Showing 1 - 21 of 21 results
Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.
We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
An Open-Source Plate Reader.
Microplate readers are foundational instruments in ex-perimental biology and bioengineering that enable mul-tiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construc-tion, validation, and benchmarking of an open-source microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs <$3500, a fraction of the cost of commer-cial plate readers, and can detect the fluorescence of common dyes down to ~10 nanomolar concentration. Functional capabilities were demonstrated in context of synthetic biology, optoge¬netics, and photosensory biol-ogy: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing, and by flavin photocycling kinetic measure-ments of a LOV (light-oxygen-voltage) domain photo-receptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and au-tomating it using the custom Python-based API (Appli-cation Program Interface) are provided. This work con-tributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid proto-typing capabilities and low-cost electronics, optoelec-tronics, and microcomputers.
Optogenetic Reconstitution for Determining the Form and Function of Membraneless Organelles.
It has recently become clear that large-scale macromolecular self-assembly is a rule, rather than an exception, of intracellular organization. A growing number of proteins and RNAs have been shown to self-assemble into micrometer-scale clusters that exhibit either liquid-like or gel-like properties. Given their proposed roles in intracellular regulation, embryo development, and human disease, it is becoming increasingly important to understand how these membraneless organelles form and to map their functional consequences for the cell. Recently developed optogenetic systems make it possible to acutely control cluster assembly and disassembly in live cells, driving the separation of proteins of interest into liquid droplets, hydrogels, or solid aggregates. Here we propose that these approaches, as well as their evolution into the next generation of optogenetic biophysical tools, will allow biologists to determine how the self-assembly of membraneless organelles modulates diverse biochemical processes.
Hydrogen Bonding Environment of the N3-H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins.
The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light-induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3-H group of flavin mononucleotide (FMN) of the LOV2 domain from Adiantum neochrome (neo) 1 was investigated by low-temperature Fourier transform infrared spectroscopy. Using specifically (15)N-labeled FMN, [1,3-(15)N2]FMN, the N3-H stretch was identified at 2831 cm(-1) for the unphotolyzed state at 150 K, indicating that the N3-H group forms a fairly strong hydrogen bond. The N3-H stretch showed temperature dependence, with a shift to lower frequencies at ≤200 K and to higher frequencies at ≥250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from Arabidopsis phot1 and phot2. By contrast, the N3-H stretch of the Q1029L mutant of neo1-LOV2 and neo1-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the β-sheet region and/or the adjacent Jα helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neo1-LOV1 domain. The environment around the N3-H group was also investigated.
Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.
Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.
Molecular Mechanism of Photozipper, a Light-Regulated Dimerizing Module Consisting of the bZIP and LOV Domains of Aureochrome-1.
Aureochrome-1 (AUREO1) is a blue light (BL) receptor responsible for the BL-induced blanching of a stramenopile alga, Vaucheria frigida. The AUREO1 protein contains a central basic region/leucine zipper (bZIP) domain, and a C-terminal light-oxygen-voltage-sensing (LOV) domain. BL induces the dimerization of monomeric AUREO1, which subsequently increases the affinity of this transcription factor for its target DNA [Hisatomi, O., et al. (2014) J. Biol. Chem. 289, 17379-17391]. We constructed a synthetic gene encoding N-terminally truncated monomeric AUREO1 (designated Photozipper) to elucidate the molecular mechanism of this BL-regulated transcription factor and to develop it as an optogenetic tool. In this study, four different Photozipper (PZ) protein constructs were prepared comprising different N-terminal truncations. The monomer-dimer equilibria of the PZ constructs were investigated in the dark and light states. Dynamic light scattering and size-exclusion chromatography analyses revealed that the apparent dissociation constants of PZ dimers with and without the ZIP region were ~100 and 30 μM, respectively, indicating that the ZIP region stabilized the monomeric form in the dark state. In the light state, fluorescence resonance energy transfer analyses demonstrated that deletion of the ZIP region increased the dissociation constant from ~0.15 to 0.6 μM, suggesting that intermolecular LOV-LOV and ZIP-ZIP interactions stabilized the dimeric forms. Our results suggest that synergistic interactions between the LOV and bZIP domains stabilize the monomeric form in the dark state and the dimeric form in the light state, which possibly contributes to the function of PZ as a BL-regulated molecular switch.
Connection between absorption properties and conformational changes in Deinococcus radiodurans phytochrome.
Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interact as a red-light-sensing system. In this study, size-exclusion chromatography and light-scattering techniques are combined with UV-vis spectroscopy to investigate light-induced changes in dimeric Deinococcus radiodurans bacterial phytochrome (DrBphP) and its subdomains. The photosensory unit (DrCBD-PHY) shows an unusually stable Pfr state with minimal dark reversion, whereas the histidine kinase (HK) domain facilitates dark reversion to the resting state. Size-exclusion chromatography reveals that all phytochrome fragments remain as dimers in the illuminated state and dark state. Still, the elution profiles of all phytochrome fragments differ between the illuminated and dark states. The differences are observed reliably only when the whole UV-vis spectrum is characterized along the elution profile and show more Pfr-state characteristics at later elution volumes in DrBphP and DrCBD-PHY fragments. This implies that the PHY domain has an important role in amplifying and relaying light-induced conformational changes to the HK domain. In the illuminated state, the HK domain appears partially unfolded and prone to form oligomers. The oligomerization of DrBphP can be diminished by converting the molecule back to the resting Pr state by using far-red light.
Optical control of protein-protein interactions via blue light-induced domain swapping.
The design of new optogenetic tools for controlling protein function would be facilitated by the development of protein scaffolds that undergo large, well-defined structural changes upon exposure to light. Domain swapping, a process in which a structural element of a monomeric protein is replaced by the same element of another copy of the same protein, leads to a well-defined change in protein structure. We observe domain swapping in a variant of the blue light photoreceptor photoactive yellow protein in which a surface loop is replaced by a well-characterized protein-protein interaction motif, the E-helix. In the domain-swapped dimer, the E-helix sequence specifically binds a partner K-helix sequence, whereas in the monomeric form of the protein, the E-helix sequence is unable to fold into a binding-competent conformation and no interaction with the K-helix is seen. Blue light irradiation decreases the extent of domain swapping (from Kd = 10 μM to Kd = 300 μM) and dramatically enhances the rate, from weeks to <1 min. Blue light-induced domain swapping thus provides a novel mechanism for controlling of protein-protein interactions in which light alters both the stability and the kinetic accessibility of binding-competent states.
Blue light-induced dimerization of a bacterial LOV-HTH DNA-binding protein.
With their utilization of light-driven allostery to control biochemical activities, photosensory proteins are of great interest as model systems and novel reagents for use by the basic science and engineering communities. One such protein, the light-activated EL222 transcription factor, from the marine bacterium Erythrobacter litoralis HTCC2594, is appealing for such studies, as it harnesses blue light to drive the reorientation of light-oxygen-voltage (LOV) sensory and helix-turn-helix (HTH) effector domains to allow photoactivation of gene transcription in natural and artificial systems. The protein conformational changes required for this process are not well understood, in part because of the relatively short lifetime of the EL222 photoexcited state (τ ∼ 29 s), which complicates its characterization via certain biophysical methods. Here we report how we have circumvented this limitation by creating an EL222 variant harboring V41I, L52I, A79Q, and V121I point mutations (AQTrip) that stabilizes the photoactivated state. Using the wild-type and AQTrip EL222 proteins, we have probed EL222 activation using a combination of solution scattering, nuclear magnetic resonance (NMR), and electromobility shift assays. Size-exclusion chromatography and light scattering indicate that AQTrip oligomerizes in the absence of DNA and selects for an EL222 dimer-DNA complex in the presence of DNA substrates. These results are confirmed in wild-type EL222 with a high-affinity DNA-binding site that stabilizes the complex. NMR analyses of the EL222-DNA complex confirm a 2:1 stoichiometry in the presence of a previously characterized DNA substrate. Combined, these novel approaches have validated a key mechanistic step, whereby blue light induces EL222 dimerization through LOV and HTH interfaces.
A circularly permuted photoactive yellow protein as a scaffold for photoswitch design.
