Toolkit Items

Avena sativa LOV2 domain variants are engineered insertion modules used to build thermosensitive allosteric chimeric proteins. In Escherichia coli, insertion of optimized LOV2 variants into diverse, structurally and functionally unrelated proteins produced potent thermoswitchable variants operating within a narrow 37-41 °C range.

Thermo-modulated CRISPR-Cas genome editors are engineered CRISPR-Cas constructs in mammalian systems whose genome-editing activity is directly modulated by subtle temperature changes within the physiological range. The reported work describes these as the first CRISPR-Cas genome editors with direct temperature responsiveness.

AsLOV2-Jα is the light-oxygen-voltage-2/Jα photoswitch domain from Avena sativa phototropin1. In the reported LOV-TAP fusion, ligation of AsLOV2-Jα to TrpR enables light-dependent control of DNA binding through photoinduced structural and electrostatic changes.

Human inward rectifier K+ channel Kir2.1 was used as a protein scaffold to identify engineerable allosteric sites through domain insertion permissibility mapping. Insertion of light-switchable domains into existing or latent allosteric sites, but not other positions, rendered Kir2.1 activity sensitive to light.

Phototropin is a plant blue-light receptor protein, exemplified by Avena sativa PHOT1/NPH1, that contains two FMN-binding LOV domains and a C-terminal serine/threonine kinase domain. It acts as a light-activated kinase in which LOV2-mediated conformational changes are coupled to kinase activation and signaling.

Its abstract indicates a mammalian-cell RNA condensate engineering platform built from nanostar-derived RNA scaffolds.

The abstract indicates an acyclovir-responsive allosteric switch.

Domain insertion permissibility is an experimental engineering paradigm established in the human inward rectifier K+ channel Kir2.1 to identify engineerable allosteric sites. In this framework, sites permissive to insertion of regulatory domains can be converted into functional control points, including light-sensitive regulation when light-switchable domains are inserted.

PS Intein is a photoswitchable intein engineered by allosterically modulating the small autocatalytic gp41-1 intein with a tandem Vivid photoreceptor. It enables light control of covalent protein binding and cleavage in cells.

The CRY2 C-terminal tail from Arabidopsis CRY2 contains an 80-residue NC80 motif that is sufficient to confer CRY2 physiological function. Evidence from transgenic plant studies indicates that this region participates in blue light-responsive regulation through phosphorylation-linked derepression of NC80.

The Diaphanous Autoregulatory Domain from mDia1, in this tool context, is an optogenetic fusion between the Avena sativa Phototropin1 LOV2 domain and an isolated mDia1 DAD. Blue light uncages the DAD, enabling rapid activation of endogenous diaphanous-related formins and acute actin cytoskeletal remodeling.

The light-oxygen-voltage 2 (LOV2) domain of Avena sativa Phototrophin1 was used as a blue-light-responsive caging module by fusion to an isolated Diaphanous Autoregulatory Domain (DAD) from mDia1. In this configuration, the LOV2-based construct was inactive in the dark and rapidly activated endogenous diaphanous-related formins upon blue-light illumination, producing acute actin cytoskeletal remodeling.

We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses.

The LOV2 module is a light-inducible protein domain incorporated into the FERM domain of an engineered focal adhesion kinase (FAK). In the reported 2021 Nature Communications system, it provides optogenetic input to an allosterically regulated single-protein two-input OR gate while preserving overall FAK domain architecture.

The photoactivatable inhibitor for cyclic-AMP dependent kinase (PKA) is a LOV2-based photoswitchable inhibitory peptide engineered to control endogenous PKA activity with light in living cells. It functions by coupling a kinase-inhibitory peptide to the LOV2 Jα helix so that inhibition is light dependent.

TrpR is the Escherichia coli tryptophan-repressor protein used as the DNA-binding module in the artificial light-responsive fusion protein LOV-TAP. In this construct, TrpR is ligated to the AsLOV2-Jα photoswitch to confer light-regulated control of DNA association.

