Curated Optogenetic Publication Database

Abstract: Light-sensitive proteins allow organisms to perceive and respond to their environment, and have diversified over billions of years. Among these, Light-Oxygen-Voltage (LOV) domains are widespread photosensors that control diverse physiological processes and are increasingly used in optogenetics. Yet, the evolutionary constraints that shaped their protein dynamics and thereby their functional diversity remain poorly resolved. Here we systematically characterize the dynamics of 21 natural LOV core domains, significantly extending the spectroscopically resolved catalog through the addition of 18 previously unstudied variants. Using time-resolved spectroscopy, we uncover an exceptional kinetic diversity spanning from picoseconds to days and identify distinct functional clusters within the LOV family. These clusters reflect evolutionary branching, including a divergence of ≈1.0 billion years between investigatedLOV variants from plants and ≈0.4 billion years of separation within one of these functional clusters. Individual variants with extreme photocycles emerge as promising anchor points for optogenetic applications, ranging from highly efficient adduct formation to ultrafast recovery. Beyond natural diversity, we introduce a LOV domain generated by artificial intelligence-guided protein design. Despite being sequentially remote from its maternal template, this variant retains core photocycle function while exhibiting unique biophysical properties, thereby occupying a new region on the biophysical landscape. Our work emphasizes how billions of years of evolution defined LOV protein dynamics, and how protein design can expand this repertoire, engineering next-generation optogenetic tools.

Abstract: Cellular signaling cascades rely on transfer of information from one protein to another or within a single protein. To facilitate signal integration, specific structural motifs evolved that allow signal processing and also enable modular downstream response integration, facilitating sophisticated regulatory mechanisms. On a structural level, especially coiled-coil helices are frequently observed as signaling motifs. In diguanylate cyclases (DGCs) featuring GGDEF domains, N-terminal coiled-coils frequently activate systems by rearrangements of the interdimer active site. The variety of sensory domains that modulate this structural equilibrium in response to different stimuli highlights the importance of DGCs in bacterial adaptation. One interesting example of sensor DGCs is blue light-activated light-oxygen-voltage (LOV)-GGDEF couples. Here, we describe molecular details of a two-stage mechanism that allows tight dark-state inhibition while enabling high enzymatic activities upon illumination, achieving fold changes exceeding 10,000-fold. Using an in vivo activity assay, we screened amino acid substitutions at the inhibitory interface and the sensor-effector linker region to identify variants that promote enzymatic activity in the dark. In combination with chimeras of LOV and GGDEF domains preventing inhibitory interface formation, we successfully stabilized elongated active-state conformations and confirmed the role of the inhibitory interface between sensor and effector in the tight dark-state inhibition. Interestingly, the initially generated chimeras are still light regulatable as long as the linker sequence is not stabilized in either inhibiting or stimulating coiled-coil register. Our results offer valuable insights for potential optogenetic applications but also demonstrate inherent challenges associated with Methylotenera sp. LOV-activated DGCs.

Abstract: Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.

Abstract: Light-Oxygen-Voltage (LOV) domains are responsible for detecting blue light (BL) and regulating the activities of effector domains in various organisms. Photozipper (PZ), an N-terminally truncated aureochrome-1 protein, contains a LOV domain and a basic leucin zipper (bZIP) domain and plays a role as a light-activatable transcription factor. PZ is monomeric in the dark state and undergoes non-covalent dimerization upon illumination with BL, subsequently increasing its affinity for the target DNA. To clarify the molecular mechanism of aureochromes, we prepared site-directed mutants of PZ and performed quantitative analyses in the dark and light states. Although the amino acid substitutions in the hinge region between the LOV core and A'α helix had minor effects on the dimerization and DNA-binding properties of PZ, the substitutions in the β-sheet region of the LOV core and in the A'α helix significantly affected these properties. We found that light signals are transmitted from the LOV core to the effector bZIP domain via the hydrophobic residues on the β-sheet. The light-induced conformational change possibly deforms the hydrophobic regions of the LOV core and induces the detachment of the A'α helix to expose the dimerization surface, likely activating the bZIP domain in a light-dependent manner.

Abstract: Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.

Abstract: Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.

Abstract: Abstract not available.