Light, Oxygen And Voltage Domains Research Articles

Solving Blue Light Riddles: New Lessons from Flavin-binding LOV Photoreceptors.

Detection of blue light (BL) via flavin-binding photoreceptors (Fl-Blues) has evolved throughout all three domains of life. Although the main BL players, that is light, oxygen and voltage (LOV), blue light sensing using flavins (BLUF) and Cry (cryptochrome) proteins, have been characterized in great detail with respect to structure and function, still several unresolved issues at different levels of complexity remain and novel unexpected findings were reported. Here, we review the most prevailing riddles of LOV-based photoreceptors, for example: the relevance of water and/or small metabolites for the dynamics of the photocycle; molecular details of light-to-signal transduction events; the interplay of BL sensing by LOV domains with other environmental stimuli, such as BL plus oxygen-mediating photodamage and its impact on microbial lifestyles; the importance of the cell or chromophore redox state in determining the fate of BL-driven reactions; the evolutionary pathways of LOV-based BL sensing and associated functions through the diverse phyla. We will discuss major novelties emerged during the last few years on these intriguing aspects of LOV proteins by presenting paradigmatic examples from prokaryotic photosensors that exhibit the largest complexity and richness in associated functions.

The Evolution and Functional Role of Flavin-based Prokaryotic Photoreceptors.

Flavin-based photoreceptor proteins of the LOV (light, oxygen and voltage) superfamily are ubiquitous and appear to be essential blue-light sensing systems not only in plants, algae and fungi, but also in prokaryotes, where they are represented in more than 10% of known species. Despite their broad occurrence, only in few cases LOV proteins have been correlated with important phenomena such as bacterial infectivity, selective growth patterns or/and stress responses; nevertheless these few known roles are helping us understand the multiple ways by which prokaryotes can exploit these soluble blue-light photoreceptors. Given the large number of sequences now deposited in databases, it becomes meaningful to define a signature for bona fide LOV domains, a procedure that facilitates identification of proteins with new properties and phylogenetic analysis. The latter clearly evidences that a class of LOV proteins from alpha-proteobacteria is the closest prokaryotic relative of eukaryotic LOV domains, whereas cyanobacterial sequences cluster with the archaeal and the other bacterial LOV domains. Distance trees built for LOV domains suggest complex evolutionary patterns, possibly involving multiple horizontal gene transfer events. Based on available data, the invivo relevance and evolution of prokaryotic LOV is discussed.

Photoinduced oligomerization of Arabidopsis thaliana phototropin 2 LOV1.

Phototropins are blue-light-sensitive photoreceptor proteins in plants. Phototropins consist of two LOV (light, oxygen, and voltage sensor) domains (LOV1 and LOV2) that undergo photochemical reactions. Although the photochemical reaction of the LOV2 domain has been investigated extensively, the reaction of the LOV1 domain remains unresolved. In this study, the reactions of the Arabidopsis phototropin 2 LOV1 (phot2LOV1) domain were revealed by the transient grating (TG) method. The TG signal showed a significant diffusion coefficient (D) change upon photoexcitation. This change was sensitive to the protein concentration and the observation time range. These observations were explained by assuming that there are reactive and nonreactive forms, and the fraction of these species is concentration dependent. From the concentration dependence of the dynamics, the monomer was found to form a dimer; however, the dimer does not exhibit an observable reaction. In the dark state, both species were in equilibrium and were not distinguishable spectroscopically. For the LOV1 domain with the hinge domain, the reaction scheme was the same as the LOV1 domain sample, but the D change was affected by the presence of the hinge region. This observation suggested that the hinge region undergoes a conformational change during the photoreaction.

From Plant Infectivity to Growth Patterns: The Role of Blue-Light Sensing in the Prokaryotic World.

