Curated Optogenetic Publication Database

Abstract: We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.

Abstract: The phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two blue light sensing Light-Oxygen-Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made progress addressing these questions by utilizing small angle X-ray scattering (SAXS) and other biophysical approaches to study multidomain phots from Chlamydomonas and Arabidopsis, leading to models where the domains have an extended linear arrangement, with the activating LOV2 domain contacting the kinase domain N-lobe. We discuss this and other advances which have improved structural and mechanistic understanding of phot regulation in this review, along with the challenges that will have to be overcome to obtain high-resolution structural information on these exciting photoreceptors. Such information will be essential to advancing fundamental understanding of plant physiology while enabling engineering efforts at both the whole plant and molecular levels.

Abstract: Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

Abstract: Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Abstract: We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Abstract: Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Abstract: Here, we describe an optogenetic gene expression system optimized for use in zebrafish. This system overcomes the limitations of current inducible expression systems by enabling robust spatial and temporal regulation of gene expression in living organisms. Because existing optogenetic systems show toxicity in zebrafish, we re-engineered the blue-light-activated EL222 system for minimal toxicity while exhibiting a large range of induction, fine spatial precision and rapid kinetics. We validate several strategies to spatially restrict illumination and thus gene induction with our new TAEL (TA4-EL222) system. As a functional example, we show that TAEL is able to induce ectopic endodermal cells in the presumptive ectoderm via targeted sox32 induction. We also demonstrate that TAEL can be used to resolve multiple roles of Nodal signaling at different stages of embryonic development. Finally, we show how inducible gene editing can be achieved by combining the TAEL and CRISPR/Cas9 systems. This toolkit should be a broadly useful resource for the fish community.

Abstract: Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.

Abstract: Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.

Abstract: 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.

Abstract: Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice. Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

Abstract: Phototropins are light-activated kinases important for plant responses to blue light. Light initiates signaling in these proteins by generating a covalent protein-flavin mononucleotide (FMN) adduct within sensory Per-ARNT-Sim (PAS) domains. We characterized the light-dependent changes of a phototropin PAS domain by solution nuclear magnetic resonance spectroscopy and found that an alpha helix located outside the canonical domain plays a key role in this activation process. Although this helix associates with the PAS core in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent bond formation to kinase activation and identifies a signaling pathway conserved among PAS domains.