Toolkit Items

15N and 1H liquid-state high-resolution NMR is an assay method used to detect light-induced photo-CIDNP signals in engineered Mr4511 flavoproteins. In the cited study, it reported nuclear hyperpolarization arising from a light-driven transient paramagnetic state in variants containing tryptophan at canonical or newly introduced positions.

The A'α/Aβ gap is a structurally defined region N-terminal to the LOV2 core of Arabidopsis thaliana phototropin1. In LOV2-serine/threonine kinase polypeptides, this region contributes to blue-light signal transmission from LOV2 to kinase activation, and conserved residues Glu474 and Lys475 are required for efficient light-induced kinase activation.

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.

All-atom replica exchange discrete molecular dynamics is a computational docking method used to generate structural models of calcium and integrin binding protein 1 (CIB1) bound to α-integrin cytoplasmic tails. In the cited CIB1 study, it predicted that multiple α-integrin tails engage the same hydrophobic binding pocket on CIB1.

The Arabidopsis CRY2 photosensory domain is a light-responsive protein domain from plant cryptochrome-2 whose active-state crystal structure was determined in a tetrameric form. Structural analysis indicates that this domain undergoes photo-induced oligomerization and contains specific structural elements and residues that participate in activation.

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.

The AsLOV2-Jα photosensor is the Avena sativa LOV2-Jα light-sensing domain used in an artificial fusion with Rac1 GTPase. In the reported AsLOV2-Jα-Rac1 construct, light initiates LOV-domain structural changes that relieve steric inhibition of Rac1 and permit effector binding.

AsLOV2-Jα-Rac1 is an artificial fusion protein that connects the Avena sativa AsLOV2-Jα photosensor to the Rac1 GTPase to create a light-responsive signaling switch. Light-triggered structural changes in the LOV2 module disrupt steric inhibition of Rac1 and permit binding of the effector protein PAK1.

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.

Bacteriophytochrome (BphP) photoreceptors are biliverdin-binding protein photoreceptors that detect red and far-red light. Upon illumination, they photoconvert between spectroscopically, structurally, and functionally distinct Pr and Pfr states.

The C-terminal extension is an exceptionally long structural feature characteristic of plant cryptochromes, which are blue-light receptors containing flavin adenine dinucleotide within a photolyase homology region. Current mechanistic models place this extension downstream of light-induced flavin reduction, with signaling culminating in dissociation of the C-terminal extension.

The catalytic/ATP-binding (CA) domain is the kinase catalytic module of the light-regulated histidine kinase EL346. In the reported structural model, its activity is regulated indirectly by LOV-domain photoactivation through changes in the DHpL/CA interface that release the CA domain from an inhibited dark-state conformation.

Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG) microparticles are injectable yellow-emitting inorganic scintillators used to convert X-ray irradiation into local optical output in vivo. In the cited study, this scintillation activated the red-shifted opsins ChRmine and GtACR1, enabling remote bidirectional control of neural activity.

Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG) microparticles are injectable yellow-emitting inorganic scintillators used as implanted transducers for X-ray-driven optogenetic control. In the cited study, they converted X-ray irradiation into local light sufficient to activate ChRmine and inhibit via GtACR1, enabling bidirectional modulation of neural activity in mice.

Channelrhodopsin-2 C128T is a point mutant of ChR2 with high-resolution structural characterization and a markedly prolonged open-state lifetime relative to wild-type ChR2. Structural comparison links residue C128 in the DC gate to retinal Schiff base interactions and coupling between the DC gate and central gate.

ChR024 is a red-shifted cation-conducting channelrhodopsin that functions as a light-gated ion channel. Structural and electrophysiological analyses place it within a pump-fold channelrhodopsin architecture while supporting passive cation conductance and color tuning.

Chrimson is a red light-activated channelrhodopsin with a reported crystal structure. It provides red-shifted optogenetic excitation and has been used with Chronos to support two-color activation of independent neural populations in mouse brain slice without detectable cross-talk.

ChRmine is only mentioned in the supplied evidence as a comparator in the context of pump-fold channelrhodopsin structural analysis. The provided source does not directly describe ChRmine’s sequence, engineering, or functional performance.

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 drosophila ELK PAS domain is the N-terminal PAS domain from a Drosophila KCNH potassium channel whose high-resolution structure has been determined. Comparative structural analysis places it within the KCNH PAS-domain family and supports a role in protein-protein interactions mediated by a conserved hydrophobic patch on the PAS beta-sheet.

