Showing 576 - 600 of 618 results
Mechanism-based tuning of a LOV domain photoreceptor.
Phototropin-like LOV domains form a cysteinyl-flavin adduct in response to blue light but show considerable variation in output signal and the lifetime of the photo-adduct signaling state. Mechanistic studies of the slow-cycling fungal LOV photoreceptor Vivid (VVD) reveal the importance of reactive cysteine conformation, flavin electronic environment and solvent accessibility for adduct scission and thermal reversion. Proton inventory, pH effects, base catalysis and structural studies implicate flavin N(5) deprotonation as rate-determining for recovery. Substitutions of active site residues Ile74, Ile85, Met135 and Met165 alter photoadduct lifetimes by over four orders of magnitude in VVD, and similar changes in other LOV proteins show analogous effects. Adduct state decay rates also correlate with changes in conformational and oligomeric properties of the protein necessary for signaling. These findings link natural sequence variation of LOV domains to function and provide a means to design broadly reactive light-sensitive probes.
LOVely enzymes - towards engineering light-controllable biocatalysts.
Light control over enzyme function represents a novel and exciting field of biocatalysis research. Blue-light photoreceptors of the Light, Oxygen, Voltage (LOV) family have recently been investigated for their applicability as photoactive switches. We discuss here the primary photochemical events leading to light activation of LOV domains as well as the proposed signal propagation mechanism to the respective effector domain. Furthermore, we describe the construction of LOV fusions to different effector domains, namely a dihydrofolate reductase from Escherichia coli and a lipase from Bacillus subtilis. Both fusion partners retained functionality, and alteration of enzyme activity by light was also demonstrated. Hence, it appears that fusion of LOV photoreceptors to functional enzyme target sites via appropriate linker structures may represent a straightforward strategy to design light controllable biocatalysts.
A genetically encoded photoactivatable Rac controls the motility of living cells.
The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.
A synthetic genetic edge detection program.
Edge detection is a signal processing algorithm common in artificial intelligence and image recognition programs. We have constructed a genetically encoded edge detection algorithm that programs an isogenic community of E. coli to sense an image of light, communicate to identify the light-dark edges, and visually present the result of the computation. The algorithm is implemented using multiple genetic circuits. An engineered light sensor enables cells to distinguish between light and dark regions. In the dark, cells produce a diffusible chemical signal that diffuses into light regions. Genetic logic gates are used so that only cells that sense light and the diffusible signal produce a positive output. A mathematical model constructed from first principles and parameterized with experimental measurements of the component circuits predicts the performance of the complete program. Quantitatively accurate models will facilitate the engineering of more complex biological behaviors and inform bottom-up studies of natural genetic regulatory networks.
Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase.
The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.
Structure and insight into blue light-induced changes in the BlrP1 BLUF domain.
BLUF domains (sensors of blue light using flavin adenine dinucleotide) are a group of flavin-containing blue light photosensory domains from a variety of bacterial and algal proteins. While spectroscopic studies have indicated that these domains reorganize their interactions with an internally bound chromophore upon illumination, it remains unclear how these are converted into structural and functional changes. To address this, we have solved the solution structure of the BLUF domain from Klebsiella pneumoniae BlrP1, a light-activated c-di-guanosine 5'-monophosphate phosphodiesterase which consists of a sensory BLUF and a catalytic EAL (Glu-Ala-Leu) domain [Schmidt et. al. (2008) J. Bacteriol. 187, 4774-4781]. Our dark state structure of the sensory domain shows that it adopts a standard BLUF domain fold followed by two C-terminal alpha helices which adopt a novel orientation with respect to the rest of the domain. Comparison of NMR spectra acquired under dark and light conditions suggests that residues throughout the BlrP1 BLUF domain undergo significant light-induced chemical shift changes, including sites clustered on the beta(4)beta(5) loop, beta(5) strand, and alpha(3)alpha(4) loop. Given that these changes were observed at several sites on the helical cap, over 15 A from chromophore, our data suggest a long-range signal transduction process in BLUF domains.
Blue light induces degradation of the negative regulator phytochrome interacting factor 1 to promote photomorphogenic development of Arabidopsis seedlings.
Phytochrome interacting factors (PIFs) are nuclear basic helix-loop-helix (bHLH) transcription factors that negatively regulate photomorphogenesis both in the dark and in the light in Arabidopsis. The phytochrome (phy) family of photoreceptors induces the rapid phosphorylation and degradation of PIFs in response to both red and far-red light conditions to promote photomorphogenesis. Although phys have been shown to function under blue light conditions, the roles of PIFs under blue light have not been investigated in detail. Here we show that PIF1 negatively regulates photomorphogenesis at the seedling stage under blue light conditions. pif1 seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12 hr light/12 hr dark) blue light conditions. Double-mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cry1, and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants under diurnal blue light conditions. Blue light induced the rapid phosphorylation, polyubiquitination, and degradation of PIF1 through the ubi/26S proteasomal pathway. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the blue light-induced degradation of PIF1. phyA played a dominant role under pulses of blue light, while phyA, phyB, and phyD induced the degradation of PIF1 in an additive manner under prolonged continuous blue light conditions. Interestingly, the absence of cry1 and cry2 enhanced the degradation of PIF1 under blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis under blue light conditions and that blue light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis.
Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis.
The ultraviolet-B (UV-B) portion of the solar radiation functions as an environmental signal for which plants have evolved specific and sensitive UV-B perception systems. The UV-B-specific UV RESPONSE LOCUS 8 (UVR8) and the multifunctional E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are key regulators of the UV-B response. We show here that uvr8-null mutants are deficient in UV-B-induced photomorphogenesis and hypersensitive to UV-B stress, whereas overexpression of UVR8 results in enhanced UV-B photomorphogenesis, acclimation and tolerance to UV-B stress. By using sun simulators, we provide evidence at the physiological level that UV-B acclimation mediated by the UV-B-specific photoregulatory pathway is indeed required for survival in sunlight. At the molecular level, we demonstrate that the wild type but not the mutant UVR8 and COP1 proteins directly interact in a UV-B-dependent, rapid manner in planta. These data collectively suggest that UV-B-specific interaction of COP1 and UVR8 in the nucleus is a very early step in signalling and responsible for the plant's coordinated response to UV-B ensuring UV-B acclimation and protection in the natural environment.
Oligomeric structure of LOV domains in Arabidopsis phototropin.
Oligomeric structures of the four LOV domains in Arabidopsis phototropin1 (phot1) and 2 (phot2) were studied using crosslinking. Both LOV1 domains of phot1 and phot2 form a dimer independently on the light conditions, suggesting that the LOV1 domain can be a stable dimerization site of phot in vivo. In contrast, phot1-LOV2 is in a monomer-dimer equilibrium and phot2-LOV2 exists as a monomer in the dark. Blue light-induced a slight increase in the monomer population in phot1-LOV2, suggesting a possible blue light-inducible dissociation of dimers. Furthermore, blue light caused a band shift of the phot2-LOV2 monomer. CD spectra revealed the unfolding of helices and the formation of strand structures. Both light-induced changes were reversible in the dark.
A conserved glutamine plays a central role in LOV domain signal transmission and its duration.
Light is a key stimulus for plant biological functions, several of which are controlled by light-activated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, light-oxygen-voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internally bound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jalpha helix and, ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue [Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain] switches its hydrogen bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the Ibeta strand that interacts with the Jalpha helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jalpha helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudodark state (Q513L) or a pseudolit state (Q513N). Further, both mutations changed the photochemical properties of this receptor, in particular the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from the core of the LOV domain through the Ibeta strand to the peripheral Jalpha helix.
A light-independent allele of phytochrome B faithfully recapitulates photomorphogenic transcriptional networks.
Dominant gain-of-function alleles of Arabidopsis phytochrome B were recently shown to confer light-independent, constitutive photomorphogenic (cop) phenotypes to transgenic plants (Su and Lagarias, 2007). In the present study, comparative transcription profiling experiments were performed to assess whether the pattern of gene expression regulated by these alleles accurately reflects the process of photomorphogenesis in wild-type Arabidopsis. Whole-genome transcription profiles of dark-grown phyAphyB seedlings expressing the Y276H mutant of phyB (YHB) revealed that YHB reprograms about 13% of the Arabidopsis transcriptome in a light-independent manner. The YHB-regulated transcriptome proved qualitatively similar to but quantitatively greater than those of wild-type seedlings grown under 15 or 50 micromol m(-2) m(-1) continuous red light (Rc). Among the 2977 genes statistically significant two-fold (SSTF) regulated by YHB in the absence of light include those encoding components of the photosynthetic apparatus, tetrapyrrole/pigment biosynthetic pathways, and early light-responsive signaling factors. Approximately 80% of genes SSTF regulated by Rc were also YHB-regulated. Expression of a notable subset of 346 YHB-regulated genes proved to be strongly attenuated by Rc, indicating compensating regulation by phyC-E and/or other Rc-dependent processes. Since the majority of these 346 genes are regulated by the circadian clock, these results suggest that phyA- and phyB-independent light signaling pathway(s) strongly influence clock output. Together with the unique plastid morphology of dark-grown YHB seedlings, these analyses indicate that the YHB mutant induces constitutive photomorphogenesis via faithful reconstruction of phyB signaling pathways in a light-independent fashion.