The uniRapR module is a rapamycin-inducible protein domain inserted into the kinase domain of engineered focal adhesion kinase (FAK). In the reported 2021 system, it provided allosteric chemical control as one input of a single-protein two-input OR gate that also contained a light-responsive LOV2 module in the FERM domain.

A chemoreceptor domain was incorporated as an alternative thermosensing module in modular thermoresponsive allosteric proteins. In this context, the domain is used to confer temperature-responsive control, supporting receptor domains as interchangeable thermosensory elements.

A chemoreceptor domain was reported as an alternative thermosensing module in a modular thermo-responsive allosteric protein engineering framework. The available evidence indicates that this domain can be incorporated into engineered proteins to confer temperature-dependent control.

Linker-mediated LOV fusion to enzyme target sites is a protein engineering strategy in which a LOV photoreceptor domain is fused to functional sites within an enzyme effector using an appropriate linker. The reported goal is to retain effector functionality while enabling light-dependent modulation of enzyme activity, thereby creating light-controllable biocatalysts.

Synthetic biology approaches for opto-protein development are protein engineering strategies for creating and optimizing non-neuronal light-regulatable proteins. The cited review describes modifying photosensitive domains and fusing them to effector domains to generate light-controllable protein functions.

Allosteric Cre regulation with NS3 ligands is a chemical multi-component recombination switch in which an NS3-based ligand-responsive system is used to allosterically regulate Cre recombinase. It was reported as an orthogonal recombination control strategy in eukaryotic cells and as a way to control prokaryotic recombinase activity across divergent organisms.

The dimerization/histidine phosphotransfer-like (DHpL) domain is a regulatory domain element within the blue-light-responsive histidine kinase EL346. Structural evidence indicates that, in the dark, interactions involving the LOV sensor domain and the DHpL domain stabilize an inhibited kinase conformation and suppress dimerization, while photoactivation weakens these contacts to promote activation.

The Vivid (VVD) LOV domain is a photosensitive allosteric light, oxygen, voltage domain from a fungal circadian clock photoreceptor. It responds to blue-light-driven covalent bond formation with a large N-terminal conformational change, and its atomistic allosteric mechanism has been analyzed computationally.

The DHFR/LOV2 fusion is an engineered photoswitch in which the LOV2 light-sensing module was used to create a light-regulated dihydrofolate reductase (DHFR) enzyme. Source evidence indicates that light activation modulates DHFR catalysis through allosteric effects associated with local disorder and altered transition-state thermodynamics.

The engineered focal adhesion kinase (FAK) is a single-protein, two-input logic OR gate that integrates chemogenetic and optogenetic control within the native FAK domain architecture. It places a rapamycin-inducible uniRapR module in the kinase domain and a light-inducible LOV2 module in the FERM domain to allosterically regulate FAK activity.

LOV-TAP is an artificial light-activable allosteric protein constructed by ligating the AsLOV2-Jα photoswitch to the tryptophan repressor TrpR. It is designed to regulate protein-DNA association by coupling light-triggered changes in the LOV module to structural and electrostatic changes in the interdomain region that alter DNA binding.

OptoCRAC is an optogenetic calcium-signaling platform built from engineered CRAC-channel components, including OptoSTIM1, to control Ca2+ influx with light. It has been used to regulate Ca2+/NFAT-dependent transcription and to reprogram endogenous gene expression when coupled to CRISPR/Cas9.

OptoORAI1 is a photoswitchable CRAC channel engineered from ORAI1 by insertion of a LOV2 photosensory domain into an ORAI1 loop region. In this design, LOV2 functions as an allosteric light-responsive switch that opens the channel, enabling optical control of calcium signaling.

The photoactivatable diaphanous autoregulatory domain is an optogenetic fusion of the Avena sativa Phototropin1 LOV2 domain to an isolated mDia1 diaphanous autoregulatory domain. In blue light, this caged construct rapidly activates endogenous diaphanous-related formins, whereas it is inactive in the dark.