Flavin-based photoreceptor proteins of the LOV (Light, Oxygen, and Voltage) and BLUF (Blue Light sensing Using Flavins) superfamilies are ubiquitous among the three life domains and are essential blue-light sensing systems, not only in plants and algae, but also in prokaryotes. Here we review their biological roles in the prokaryotic world and their evolution pathways. An unexpected large number of bacterial species possess flavin-based photosensors, amongst which are important human and plant pathogens. Still, few cases are reported where the activity of blue-light sensors could be correlated to infectivity and/or has been shown to be involved in the activation of specific genes, resulting in selective growth patterns. Metagenomics and bio-informatic analysis have only recently been initiated, but signatures are beginning to emerge that allow definition of a bona fide LOV or BLUF domain, aiming at better selection criteria for novel blue-light sensors. We also present here, for the first time, the phylogenetic tree for archaeal LOV domains that have reached a statistically significant number but have not at all been investigated thus far.

Distance-tree analysis, distribution and co-presence of bilin- and flavin-binding prokaryotic photoreceptors for visible light

In recent years it has become increasingly evident that prokaryotic organisms can sense and react to light stimuli via a variety of photosensory receptors and signal transduction pathways. There are two main superfamilies of non-membrane-bound photoreceptors: the bilin-binding phytochrome-related proteins based on GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and transcription activator FhlA) domains (bilin-GAF proteins), and the flavin-binding proteins (FL-Blues), photoperceptive thanks to their LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using Flavins) domains. In this manuscript we present a comprehensive scenario of the existence of bilin-GAF, LOV and BLUF proteins in the prokaryotic world and inspect possible phylogenetic pathways, also defining novel criteria for identifying gene (and protein) sequences based on experimentally assessed photochemical events. As a whole we have inspected almost 2000 proteins recovered in 985 bacteria and 16 archaea. For LOV and BLUF proteins, ten and, respectively, twelve superconserved amino acids have been identified, which were used as criterion for selection. A similarly strict parameter cannot be applied to the more variegate family of bilin-GAF domains. The co-presence of bilin-GAF and FL-Blues occurs in 22% of the analyzed bacteria, with emphasis on the bilin-GAF/LOV co-presence in cyanobacteria and of bilin-GAF/BLUF in the Bacteroidetes/Chlorobi group. For construction of phylogeny/distance-trees we used the neighboring-method to obtain a branching pattern, limited to photosensing domains. We observed that in many cases organisms belonging to the same phylum are neighbors, but clustering mostly occurs according to the type of functional domain associated with the photosensing modules.

Applications of vibrational spectroscopy in the study of flavin-based photoactive proteins

Flavin cofactor is known to perform diverse biological functions. Recently, its role as a photoreceptor has been identified. So far, three classes of photoactive flavoproteins have been recognized: phototropin with LOV (Light, Oxygen and Voltage) domain, blue light sensory protein with BLUF (Blue Light sensing Using Flavin adenine dinucleotide) domain and photolyase/cryptochrome protein with PHR (Photolyase Homology Region) domain. Photochemistry of flavin is the key to unravel the reaction mechanisms of photoactive flavoproteins in their biological functions such as DNA repair or signal transduction. Vibrational (Infrared and Raman) spectroscopy is a useful and sensitive tool to investigate the photochemistry of flavin in protein environments and has significantly contributed to elucidate the reaction mechanisms of these photoactive proteins. This study will survey recent advances in vibrational spectroscopic studies on this topic and remaining questions to be answered.

Applications of vibrational spectroscopy in the study of flavin-based photoactive proteins

Flavin cofactor is known to perform diverse biological functions. Recently, its role as a photoreceptor has been identified. So far, three classes of photoactive flavoproteins have been recognized: phototropin with LOV (Light, Oxygen and Voltage) domain, blue light sensory protein with BLUF (Blue Light sensing Using Flavin adenine dinucleotide) domain and photolyase/cryptochrome protein with PHR (Photolyase Homology Region) domain. Photochemistry of flavin is the key to unravel the reaction mechanisms of photoactive flavoproteins in their biological functions such as DNA repair or signal transduction. Vibrational (Infrared and Raman) spectroscopy is a useful and sensitive tool to investigate the photochemistry of flavin in protein environments and has significantly contributed to elucidate the reaction mechanisms of these photoactive proteins. This study will survey recent advances in vibrational spectroscopic studies on this topic and remaining questions to be answered.