Design and signaling mechanism of light-regulated histidine kinases.
Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by approximately 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40 degrees -60 degrees rotational movement within an alpha-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by alpha-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.
Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness.
An important contributing factor to the success of terrestrial flowering plants in colonizing the land was the evolution of a developmental strategy, termed skotomorphogenesis, whereby postgerminative seedlings emerging from buried seed grow vigorously upward in the subterranean darkness toward the soil surface.
Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.
Cryptochromes (CRY) are photolyase-like blue-light receptors that mediate light responses in plants and animals. How plant cryptochromes act in response to blue light is not well understood. We report here the identification and characterization of the Arabidopsis CIB1 (cryptochrome-interacting basic-helix-loop-helix) protein. CIB1 interacts with CRY2 (cryptochrome 2) in a blue light-specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 binds to G box (CACGTG) in vitro with a higher affinity than its interaction with other E-box elements (CANNTG). However, CIB1 stimulates FT messenger RNA expression, and it interacts with chromatin DNA of the FT gene that possesses various E-box elements except G box. We propose that the blue light-dependent interaction of cryptochrome(s) with CIB1 and CIB1-related proteins represents an early photoreceptor signaling mechanism in plants.
Surface sites for engineering allosteric control in proteins.
Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites.
Photodynamics of blue-light-regulated phosphodiesterase BlrP1 protein from Klebsiella pneumoniae and its photoreceptor BLUF domain.
The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain
and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF
domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor
FAD in its oxidized redox state (FADox) is brought from the dark-adapted receptor state to the 10-nm
red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs
with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for
BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FADox cofactor is small due to
photo-induced reductive electron transfer. Prolonged light exposure converts FADox in the signalling state
to the fully reduced hydroquinone form FADredH and causes low-efficient chromophore release with
subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state
and in the signalling state are discussed.
Transposing phytochrome into the nucleus.
To control many physiological responses, phytochromes directly modulate gene expression. A key regulatory event in this signal transduction pathway is the light-controlled translocation of the photoreceptor from the cytoplasm into the nucleus. Recent publications are beginning to shed light on the molecular mechanisms underlying this central control point. Interestingly, there is a specific mechanism for phytochrome A (phyA) nuclear accumulation. The dedicated phyA nuclear import pathway might be important for the distinct photosensory specificity of this atypical phytochrome. Recent studies in the field also provide a starting point for investigating how the different subcellular pools of phytochrome can control distinct responses to light.
Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway.
General methods to engineer genetically encoded, reversible, light-mediated control over protein function would be useful in many areas of biomedical research and technology. We describe a system that yields such photo-control over actin assembly. We fused the Rho family GTPase Cdc42 in its GDP-bound form to the photosensory domain of phytochrome B (PhyB) and fused the Cdc42 effector, the Wiskott-Aldrich Syndrome Protein (WASP), to the light-dependent PhyB-binding domain of phytochrome interacting factor 3 (Pif3). Upon red light illumination, the fusion proteins bind each other, activating WASP, and consequently stimulating actin assembly by the WASP target, the Arp2/3 complex. Binding and WASP activation are reversed by far-red illumination. Our approach, in which the biochemical specificity of the nucleotide switch in Cdc42 is overridden by the light-dependent PhyB-Pif3 interaction, should be generally applicable to other GTPase-effector pairs.
PixE promotes dark oligomerization of the BLUF photoreceptor PixD.
Cyanobacteria perceive and move (phototax) in response to blue light. In this study, we demonstrate that the PixD blue light-sensing using FAD (BLUF) photoreceptor that governs this response undergoes changes in oligomerization state upon illumination. Under dark conditions we observed that PixD forms a large molecular weight complex with another protein called PixE. Stoicheometric analyses, coupled with sedimentation equilibrium and size exclusion chromatography, demonstrates that PixE drives aggregation of PixD dimers into a stable PixD(10)-PixE(5) complex under dark conditions. Illumination of a flavin chromophore in PixD destabilizes the PixD(10)-PixE(5) complex into monomers of PixE and dimers of PixD. A crystallographic structure of PixD, coupled with Gibbs free energy calculation between interacting faces of PixD, lends to a model in which a light induces a conformational change in a critical PixD-interfacing loop that results in destabilization of the PixD(10)-PixE(5) complex.
Light-activated DNA binding in a designed allosteric protein.
An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an alpha-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical "allosteric lever arm" is a general scheme for coupling the function of two proteins.
Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein.
