Conformational Uncaging

LiCre is a single-chain light-inducible Cre recombinase optogenetic switch. The supplied evidence supports that blue light can activate Cre-dependent recombination outputs, including induction of antibiotic resistance gene expression in Escherichia coli.

Cry2 is a blue-light photoreceptor cryptochrome from Arabidopsis used as a light-responsive multi-component optogenetic switch. The supplied evidence supports blue-light-dependent photoactivation linked to regulation of transcription factor control and to CRY2 degradation.

UNC10245092 is a previously identified linear peptide inhibitor that binds calcium and integrin binding protein 1 (CIB1). It has been used as a reference peptide in structural and in silico studies of CIB1-targeting decoy peptide design.

iLID/SspB is a blue-light-inducible heterodimerization system built from an engineered iLID module and the SspB binding partner. It is used to reversibly recruit proteins in cells for control of localization and signaling, including membrane recruitment, neurotrophin receptor construction, microtubule plus-end targeting, and perturbation of small GTPase pathways.

The abstract indicates an acyclovir-responsive allosteric switch.

The alpha-helical domain linker is a construct pattern in which a rigid alpha-helical segment is placed between fused protein domains to couple their functions. In the cited design context, it is proposed to act as a helical allosteric lever arm that transmits conformational information between domains.

Bacteriophage-derived peptides are peptide inhibitors reported to allosterically inhibit CRISPR-Cas9. Based on the supplied evidence, they act as anti-CRISPR-like modulators of Cas9 activity in genome editing contexts.

The biosensors (termed FLIPs) offer an extremely simple design, high sensitivity, multiplexing capability, ratiometric readout, and other advantages, without requiring modifications to their targets.

We investigated the G-quadruplex (G4) binding selectivity of short aromatic oligoamide helical foldamers comprising quinoline (Q) and pyridine (P) units... Mixed quinoline-pyridine foldamers are thus a promising class of selective G4 ligands

Here we achieve fully reversible in-situ mechano-optoelectronic switching in self-assembled monolayers (SAMs) of tetraphenylethylene molecules by bending their supporting electrodes to maximize aggregation-induced emission (AIE).

Here we use genetic code expansion and bioorthogonal labelling to generate a panel of fluorescence-based biosensors for a prototypical GPCR, the M2 muscarinic acetylcholine receptor (M2R). These biosensors enable real-time monitoring of agonist-promoted conformational changes across the receptor's extracellular surface in intact cells.

LOV2-based photoswitches are optogenetic switches engineered from the LOV2 photoreceptor domain to control biological activities with light. They repurpose endogenous light-induced conformational changes in LOV2 to generate new cellular outputs and have been developed on the basis of detailed biophysical characterization of the isolated domain.

we previously developed a Francisella novicida Cas12a variant (FnoCas12a) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH)

Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions.

The development of constrained peptides represents an emerging strategy to generate peptide-based PPI inhibitors, typically mediated by α-helices.

"Joining proteins in creative ways" is a protein engineering construct pattern in which protein domains are fused or otherwise combined to create stimulus-coupled conformational switching in proteins that previously lacked such behavior. The cited literature presents this strategy as a route to generate switchable proteins for biosensing and regulated biological function.

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.

a spatially controllable strategy via the photo-patterned uncaging of an o-nitrobenzyl-caged fluorescein conjugate

we developed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate cells while preserving viability

The web research summary states that the anchor paper describes MRS7145 as the first light-dependent adenosine A2A receptor antagonist and identifies it as the photo-controlled/photocaged adenosine A2A receptor antagonist used for light-dependent blockade in cells and behaving animals.

Here we report the 2.7 Å-resolution cryo-electron microscopy structure of a ChR from Klebsormidium nitens (KnChR), which is one of the most blue-shifted ChRs.

Light-regulated protein-protein interaction is a construct pattern in optogenetics that controls signaling protein function by coupling light input to regulated protein-protein interactions or to caging/uncaging events. It is presented as a general strategy for the spatiotemporal control of signal transduction pathways.

