Blue Light Using FAD Research Articles

Effects of N- and C-terminal regions on oligomeric formation of blue light sensor protein SyPixD.

A sensor of blue-light using flavin adenine dinucleotide (BLUF) is a typical blue light photoreceptor domain that is found in many photosensor proteins in bacteria and some eukaryotic algae. SyPixD in Synechocystis is one of the well-studied BLUF proteins. In the dark state, it forms a decamer and, upon photoexcitation, a dissociation reaction takes place to yield dimers. Such change in the intermolecular interactions of the protomers is important for the biological function. The effect of the N- and C-terminal sequences on the stability of SyPixD oligomeric states and photoreactions of SyPixD were studied to understand how the oligomeric form is maintained with weak interaction. It was found that a few residues that frequently persist at the N-terminus after removing a tag for purification are sensitive to the stability of the decamer structure. Even two or three residues at the N-terminus considerably reduces decamer stability, whereas four or more residues completely prevent decamer formation. Unexpectedly, truncating C-terminal sequences, which locate far from any protomer interface and of which structure is undetermined in crystal structure, also destabilizes the decamer structure. This destabilization is also apparent from the dissociation reaction dynamics detected by the transient grating and transient absorption measurements. The dissociation reaction is faster and the yield increases when the C-terminus does not contain seven amino acid residues. Photoexcitation induces a conformational change in the C-terminus of the decamer but not the dimer.

Femtosecond Time-Resolved Absorption Study of Signaling State of a BLUF Protein PixD from the Cyanobacterium Synechocystis: Hydrogen-Bond Rearrangement Completes during Forward Proton-Coupled Electron Transfer.

Femtosecond time-resolved absorption measurements were carried out for the dark and signaling states of a BLUF (Blue Light Using FAD) protein, PixD, from the cyanobacterium Synechocystis. When the dark state was excited, FAD semiquinone radical (FADH•) was produced from the S1 state, and FADH• led to the signaling state. On the other hand, photoexcitation of the signaling state generated FADH• and FAD anion radical (FAD•-), and they decayed back to the original signaling state. In both cases, FADH• was formed and decayed with a proton-coupled electron transfer (PCET) via the hydrogen-bond network that involves FAD, Gln50, and Tyr8, and hence the kinetics of FADH• directly reflects the hydrogen-bond structure in the FAD-binding sites. It was found that the formation rate of FADH• was significantly different between the dark and signaling states, whereas the decay rate was the same. This indicates that the hydrogen-bond network of FAD-Gln50-Tyr8 in the dark and signaling states is initially different but it becomes indistinguishable after FADH• is formed, implying that the FAD-Gln50-Tyr8 hydrogen-bond network is rearranged during the PCET to generate FADH•. The present results best agree with the model in which the Gln tautomerizes without rotation in the signaling-state formation.

A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light.

Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.

Photoreaction Mechanisms of Flavoprotein Photoreceptors and Their Applications.

Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.

BlsA Is a Low to Moderate Temperature Blue Light Photoreceptor in the Human Pathogen Acinetobacter baumannii.

Light is an environmental signal that produces extensive effects on the physiology of the human pathogen Acinetobacter baumannii. Many of the bacterial responses to light depend on BlsA, a bluelight using FAD (BLUF)-type photoreceptor, which also integrates temperature signals. In this work, we disclose novel mechanistic aspects of the function of BlsA. First, we show that light modulation of motility occurs only at temperatures lower than 24°C, a phenotype depending on BlsA. Second, blsA transcript levels were significantly reduced at temperatures higher than 25°C, in agreement with BlsA protein levels in the cell which were undetectable at 26°C and higher temperatures. Also, quantum yield of photo-activation of BlsA (lBlsA) between 14 and 37°C, showed that BlsA photoactivity is greatly compromised at 25°C and absent above 28°C. Fluorescence emission and anisotropy of the cofactor together with the intrinsic protein fluorescence studies suggest that the FAD binding site is more susceptible to structural changes caused by increments in temperature than other regions of the protein. Moreover, BlsA itself gains structural instability and strongly aggregates at temperatures above 30°C. Overall, BlsA is a low to moderate temperature photoreceptor, whose functioning is highly regulated in the cell, with control points at expression of the cognate gene as well as photoactivity.

Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues.

The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.