Cyanobacteriochromes are a newly recognized group of photoreceptors that are distinct relatives of phytochromes but are found only in cyanobacteria. A putative cyanobacteriochrome, CcaS, is known to chromatically regulate the expression of the phycobilisome linker gene (cpcG2) in Synechocystis sp. PCC 6803. In this study, we isolated the chromophore-binding domain of CcaS from Synechocystis as well as from phycocyanobilin-producing Escherichia coli. Both preparations showed the same reversible photoconversion between a green-absorbing form (Pg, lambda(max) = 535 nm) and a red-absorbing form (Pr, lambda(max) = 672 nm). Mass spectrometry and denaturation analyses suggested that Pg and Pr bind phycocyanobilin in a double-bond configuration of C15-Z and C15-E, respectively. Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by preirradiation with green light. Similarly, phosphotransfer to the cognate response regulator, CcaR, was higher in Pr than in Pg. From these results, we conclude that CcaS phosphorylates CcaR under green light and induces expression of cpcG2, leading to accumulation of CpcG2-phycobilisome as a chromatic acclimation system. CcaS is the first recognized green light receptor in the expanded phytochrome superfamily, which includes phytochromes and cyanobacteriochromes.
Photoregulation in prokaryotes.
The spectroscopic identification of sensory rhodopsin I by Bogomolni and Spudich in 1982 provided a molecular link between the light environment and phototaxis in Halobacterium salinarum, and thus laid the foundation for the study of signal transducing photosensors in prokaryotes. In recent years, a number of new prokaryotic photosensory receptors have been discovered across a broad range of taxa, including dozens in chemotrophic species. Among these photoreceptors are new classes of rhodopsins, BLUF-domain proteins, bacteriophytochromes, cryptochromes, and LOV-family photosensors. Genetic and biochemical analyses of these receptors have demonstrated that they can regulate processes ranging from photosynthetic pigment biosynthesis to virulence.
Activation of protein splicing with light in yeast.
Spatiotemporal regulation of protein function is a key feature of living systems; experimental tools that provide such control are of great utility. Here we report a genetically encoded system for controlling a post-translational process, protein splicing, with light. Studies in Saccharomyces cerevisiae demonstrate that fusion of a photodimerization system from Arabidopsis thaliana to an artificially split intein permits rapid activation of protein splicing to yield a new protein product.
N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa.
Light sensing by photoreceptors controls phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Understanding the molecular mechanism by which these processes are regulated requires a quantitative description of photoreceptor dynamics. We focus on a light-driven signal transduction mechanism in the LOV2 domain (LOV, light, oxygen, voltage) of the blue light photoreceptor phototropin 1 from Avena sativa (oat). High-resolution crystal structures of the dark and light states of an oat LOV2 construct including residues Leu404 through Leu546 (LOV2 (404-546)) have been determined at 105 and 293 K. In all four structures, LOV2 (404-546) exhibits the typical Per-ARNT-Sim (PAS) fold, flanked by an additional conserved N-terminal turn-helix-turn motif and a C-terminal flanking region containing an amphipathic Jalpha helix. These regions dock on the LOV2 core domain and bury several hydrophobic residues of the central beta-sheet of the core domain that would otherwise be exposed to solvent. Light structures of LOV2 (404-546) reveal that formation of the covalent bond between Cys450 and the C4a atom of the flavin mononucleotide (FMN) results in local rearrangement of the hydrogen-bonding network in the FMN binding pocket. These rearrangements are associated with disruption of the Asn414-Asp515 hydrogen bond on the surface of the protein and displacement of the N- and C-terminal flanking regions of LOV2 (404-546), both of which constitute a structural signal.
Dual role for a bacteriophytochrome in the bioenergetic control of Rhodopseudomonas palustris: enhancement of photosystem synthesis and limitation of respiration.
In the purple photosynthetic bacterium Rhodopseudomonas palustris, far-red illumination induces photosystem synthesis via the action of the bacteriophytochrome RpBphP1. This bacteriophytochrome antagonizes the repressive effect of the transcriptional regulator PpsR2 under aerobic condition. We show here that, in addition to photosystem synthesis, far-red light induces a significant growth rate limitation, compared to cells grown in the dark, linked to a decrease in the respiratory activity. The phenotypes of mutants inactivated in RpBphP1 and PpsR2 show their involvement in this regulation. Based on enzymatic and transcriptional studies, a 30% decrease in the expression of the alpha-ketoglutarate dehydrogenase complex, a central enzyme of the Krebs cycle, is observed under far-red light. We propose that this decrease is responsible for the down-regulation of respiration in this condition. This regulation mechanism at the Krebs cycle level still allows the formation of the photosynthetic apparatus via the synthesis of key biosynthesis precursors but lowers the production of NADH, i.e. the respiratory activity. Overall, the dual action of RpBphP1 on the regulation of both the photosynthesis genes and the Krebs cycle allows a fine adaptation of bacteria to environmental conditions by enhancement of the most favorable bioenergetic process in the light, photosynthesis versus respiration.