Toolkit Items

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.

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.

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.

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 abstract indicates an acyclovir-responsive allosteric switch.

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 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.

The review title directly names atomic force microscopy, and the supplied summary states that the review emphasizes AFM modalities for mapping dynamic mechanical properties of biological samples.

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 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.

This computational design strategy combines in silico design with biophysical experiments to improve the response kinetics of protein conformational switches. In the cited 2018 Nature Communications study, it was applied to an engineered protein-based Ca2+ sensor and rationally accelerated its response into the range of fast physiological Ca2+ fluctuations.

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.

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

Glass nanopipette-based single-cell extraction is an ex situ single-cell sampling method that removes material from individual cells for downstream analysis. In the cited Chemical Science study, it was coupled to SiNx solid-state nanopores to identify LOV2 and monitor its conformational changes from single-cell extracts.

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.

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

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

In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action.

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.

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.

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.

Co-opting natural allosteric coupling is a protein engineering method that converts proteins into conformational switches by leveraging pre-existing allosteric relationships. The cited literature describes its use to generate proteins that respond to signaling events and thereby enable biosensing or regulated biological function.

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.

Furthermore, epitope analysis reveals that linear, as opposed to conformational epitopes, are prominent; however, single amino-acid substitutions of IgE-binding sites often lead to a loss of binding to these epitopes, a finding with obvious therapeutic implications.

Imaging molecular interactions in living cells by FRET microscopy. FRET imaging provides information about all these molecular processes with high specificity and sensitivity via probes expressed by or introduced from the external medium into the cell, tissue or organism.

"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.

Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers...

Here, we review a broad spectrum of single-molecule tools and techniques such as ... nanopore tweezers...

New switching mechanisms are a protein engineering approach that introduces stimulus-responsive conformational switching into proteins that previously lacked such behavior. In the cited review, these engineered switches are described as enabling reagent-free biosensing and regulated biological function.

Here, we review a broad spectrum of single-molecule tools and techniques such as ... single-molecule fluorescence resonance energy transfer (smFRET)...

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 atomic force sensing technique is an assay method for dynamically probing protein conformational changes with microsecond time resolution. In the cited 1997 study, it was applied to light-induced conformational changes in bacteriorhodopsin.

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.