The Drosophila PERIOD PAS domain fragment is a dPER protein segment comprising the PAS-A and PAS-B domains. Structural and functional analyses indicate that this fragment participates in PAS-mediated protein interactions, with the PAS-B beta-sheet surface mediating heterodimer formation with Drosophila TIMELESS (dTIM).

EF-II refers to the second EF-hand region within calcium- and integrin-binding protein 1 (CIB1), with the supplied extraction specifically noting an F91W variant. The available evidence describes EF-II as part of full-length CIB1 structural dynamics rather than as a standalone engineered tool with a defined application.

EL346 is a full-length blue light-activated histidine kinase from Erythrobacter litoralis HTCC2594 that contains a LOV photosensor domain. Structural evidence indicates that the LOV domain regulates kinase state by controlling intramolecular domain interactions, maintaining an inhibited dark-state conformation, and releasing that inhibition upon photoactivation.

The elastic network model (ENM) is a computational method for analyzing protein structural dynamics from an elastic network representation. In the cited PAS domain superfamily study, ENM was used to quantify residue fluctuations and vibrational patterns and to relate these dynamic features to sequence conservation, structural features, and biological function.

Electron-electron double resonance (ELDOR) spectroscopy is a structural assay method that, when combined with site-directed spin labelling, was used to chart light-induced structural transitions in the engineered LOV histidine kinase YF1. In the cited study, it provided pairwise distance information used to model blue-light-driven quaternary rearrangements in a signaling photoreceptor.

FRASE, also described as FRASE-bot, is a computational fragment-based ligand discovery method that mines 3D ligand–protein complex structures to build a database of fragments in structural environments. It screens this database against a target protein, seeds the target structure with relevant ligand fragments, and uses a neural network to prioritize fragments with the highest likelihood of being native binders.

FRASE-bot is an in silico fragment-based hit-finding method for drug discovery against unconventional therapeutic targets. It mines thousands of 3D protein-ligand complex structures to build a fragment-in-structural-environment database, matches target protein environments to that database, and uses machine learning to prioritize seeded fragments as candidate binders.

Free-energy calculations are an in silico prediction method used in the rational design of human Caspase-2 mutants. In the cited study, they were applied alongside sequence and structural comparisons of Caspase-2 and Caspase-3 to predict effects of active-site mutations on substrate recognition and to support engineering of broader amino-acid acceptance.

The heme PAS domain of Escherichia coli direct oxygen sensor (Ec DOS) is a bacterial heme-binding sensor domain structurally characterized in inactive Fe(3+) and active Fe(2+) states. It acts as a redox-responsive molecular switch in which changes in heme coordination are coupled to conformational rearrangements within the PAS domain.

The human ERG PAS domain is the N-terminal Per-Arnt-Sim domain from the KCNH/hERG potassium channel with a reported high-resolution structure. Structural analysis indicates that it preserves canonical PAS-domain architecture, including a conserved hydrophobic beta-sheet patch implicated in protein-protein contacts and N-terminal helices linked to channel function.

Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) is an assay method used to complement structural characterization of light-activated photoreceptors. It reports on protein conformational dynamics in solution and can probe multiple functionally relevant states.

The integrin αIIb cytoplasmic domain is a platelet integrin tail segment that functions as a specific binding partner for the calcium- and integrin-binding protein CIB1. Solution structural analysis indicates that this interaction involves EF-hand III of CIB1 and is associated with a CIB1 conformational response distinct from that of related EF-hand proteins.

The Jα-helix of LOV2 is a C-terminal helix located downstream of the LOV2 domain in Arabidopsis phototropin1. Evidence from blue-light-dependent trypsin digestion indicates that this helix undergoes a light-responsive structural change and participates in intramolecular signal transmission toward the kinase region.

The KCNH channel PAS domain is an N-terminal Per-Arnt-Sim (PAS) domain found in KCNH potassium channels, including mouse EAG, Drosophila ELK, and human ERG channel proteins. Structural analysis indicates that it retains canonical PAS-domain features associated with cell signaling and contains conserved elements linked to channel function.