Novel blue light‐sensitive proteins from a metagenomic approach

A microarray-based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor-encoding genes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2-5 microg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 microg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid-cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per-Arnt-Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht-met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht-met1 is followed by a second open reading frame encoding a Fe-chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht-met1 was subcloned and expressed yielding a fully functional, flavin-containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin-protein photoadduct. The dark recovery lifetime was found as tau(REC) = 120 s (20 degrees C) and is among the fastest ones determined so far for bacterial LOV domains.

In Vivo Mutational Analysis of YtvA from Bacillus subtilis: MECHANISM OF LIGHT ACTIVATION OF THE GENERAL STRESS RESPONSE

The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates sigma(B) through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light, oxygen, and voltage) domain and a C-terminal STAS (sulfate transporter and anti-sigma factor) domain and shows light-modulated GTP binding in vitro. Here, we examine the mechanism of YtvA-mediated activation of sigma(B) in vivo with site-directed mutagenesis. Constitutive off and constitutive on mutations have been identified. Disruption of GTP binding in the STAS domain eliminates light activation of sigma(B). In contrast, modification of sites relevant for phosphorylation of STAS domains does not affect the stress response significantly. The data obtained are integrated into a model for the structure of full-length YtvA, which presumably functions as a dimer.

Characterization of an Unusual LOV Domain Protein in the α‐Proteobacterium Rhodobacter sphaeroides

The facultatively phototrophic purple bacterium Rhodobacter sphaeroides 2.4.1 harbors a LOV (light, oxygen and voltage) domain protein, which shows a particular structure. LOV domains perceive blue light by a noncovalently bound flavin and transmit the signal to various coupled output domains. Proteins, that harbor a LOV core, function e.g. as phototropins or circadian clock regulators. Jalpha helices, which act as linker between the LOV core and the output domain, were shown to be involved in the light-dependent activation of the output domain. Like PpSB2 from Pseudomonas putida, the LOV domain protein of R. sphaeroides is not coupled to an effector domain and harbors an extended C-terminal alpha helix. We expressed the R. sphaeroides LOV domain recombinantly in Escherichia coli. The protein binds an FMN as a cofactor and shows a photocycle typical for LOV domain containing proteins. In R. sphaeroides, we detected the protein as well in the cytoplasm as in the membrane fraction, which was not reported for other bacterial LOV domain proteins.

Estimation of the available free energy in a LOV2-Jα photoswitch

Protein photosensors are versatile tools for studying ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins--plant serine/threonine kinases that are activated by blue light via conserved LOV (light, oxygen and voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in a light-dependent fashion with an adjacent alpha-helix (J alpha) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain-J alpha-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by approximately 3.8 kcal mol(-1), thus quantifying the energy available through LOV-J alpha for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-J alpha interaction.

Flavin‐based Blue‐light Photosensors: A Photobiophysics Update

This review deals with the biophysical aspects of flavin-based photosensors, comprising cryptochromes, LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using FAD) proteins. Special emphasis is given to structural issues, photocycle quantum yields and energetics, mechanism of the light-triggered reactions, early stages in signal transduction and oligomeric states of the light sensing protein modules. For BLUF and LOV domains important parallels are emerging, despite their different alpha/beta fold arrangement, whereas there is increasing evidence for a mechanicistic and functional splitting of the cryptochrome family.

NTP-binding properties of the blue-light receptor YtvA and effects of the E105L mutation

YtvA is a blue-light-sensing protein from Bacillus subtilis related to plant phototropins. It carries a LOV (light, oxygen and voltage) domain, binding FMN (flavin mononucleotide) as chromophore, and a STAS (sulphate transporters and antisigma-factor antagonists) domain with poorly characterized function. We have recently shown that YtvA binds triphosphate nucleotides (NTP) and highlighted a structural similarity between the STAS domain and small GTP-binding proteins. In this work we further investigated the NTP-binding properties of YtvA, employing a fluorescent derivative of GTP (GTP(TR)) and mutagenesis experiments. The main results are as follows: (a) competition experiments indicate that the affinity of YtvA for GTP is much higher than that for GDP and GMP. (b) Blue-light-induced structural changes are transmitted from the LOV core to the NTP-binding cavity, establishing a possible intraprotein signal-transduction pathway. (c) A mutation in the central beta-scaffold of the LOV core, E105L, impairs the light-driven spectroscopic changes of bound GTP(TR). This result is supported by circular dichroism data, in that YtvA-E105L does not show the light-induced conformational change in the turn fraction that characterizes YtvA, implying that E105 is functionally important. (d) In the structural model of the LOV-STAS complex, based on docking algorithms, the interface includes the Ibeta-Hbeta loop on the LOV core, as well as parts of the central beta-scaffold. E105 is predicted to interact with the LOV-STAS linker region, suggested to play a role in phototropin signaling.