LOV-based optogenetic devices are engineered tools built from small, light-responsive LOV modules to confer photoregulated control of cellular signaling. The supplied evidence supports their role as a general platform for optogenetic tool construction but does not specify individual device architectures or target proteins.

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 two-input protein logic OR gate is an engineered single-protein focal adhesion kinase (FAK) system designed to integrate chemical and optical inputs within the native FAK domain architecture. It functions as an allosterically regulated OR gate by combining a rapamycin-inducible uniRapR module in the kinase domain with a light-inducible LOV2 module in the FERM domain.

Our predictions guided the engineering of four variants (R20A, R22A, D103A, and R20A-R22A), all of which impaired GAL-7-induced apoptosis in human T cells.

A protein conformational switch is an engineered protein system in which a signaling event induces a conformational change. Reported uses include reagent-free biosensing and regulation of biological function.

Self-assembled proteomimetic (SAP) is a multi-component binding scaffold built from short PNA-peptide conjugates that assemble into a proteomimetic three-helix-bundle-like architecture. In the cited study, SAPs were generated against HER2 and the SARS-CoV-2 spike receptor-binding domain, and an RBD-targeting SAP inhibited viral entry with an IC50 of 2.8 nM.

The AUREO1 bZIP-LOV truncated construct (ZL) is an N-terminally truncated aureochrome-1 derivative that retains the bZIP DNA-binding region and the LOV photosensory domain. It binds DNA in a sequence-specific manner and undergoes a blue-light-induced conformational response measurable as an approximately 5% increase in hydrodynamic radius without a detectable change in secondary structure.

BLISS is a blue-light-inducible SpyTag system generated by inserting SpyTag into different positions of the AsLOV2 Jα-helix. In this design, blue light exposure enables conditional SpyTag reactivity with SpyCatcher, allowing light-gated coupling.

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.

Hybrid phototropin LOV2 domains were engineered to incorporate the BID Bcl homology region 3 (BH3), creating a light-dependent optogenetic switch. Illumination induces LOV2 conformational changes that expose the BH3 element and modulate binding to the anti-apoptotic Bcl-2 family protein Bcl-xL.

LOV-LexA is a light-gated LexA-based expression system for Drosophila that fuses the bacterial LexA transcription factor to a plant-derived LOV photosensitive domain and a fluorescent protein. Blue light uncages a nuclear localization signal, drives nuclear translocation, and initiates LexAop transgene expression with spatial and temporal control.

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.

Caging/uncaging events are optogenetic control strategies in which light is used to regulate the functional state of signaling proteins. In the cited review, they are presented as one of two broad routes, alongside light-regulated protein-protein interactions, for steering signal transduction.

Since crystal structures of both phytochromes are known, the phytochrome system of A. fabrum provides a tool for following the entire signal transduction cascade starting from light induced conformational changes to protein interaction and the triggering of DNA transfer processes.

Allosteric transcription factor (aTF)-based in vitro biosensors constitute a class of detection tools formed by the functional coupling of the ligand-binding domain of aTFs with a reporter system.

We present the cell-free recombinase-integrated Boolean output system (CRIBOS), a site-specific recombinase-based multiplex genetic circuit platform designed for cell-free environments.

Allosteric and dualsteric photoswitchable ligands, the latter targeting both orthosteric and allosteric sites, are being developed.

Here, we report the design of a protease-responsive conformationally inhibited system (PRCIS). By intramolecularly linking the free DNA-binding domains of ATF to confined dimerized regions, the transcriptional binding is conformationally inactivated. The function of DNA binding is reinstated upon proteolytic cleavage of linkages, activating the downstream gene expressions.

AsLOV2 is the light-oxygen-voltage 2 photosensory domain from Avena sativa phototropin 1 used as a blue-light-responsive actuator in engineered fusion proteins. Blue-light activation drives allosteric conformational extension involving sequential unfolding of the N-terminal A'α helix and the C-terminal Jα helix, enabling conformational uncaging and related optogenetic control.