How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

Signaling-State Formation Mechanism of a BLUF Protein PapB from the Purple Bacterium Rhodopseudomonas palustris Studied by Femtosecond Time-Resolved Absorption Spectroscopy.

We studied the signaling-state formation of a BLUF (blue light using FAD) protein, PapB, from the purple bacterium Rhodopseudomonas palustris, using femtosecond time-resolved absorption spectroscopy. Upon photoexcitation of the dark state, FADH(•) (neutral flavin semiquinone FADH radical) was observed as the intermediate before the formation of the signaling state. The kinetic analysis based on singular value decomposition showed that FADH(•) mediates the signaling-state formation, showing that PapB is the second example of FADH(•)-mediated formation of the signaling state after Slr1694 (M. Gauden et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10895-10900). The mechanism of the signaling-state formation is discussed on the basis of the comparison between femtosecond time-resolved absorption spectra of the dark state and those obtained by exciting the signaling state. FADH(•) was observed also with excitation of the signaling state, and surprisingly, the kinetics of FADH(•) was indistinguishable from the case of exciting the dark state. This result suggests that the hydrogen bond environment in the signaling state is realized before the formation of FADH(•) in the photocycle of PapB.

Light Detection and Signal Transduction in the BLUF Photoreceptors.

BLUF (sensor of blue light using FAD) domain-containing proteins are one of three types of flavin-binding, blue-light-sensing proteins found in many bacteria and some algae. The other types of blue-light-sensing proteins are the cryptochromes and the light, oxygen, voltage (LOV) domain-containing proteins. BLUF proteins control a wide variety of light-dependent physiological activities including photosystem synthesis, biofilm formation and the photoavoidance response. The BLUF domain photochemical reaction is unique in that only small chromophore structural changes are involved in the light activation process, because the rigid flavin moiety is involved, rather than an isomerizable chromophore (e.g. phytochromobilin in phytochromes and retinal in rhodopsins). Recent spectroscopic, biochemical and structural studies have begun to elucidate how BLUF domains transmit the light-induced signal and identify related, subsequent changes in the domain structures. Herein, I review progress made to date concerning the physiological functions and the phototransduction mechanism of BLUF proteins.

Characterization of Elements Involved in Allosteric Light Regulation of Phosphodiesterase Activity by Comparison of Different Functional BlrP1 States

Bacteria have evolved dedicated signaling mechanisms that enable the integration of a range of environmental stimuli and the accordant modulation of metabolic pathways. One central signaling molecule in bacteria is the second messenger cyclic dimeric GMP (c-di-GMP). Complex regulatory mechanisms for modulating c-di-GMP concentrations have evolved, in line with its importance for maintaining bacterial fitness under changing environmental conditions. One interesting example in this context is the blue-light-regulated phosphodiesterase 1 (BlrP1) of Klebsiella pneumoniae. This covalently linked system of a sensor of blue light using FAD (BLUF) and an EAL phosphodiesterase domain orchestrates the light-dependent down-regulation of c-di-GMP levels. To reveal details of light-induced structural changes involved in EAL activity regulation, we extended previous crystallographic studies with hydrogen–deuterium exchange experiments and small-angle X-ray scattering analysis of different functional BlrP1 states. The combination of hydrogen–deuterium exchange and small-angle X-ray scattering allows the integration of local and global structural changes and provides an improved understanding of light signaling via an allosteric communication pathway between the BLUF and EAL domains. This model is supported by results from a mutational analysis of the EAL dimerization region and the analysis of metal-coordination effects of the EAL active site on the dark-state recovery kinetics of the BLUF domain. In combination with structural information from other EAL domains, the observed bidirectional communication points to a general mechanism of EAL activity regulation and suggests that a similar allosteric coupling is maintained in catalytically inactive EAL domains that retain a regulatory function.