Blue news: NTP binding properties of the blue-light sensitive YtvA protein from Bacillus subtilis

The blue-light sensitive protein YtvA from Bacillus subtilis is built of a photoactive, flavin-binding LOV (Light, Oxygen and Voltage) domain and a STAS domain with unknown function. Here we show that YtvA binds a fluorescent derivative of guanosine triphosphate (GTP TR) that can be displaced by both GTP or ATP. Unspecific NTP (N = G or A) binding is supported by the molecular model of YtvA-STAS. Blue-light activation of YtvA results in small and dark-reversible spectroscopic changes for GTP TR, suggesting that light-driven conformational changes are transmitted from the LOV core to the GTP TR binding site. These results support the idea that STAS domains may have a general NTP binding role and open a way to investigate the molecular functionality of YtvA-STAS.

Quaternary structure of LOV-domain containing polypeptide of Arabidopsis FKF1 protein

Flavin-binding, Kelch repeat, F-box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)-sensing domain binding a flavin mononucleotide. The photo-activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV-containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small-angle X-ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic-shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27Å and the maximum dimension of 94Å. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue-light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino-acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.

Autophosphorylation, Electrophoretic Mobility and Immunoreaction of Oat Phototropin 1 Under UV and Blue Light¶

Phototropins are UV-A/blue light photoreceptors containing two flavin mononucleotide (FMN)-binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light-dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rudiger [2003] Biochemistry 42, 4217-4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV-C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV-C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV-C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.

Autophosphorylation, Electrophoretic Mobility and Immunoreaction of Oat Phototropin 1 Under UV and Blue Light¶

ABSTRACTPhototropins are UV‐A/blue light photoreceptors containing two flavin mononucleotide (FMN)–binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light‐dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rüdiger [2003] Biochemistry42, 4217–4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV‐C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV‐C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV‐C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.

Tryptophan Fluorescence in the Bacillus subtilis Phototropin‐related Protein YtvA as a Marker of Interdomain Interaction¶

ABSTRACTThe Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N‐terminal light, oxygen and voltage (LOV) domain. The blue light‐triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue. YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot‐LOV domains. In this study, we show that the fluorescence parameters of W103 in YtvA‐LOV are markedly different from those observed in the full‐length YtvA. The fluorescence quantum yields are ca 0.03 and 0.08, respectively. In YtvA‐LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns). These data indicate that W103 is located in a site of tight contact between the two domains of YtvA. In the FMN‐cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state. On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence. This effect is reversible in the dark and might arise from a light‐regulated energy transfer process from a yet unidentified tyrosine to W103.

Tryptophan fluorescence in the Bacillus subtilis phototropin-related protein YtvA as a marker of interdomain interaction.

The Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N-terminal light, oxygen and voltage (LOV) domain. The blue light-triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue. YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot-LOV domains. In this study, we show that the fluorescence parameters of W103 in YtvA-LOV are markedly different from those observed in the full-length YtvA. The fluorescence quantum yields are ca 0.03 and 0.08, respectively. In YtvA-LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns). These data indicate that W103 is located in a site of tight contact between the two domains of YtvA. In the FMN-cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state. On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence. This effect is reversible in the dark and might arise from a light-regulated energy transfer process from a yet unidentified tyrosine to W103.

Autophosphorylation, electrophoretic mobility and immunoreaction of oat phototropin 1 under UV and blue Light.

Phototropins are UV-A/blue light photoreceptors containing two flavin mononucleotide (FMN)-binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light-dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rudiger [2003] Biochemistry 42, 4217-4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV-C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV-C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV-C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.