Upon blue light irradiation, photoactive yellow protein (PYP) undergoes a conformational change that involves large movements at the N-terminus of the protein. We reasoned that this conformational change might be used to control other protein or peptide sequences if these were introduced as linkers connecting the N- and C-termini of PYP in a circular permutant. For such a design strategy to succeed, the circularly permuted PYP (cPYP) would have to fold normally and undergo a photocycle similar to that of the wild-type protein. We created a test cPYP by connecting the N- and C-termini of wild-type PYP (wtPYP) with a GGSGGSGG linker polypeptide and introducing new N- and C-termini at G115 and S114, respectively. Biophysical analysis indicated that this cPYP adopts a dark-state conformation much like wtPYP and undergoes wtPYP-like photoisomerization driven by blue light. However, thermal recovery of dark-state cPYP is ∼10-fold faster than that of wtPYP, so that very bright light is required to significantly populate the light state. Targeted mutations at M121E (M100 in wtPYP numbering) were found to enhance the light sensitivity substantially by lengthening the lifetime of the light state to ∼10 min. Nuclear magnetic resonance (NMR), circular dichroism, and UV-vis analysis indicated that the M121E-cPYP mutant also adopts a dark-state structure like that of wtPYP, although protonated and deprotonated forms of the chromophore coexist, giving rise to a shoulder near 380 nm in the UV-vis absorption spectrum. Fluorine NMR studies with fluorotryptophan-labeled M121E-cPYP show that blue light drives large changes in conformational dynamics and leads to solvent exposure of Trp7 (Trp119 in wtPYP numbering), consistent with substantial rearrangement of the N-terminal cap structure. M121E-cPYP thus provides a scaffold that may allow a wider range of photoswitchable protein designs via replacement of the linker polypeptide with a target protein or peptide sequence.
A predicted structure for the PixD-PixE complex determined by homology modeling, docking simulations, and a mutagenesis study.
PixD is a blue light-using flavin (BLUF) photoreceptor that controls phototaxis in the cyanobacterium Synechocystis sp. PCC6803. PixD interacts with the response regulator-like protein PixE in a light-dependent manner, and this interaction is critical for light signal transduction in vivo. However, the structure of the PixD-PixE complex has not been determined. To improve our understanding of how PixD transmits its captured light signal to PixE, we used blue-native polyacrylamide gel electrophoresis to characterize the molecular mass of a recombinant PixD-PixE complex purified from Escherichia coli and found it to be 342 kDa, suggesting that the complex contains 10 PixD and 4 PixE monomers. The stoichiometry of the complex was confirmed by Western blotting. Specifically, three intermediate states, PixD(10)-PixE(1), PixD(10)-PixE(2), and PixD(10)-PixE(3), were detected. The apparent dissociation constant for PixE and PixD is ~5 μM. A docking simulation was performed using a modeled PixE structure and the PixD(10) crystal structure. The docking simulation showed how the molecules in the PixD(10)-PixE(4) structure interact. To verify the accuracy of the docked model, a site-directed mutagenesis study was performed in which Arg80 of PixE, which appears to be capable of interacting electrostatically with Asp135 of PixD in the predicted structure, was shown to be critical for complex formation as mutation of PixE Arg80 to Asp or Ala prevented PixD-PixE complex formation. This study provides a structural basis for future investigations of the light signal transduction mechanism involving PixD and PixE.
Identification of natural and artificial DNA substrates for light-activated LOV-HTH transcription factor EL222.
Light-oxygen-voltage (LOV) domains serve as the photosensory modules for a wide range of plant and bacterial proteins, conferring blue light-dependent regulation to effector activities as diverse as enzymes and DNA binding. LOV domains can also be engineered into a variety of exogenous targets, allowing similar regulation for new protein-based reagents. Common to these proteins is the ability for LOV domains to reversibly form a photochemical adduct between an internal flavin chromophore and the surrounding protein, using this to trigger conformational changes that affect output activity. Using the Erythrobacter litoralis protein EL222 model system that links LOV regulation to a helix-turn-helix (HTH) DNA binding domain, we demonstrated that the LOV domain binds and inhibits the HTH domain in the dark, releasing these interactions upon illumination [Nash, A. I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 9449-9454]. Here we combine genomic and in vitro selection approaches to identify optimal DNA binding sites for EL222. Within the bacterial host, we observe binding at several genomic sites using a 12 bp sequence consensus that is also found by in vitro selection methods. Sequence-specific alterations in the DNA consensus reduce EL222 binding affinity in a manner consistent with the expected binding mode, a protein dimer binding to two repeats. Finally, we demonstrate the light-dependent activation of transcription of two genes adjacent to an EL222 binding site. Taken together, these results shed light on the native function of EL222 and provide useful reagents for further basic and applications research of this versatile protein.
Red/green cyanobacteriochromes: sensors of color and power.