Light-induced Fourier transform infrared (FTIR) difference spectroscopy is an assay method for detecting light-triggered structural changes associated with signaling-state formation in photoreceptor proteins. In the cited literature, it was applied to blue-light sensing LOV and BLUF/FAD systems to measure protein- and chromophore-associated spectral changes after illumination.

The Light-Oxygen-Voltage (LOV) domain is a small blue-light-sensing protein domain used as an optogenetic input module. It binds flavin nucleotides and undergoes blue-light-induced structural rearrangements that can regulate linked effector domains, including in phototropins where LOV1 and LOV2 are coupled to a C-terminal serine/threonine kinase.

The light-oxygen-voltage (LOV) sensor domain is a light-responsive regulatory protein domain characterized in the monomeric histidine kinase EL346. Structural evidence indicates that it binds one side of the DHpL domain to control kinase output, prevent dimerization, and release the catalytic apparatus from an inhibited conformation upon photoactivation.

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.

LOV2 is a blue-light-sensing Light-Oxygen-Voltage domain from phototropins, which are multidomain plant photoreceptors containing LOV1, LOV2, and a C-terminal serine/threonine kinase domain. In this native context, light activation of LOV2 induces structural changes that are transmitted to the kinase domain to regulate signaling.

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.

Magnetic-field dependent 13C NMR spectroscopy is a solution-state photo-CIDNP assay in which 13C NMR spectra are acquired across multiple magnetic fields under light activation. In the cited 2014 study, it was applied to the LOV2-C450A domain of phototropin over 4.7–11.8 T to detect field-dependent 13C photo-CIDNP signals and support interpretation involving a novel triplet mechanism.

Markov State Modeling (MSM) is a computational method applied with molecular dynamics simulations to resolve conformational dynamics in the AsLOV2 photosensory domain. In the cited 2023 study, MSM was used to explain blue-light-induced stepwise unfolding of the C-terminal Jα-helix and to identify seven structurally distinguishable unfolding states spanning initiation and post-initiation phases.

Molecular dynamics simulations combined with Markov state modeling were used to characterize blue-light-induced conformational switching in the Avena sativa LOV2 (AsLOV2) domain. This computation method resolved C-terminal Jα-helix unfolding into seven structurally distinguishable steps spanning initiation and post-initiation phases.

Mr4511 is a light-responsive LOV domain from Methylobacterium radiotolerans that has been used as a protein scaffold for engineered flavoprotein spin machines. In designed variants, tryptophan insertion at canonical or novel positions enabled illumination-dependent nuclear hyperpolarization detectable by 15N and 1H liquid-state high-resolution NMR.

The oat phy-CAT fusion gene is a heterologous reporter construct in which the 5'-flanking sequence and part of the structural region of an oat phytochrome gene are fused to chloramphenicol acetyltransferase (CAT). It is used to measure light-regulated transcriptional activity of the oat phy promoter after transient introduction into plant cells.

The optogenetic inhibitor of c-Jun NH2-terminal kinase (JNK) is a light-controlled construct used to inhibit JNK signaling. In the cited eNeuro study, it was used to interrogate local JNK function in dendritic spine heads and to modulate stress-associated AMPAR internalization, actin reorganization, and spine structural remodeling.

Order-disorder transitions are a light-regulated design principle identified in a 2015 survey of engineered photoreceptors. In this framework, light-driven structural transitions are used as a versatile basis for building optogenetic tools within photoreceptor engineering.

The Arabidopsis thaliana phototropin 1 LOV2 domain is a blue-light-sensing protein domain from phototropin 1 whose dark-adapted crystal structure has been determined. In this state, the domain is dimeric and contains an N-terminal A'α helix and a C-terminal Jα helix that contribute to coiled-coil-mediated dimerization.

PpSB1-LOV is a bacterial short LOV photosensory domain from Pseudomonas putida KT2440 with a light-induced flavin-cysteinyl photo-adduct and exceptionally slow dark recovery. It has been characterized as a compact LOV building block whose photocycle kinetics can be tuned by conserved hydrophobic-pocket mutation, including the I48T variant that accelerates adduct rupture while remaining structurally and mechanistically benign.

The QM(B3LYP/cc-pVDZ)/MM(AMBER) approach is a hybrid quantum mechanics/molecular mechanics computational method used for geometry optimization and vibrational frequency calculations in flavin-binding photoreceptor proteins. In the cited BLUF photoreceptor study, it was used to model light-induced structural changes and associated spectral shifts.