SspB is the binding partner used in the iLID blue-light-inducible dimerization system. Upon blue-light activation of iLID, the exposed SsrA peptide binds SspB, enabling light-controlled recruitment and localization of SspB-fused cargo proteins.

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.

Light-harvesting complex II (LHCII) is the major chlorophyll a/b-binding photosynthetic antenna complex of plants that has been studied in isolated native and recombinant forms. The cited literature indicates that light induces reversible conformational changes in LHCII that expose its N-terminal phosphorylation site and can also promote formation of dimeric LHCII states with distinct chlorophyll excitation-quenching properties.

YtvA is a blue-light-sensing LOV-STAS photoreceptor from Bacillus subtilis whose LOV domain has been structurally analyzed for LOV-LOV dimerization and interdomain interactions. Homologous mutations in a conserved LOV hydrophobic pocket alter activation-state kinetics, supporting YtvA as a tunable LOV sensor domain relevant to optogenetic design.

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.

The Avena sativa LOV2 domain is a blue-light-sensing photosensory domain used as a photoswitchable scaffold for engineered control of protein interactions. In the iLID design, the bacterial SsrA peptide is embedded in the LOV2 C-terminal helix so that blue light triggers helix undocking and enables binding to SspB.

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.

The Avena sativa phototropin-1 LOV2 domain is a blue-light-sensing flavin-binding photosensory domain used as a module for light-controlled conformational uncaging. Available evidence shows that its dark-state recovery follows a base-catalyzed mechanism and that its light responsiveness is influenced by the flavin redox state.

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

Here, we describe the development of a sequence-activated fluorescent RNA (SaFR) technique.

The Aer PAS domain is the FAD-binding sensory domain from the dimeric Escherichia coli aerotaxis receptor Aer. It monitors cellular respiration through a redox-sensitive flavin cofactor and is structurally characterized in the Aer-PAS-GVV variant at 2.4 Å resolution.

The CIB1 helix 10 pocket is a ligandable site in calcium and integrin binding protein 1 (CIB1) identified as the binding site for the peptide inhibitor UNC10245092. In the cited 2020 ACS Chemical Biology study, UNC10245092 bound this pocket with low nanomolar affinity and acted as a first-in-class chemical tool site for CIB1 inhibition in cell culture.

The LOV2 domain C450A variant is a mutant form of the LOV2 photosensory domain that has been examined for its conformational properties across a broad pH range. The available evidence supports biophysical characterization of this variant rather than a demonstrated engineered application.

SaFR undergoes conformational reorganization and transforms into the fluorogenic conformation of Pepper, enabling the activation of fluorophores to produce fluorescent signals.

Anion channelrhodopsins (ACRs) are natural light-gated anion-conducting microbial rhodopsins from cryptophyte algae used as optogenetic actuators. In cultured neonatal rat ventricular cardiomyocytes, their expression enables light-evoked inhibitory currents, suppression of electrical activity, and shortening of action potential duration when illumination is applied during repolarization.

This review describes the means by which to achieve these goals using nucleobase replacement or modification with advanced fluorescent dyes that respond by the changing of their fluorescence parameters to their local environment.

FtsZ is a prokaryotic filamentous cell-division protein that was adapted as a light-responsive protein domain by site-specific incorporation of a photocaged tyrosine. In this engineered form, UV-mediated uncaging at tyrosine 222 was used to control FtsZ self-organization, GTPase regulation, and treadmilling-related dynamics.

The LHCII N-terminal domain is the region of the light-harvesting complex II chlorophyll-protein substrate that contains the phosphothreonine site. In thylakoid studies, illumination induces a reversible conformational change that increases exposure of this N-terminal region, enabling access by endogenous thylakoid protein kinase(s) and increasing susceptibility to tryptic cleavage.

Light-Oxygen-Voltage (LOV) domains are small, light-responsive protein modules found in algae, plants, bacteria, and fungi that function as blue-light sensors controlling cellular responses to light. They are presented as a platform for constructing optogenetic tools that confer photoregulated control of cellular signaling.