A LOV-domain-mediated blue-light-activated adenylate (adenylyl) cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420

Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2kDa in size) characteristic of a BL (blue light)-regulated adenylate (adenylyl) cyclase function. The photoreceptive part showed signatures of a LOV (light, oxygen, voltage) domain. The gene product, mPAC (Microcoleus photoactivated adenylate cyclase), exhibited the LOV-specific three-peaked absorption band (λmax=450nm) and underwent conversion into the photoadduct form (λmax=390nm) upon BL-irradiation. The lifetime for thermal recovery into the parent state was determined as 16s at 20°C (25s at 11°C). The adenylate cyclase function showed a constitutive activity (in the dark) that was in-vitro-amplified by a factor of 30 under BL-irradiation. Turnover of the purified protein at saturating light and pH8 is estimated to 1 cAMP/mPAC per s at 25°C (2 cAMP/mPAC per s at 35°C). The lifetime of light-activated cAMP production after a BL flash was ~14s at 20°C. The temperature optimum was determined to 35°C and the pH optimum to 8.0. The value for half-maximal activating light intensity is 6 W/m2 (at 35°C). A comparison of mPAC and the BLUF (BL using FAD) protein bPAC (Beggiatoa PAC), as purified proteins and expressed in Xenopus laevis oocytes, yielded higher constitutive activity for mPAC in the dark, but also when illuminated with BL.

Glutamine Rotamers in BLUF Photoreceptors: A Mechanistic Reappraisal

The blue light using FAD (BLUF) photosensory protein domain is activated by a unique photoreaction that results in a hydrogen-bond rearrangement around the flavin chromophore. The chemical structure of the hydrogen bond switch is a long-standing debate: The two main hypotheses postulate rotation as opposed to tautomerization of a conserved glutamine residue. Attempts to resolve the debate were inconclusive so far, despite numerous experimental and computational studies. Here we propose physical criteria for the dark and light state structures as well as for the light-activation process to evaluate existing models of BLUF using quantum-chemical calculations. The glutamine rotamer assignment of the crystal structure with the pdb code 1YRX does not satisfy our criteria because after equilibrating the intermolecular forces the glutamine rotamer in 1YRX is incompatible with the experimental density. We identified the root of the mechanistic controversy in the incorrect glutamine rotamer assignment of 1YRX . Furthermore, we show that the glutamine side chain may rotate without light activation in the BLUF dark state. Finally, we demonstrate that the tautomerized glutamine is consistent with our criteria and observations of the BLUF light state.

Light Detection and Signal Transduction in the BLUF Photoreceptors

BLUF (sensor of blue light using FAD) domain-containing proteins are one of three types of flavin-binding, blue-light-sensing proteins found in many bacteria and some algae. The other types of blue-light-sensing proteins are the cryptochromes and the light, oxygen, voltage (LOV) domain-containing proteins. BLUF proteins control a wide variety of light-dependent physiological activities including photosystem synthesis, biofilm formation and the photoavoidance response. The BLUF domain photochemical reaction is unique in that only small chromophore structural changes are involved in the light activation process, because the rigid flavin moiety is involved, rather than an isomerizable chromophore (e.g. phytochromobilin in phytochromes and retinal in rhodopsins). Recent spectroscopic, biochemical and structural studies have begun to elucidate how BLUF domains transmit the light-induced signal and identify related, subsequent changes in the domain structures. Herein, I review progress made to date concerning the physiological functions and the phototransduction mechanism of BLUF proteins.

Mutational and Structural Studies of the PixD BLUF Output Signal That Affects Light-Regulated Interactions with PixE

PixD (Slr1694) is a BLUF (blue-light-using FAD) photoreceptor used by the cyanobacterium Synechocystis sp. PCC6803 to control phototaxis toward blue light. In this study, we probe the involvement of a conserved Tyr8-Gln50-Met93 triad in promoting an output signal upon blue light excitation of the bound flavin. Analysis of acrylamide quenching of Trp91 fluorescence shows that the side chain of this residue remains partially solvent exposed in both the lit and dark states. Mutational analysis demonstrates that substitution mutations at Tyr8 and Gln50 result in the loss of the photocycle while a mutation of Met93 does not appreciably disturb the formation of the light-excited state and only minimally accelerates its decay from 5.7 to 4.5 s. However, mutations of Tyr8, Gln50, and Met93 disrupt the ability of PixD dimers to interact with PixE to form a higher-order PixD(10)-PixE(5) complex, which is indicative of a lit conformational state. Solution nuclear magnetic resonance spectroscopy and X-ray crystallographic analyses confirm that a Tyr8 to Phe mutation is locked in a pseudo-light-excited state revealing flexible areas in PixD that likely constitute part of an output signal upon light excitation of wild-type PixD.

