the N(5) atom of FMN, and such strong interaction presumably drives adduct formation in a microsecond timescale.