LOV1 is a blue-light-sensing Light-Oxygen-Voltage photosensor domain from Chlamydomonas reinhardtii that occurs in phototropin receptors. In phototropins, LOV1 is paired with LOV2 and a C-terminal serine/threonine kinase domain within a multidomain light-regulated signaling protein.

NC80 is an 80-residue motif from Arabidopsis CRY2 that is sufficient to confer CRY2 physiological function. Source evidence indicates that blue light activates CRY2 by a phosphorylation-associated conformational change that derepresses the NC80 motif.

Phosphorothioate-caged antisense oligonucleotides are mixed-backbone antisense oligonucleotides in which phosphorothioate linkages are modified with 2-nitroveratryl photocages. In the caged state, these modifications suppress target RNA duplex formation and RNase H activity, and UV uncaging restores antisense function to enable light-controlled knockdown of cell-free protein synthesis.

The photo-N-degron is a peptide tag for optogenetic control of protein function in vivo through light-mediated protein degradation. It was reported to direct light-dependent degradation in Saccharomyces cerevisiae and Drosophila melanogaster, including light-dependent loss of Cactus function in developing Drosophila embryos.

Photoswitchable inhibitory peptides are LOV2-based, light-dependent analogues of kinase inhibitory peptides engineered to control kinase inhibition with illumination. In the reported implementation, inhibitory peptides appended to the LOV2 Jα helix were used to modulate endogenous signaling in living cells, including systems targeting protein kinase A and myosin light chain kinase.

Zdk2-AsLOV2 is an optogenetic protein-domain construct built around the Avena sativa LOV2 photosensory module. The supplied evidence supports blue-light activation through canonical AsLOV2 photochemistry, including cysteinyl-FMN adduct formation and Jα-helix unfolding, but does not describe the specific functional role contributed by the Zdk2 fusion in cells.

EF-IV is the C-terminal EF-hand IV region of calcium- and integrin-binding protein 1 (CIB1), represented here with the F173W variant. Available evidence indicates that this region is partially unfolded and dynamic in apo CIB1, and becomes more ordered in Mg2+-bound CIB1 while retaining some conformational heterogeneity.

The Avena sativa phototropin LOV2 domain is a recombinant blue-light-sensing protein domain that binds flavin mononucleotide (FMN) and undergoes a reversible light-triggered conformational change. Blue light induces formation of a cysteinyl-FMN adduct, and the adduct spontaneously reverses in the dark, enabling optomechanical signal transduction.

The LOV2/Jα domain from Avena sativa phototropin 1 is a light-responsive protein domain examined for conformational heterogeneity and propagation of structural changes. The supplied evidence specifically supports its structural characterization by temperature-dependent FTIR spectroscopy.

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 AUREO1 LOV-only construct is an isolated LOV photosensory domain derived from aureochrome-1. In the cited study, blue light induced a secondary-structure shift from increased alpha-helical to increased beta-sheet character in this LOV-only construct without a detectable change in hydrodynamic radius.

Optical control elements can be classified according to their molecular reversibility as non-reversible phototriggers where light breaks a chemical bond (e.g. caged ligands)

The supplied web research summary identifies pdDronpa as a photodissociable dimeric Dronpa component used to cage and uncage protein activity in photoswitchable kinases.

Allosteric and dualsteric photoswitchable ligands ... are being developed ... The novel ligands can photocontrol even more refined GPCR functions, like signal bias and degrees of partial agonism.

Photoswitchable glycoconjugates have emerged as promising investigational tools for probing carbohydrate-protein recognition and for controlling bacterial adhesion.

The SiNx solid-state nanopore single-cell protein analysis approach combines glass nanopipette-based single-cell extraction with silicon nitride nanopore sensing for single-molecule protein analysis from complex cellular samples. In the cited Chemical Science study, this workflow identified the LOV2 protein and monitored its conformational changes directly from single-cell extracts.

The cis- and trans-isomers of one of our peptidomimetics, termed TOP271, exhibit a four-fold difference in NPR-A mediated cGMP synthesis in vitro.