Strong Donation of the Hydrogen Bond of Tyrosine during Photoactivation of the BLUF Domain

In the photoreaction of BLUF (sensor of blue light using FAD) domains, conserved Tyr/Gln nearby the FAD chromophore plays a crucial role in the formation of a red-shifted intermediate. The intermediate appears accompanying a hydrogen-bonding alteration of the Tyr/Gln. In order to reveal the hydrogen-bonding structure of the Tyr/Gln active center and FAD, FTIR spectroscopy was applied in the 4000–1800 cm–1 region, where X–H stretches appear. The present FTIR data show an unusually low O–H stretching vibration of tyrosine at 2800–2600 cm–1. This provides direct evidence of the light-induced switch of the hydrogen-bonding network, where the conserved Tyr (Tyr21 for AppA) donates an unusually strong hydrogen bond in the intermediate.

Photoreaction in BLUF Receptors: Proton‐coupled Electron Transfer in the Flavin‐Gln‐Tyr System†

Photoinduced electron transfer from tyrosine to the flavin chromophore is involved in activation of BLUF (sensor of blue light using FAD) photoreceptors. We studied the electron transfer (ET) coupled with proton-transfer (PT) reactions, by means of XMCQDPT2//CASSCF calculations on a molecular cluster model. By defining a minimum active space in the CASSCF calculations, we could compute the entire photoreaction pathway. We find that the crossing of the locally excited and ET states is located along the flavin bond-stretching coordinate. The ET state is stabilized by a proton transfer from the electron donor to the electron acceptor. We mapped two different PT pathways from tyrosine to flavin via the conserved glutamine. These reactions generate a tautomeric form of glutamine. Along the PT coordinates, we find geometries where the ET and the electronic ground states degenerate. At the state crossing structures, either formation of the ground state biradical intermediate or a relaxation back to the Franck-Condon minimum takes places. The computed relaxation pathways reveal that the hydrogen bonds involving glutamine in the chromophore-binding pocket control BLUF photoefficiency.

Macromolecular Crowding Effects on Reactions of TePixD (Tll0078)†

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.

The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli

The blue light using FAD (BLUF)-EAL protein YcgF is a known blue-light sensor of Escherichia coli, but its direct regulatory output and physiological function have remained unknown. Here, we demonstrate that unlike other EAL domain proteins, YcgF does not degrade the signaling molecule c-di-GMP, but directly binds to and releases the MerR-like repressor YcgE from its operator DNA upon blue-light irradiation. As a consequence, a distinct regulon of eight small proteins (of 71-126 amino acids) is strongly induced. These include YmgA and YmgB, which, via the RcsC/RcsD/RcsB two-component phosphorelay system, activate production of the biofilm matrix substance colanic acid as well as acid resistance genes and the biofilm-associated bdm gene and down-regulate adhesive curli fimbriae. Thus, small proteins under YcgF/YcgE control seem to act as "connectors" that provide additional signal input into a two-component signaling pathway. Moreover, we found ycgF and ycgE expression to be strongly activated at low temperature, and we elucidate how blue light, cold, and starvation signals are integrated in the expression and activity of the YcgF/YcgE/small protein signaling pathway. In conclusion, this pathway may modulate biofilm formation via the two-component network when E. coli has to survive in an extrahost aquatic environment.

Oligomeric-State-Dependent Conformational Change of the BLUF Protein TePixD (Tll0078)

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 μs, 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 × 10 − 11 to 4.4 × 10 − 11 m 2 s − 1 upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 × 10 − 11 m 2 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.

Molecular Models Predict Light-Induced Glutamine Tautomerization in BLUF Photoreceptors

The recently discovered photoreceptor proteins containing BLUF (sensor of blue light using FAD) domains mediate physiological responses to blue light in bacteria and euglena. In BLUF domains, blue light activates the flavin chromophore yielding a signaling state characterized by a ∼10nm red-shifted absorption. We developed molecular models for the dark and light states of the BLUF domain of the Rhodobacter sphaeroides AppA protein, which are based on the crystal structures and quantum-mechanical simulations. According to these models, photon absorption by the flavin results in a tautomerization and 180° rotation of the Gln side chain that interacts with the flavin cofactor. This chemical modification of the Gln residue induces alterations in the hydrogen bond network in the core of the photoreceptor domain, which were observed in numerous spectroscopic experiments. The calculated electronic transition energies and vibrational frequencies of the proposed dark and light states are consistent with the optical and IR spectral changes observed during the photocycle. Light-induced isomerization of an amino acid residue instead of a chromophore represents a feature that has not been described previously in photoreceptors.