Curated Optogenetic Publication Database

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: Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometre precision within cells. While light-actuated chemicals such as photolabile 'caged' compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.

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: Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.

Abstract: Photoreceptor flavoproteins of the LOV, BLUF, and cryptochrome families are ubiquitous among the three domains of life and are configured as UVA/blue-light systems not only in plants-their original arena-but also in prokaryotes and microscopic algae. Here, we review these proteins' structure and function, their biological roles, and their evolution and impact in the living world, and underline their growing application in biotechnologies. We present novel developments such as the interplay of light and redox stimuli, emerging enzymatic and biological functions, lessons on evolution from picoalgae, metagenomics analysis, and optogenetics applications.

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: LOV (light, oxygen or voltage) domains are protein photosensors that are conserved in bacteria, archaea, plants and fungi, and detect blue light via a flavin cofactor. LOV domains are present in both chemotrophic and phototrophic bacterial species, in which they are found amino-terminally of signalling and regulatory domains such as sensor histidine kinases, diguanylate cyclases-phosphodiesterases, DNA-binding domains and regulators of RNA polymerase σ-factors. In this Review, we describe the current state of knowledge about the function of bacterial LOV proteins, the structural basis of LOV domain-mediated signal transduction, and the use of LOV domains as genetically encoded photoswitches in synthetic biology.

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.