Phytochromes are red/far-red photoreceptors using cysteine-linked linear tetrapyrrole (bilin) chromophores to regulate biological responses to light. Light absorption triggers photoisomerization of the bilin between the 15Z and 15E photostates. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Several subfamilies of CBCRs have been described. Representatives of one such subfamily, including AnPixJ and NpR6012g4, exhibit red/green photocycles in which the 15Z photostate is red-absorbing like that of phytochrome but the 15E photoproduct is instead green-absorbing. Using recombinant expression of individual CBCR domains in Escherichia coli, we fully survey the red/green subfamily from the cyanobacterium Nostoc punctiforme. In addition to 14 new photoswitching CBCRs, one apparently photochemically inactive protein exhibiting intense red fluorescence was observed. We describe a novel orange/green photocycle in one of these CBCRs, NpF2164g7. Dark reversion varied in this panel of CBCRs; some examples were stable as the 15E photoproduct for days, while others reverted to the 15Z dark state in minutes or even seconds. In the case of NpF2164g7, dark reversion was so rapid that reverse photoconversion of the green-absorbing photoproduct was not significant in restoring the dark state, resulting in a broadband response to light. Our results demonstrate that red/green CBCRs can thus act as sensors for the color or intensity of the ambient light environment.
Phycoviolobilin formation and spectral tuning in the DXCF cyanobacteriochrome subfamily.
Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.
Variations in protein-flavin hydrogen bonding in a light, oxygen, voltage domain produce non-Arrhenius kinetics of adduct decay.
Light, oxygen, voltage (LOV) domains utilize a conserved blue light-dependent mechanism to control a diverse array of effector domains in biological and engineered proteins. Variations in the kinetics and efficiency of LOV photochemistry fine-tune various aspects of the photic response. Characterization of the kinetics of a key aspect of this photochemical mechanism in EL222, a blue light responsive DNA binding protein from Erythrobacter litoralis HTCC2594, reveals unique non-Arrhenius behavior in the rate of dark-state cleavage of the photochemically generated adduct. Sequence analysis and mutagenesis studies establish that this effect stems from a Gln to Ala mutation unique to EL222 and homologous proteins from marine bacteria. Kinetic and spectroscopic analyses reveal that hydrogen bonding interactions between the FMN N1, O2, and ribityl hydroxyls and the surrounding protein regulate photocycle kinetics and stabilize the LOV active site from temperature-induced alteration in local structure. Substitution of residues interacting with the N1-O2 locus modulates adduct stability, structural flexibility, and sequestration of the active site from bulk solvent without perturbation of light-activated DNA binding. Together, these variants link non-Arrhenius behavior to specific alteration of an H-bonding network, while affording tunability of photocycle kinetics.
Tripping the light fantastic: blue-light photoreceptors as examples of environmentally modulated protein-protein interactions.
Blue-light photoreceptors play a pivotal role in detecting the quality and quantity of light in the environment, controlling a wide range of biological responses. Several families of blue-light photoreceptors have been characterized in detail using biophysics and biochemistry, beginning with photon absorption, through intervening signal transduction, to regulation of biological activities. Here we review the light oxygen voltage, cryptochrome, and sensors of blue light using FAD families, three different groups of proteins that offer distinctly different modes of photochemical activation and signal transduction yet play similar roles in a vast array of biological responses. We cover mechanisms of light activation and propagation of conformational responses that modulate protein-protein interactions involved in biological signaling. Discovery and characterization of these processes in natural proteins are now allowing the design of photoregulatable engineered proteins, facilitating the generation of novel reagents for biochemical and cell biological research.
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.
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.
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.
Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1.
Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.
The LOV domain family: photoresponsive signaling modules coupled to diverse output domains.
For single-cell and multicellular systems to survive, they must accurately sense and respond to their cellular and extracellular environment. Light is a nearly ubiquitous environmental factor, and many species have evolved the capability to respond to this extracellular stimulus. Numerous photoreceptors underlie the activation of light-sensitive signal transduction cascades controlling these responses. Here, we review the properties of the light, oxygen, or voltage (LOV) family of blue-light photoreceptor domains, a subset of the Per-ARNT-Sim (PAS) superfamily. These flavin-binding domains, first identified in the higher-plant phototropins, are now shown to be present in plants, fungi, and bacteria. Notably, LOV domains are coupled to a wide array of other domains, including kinases, phosphodiesterases, F-box domains, STAS domains, and zinc fingers, which suggests that the absorption of blue light by LOV domains regulates the activity of these structurally and functionally diverse domains. LOV domains contain a conserved molecular volume extending from the flavin cofactor, which is the locus for light-driven structural change, to the molecular surface. We discuss the role of this conserved volume of structure in LOV-regulated processes.