Structural Characterization

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.

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 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.

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.

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.

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.

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 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.

Reverse cross-saturation NMR methodology is an NMR assay approach that maps the binding interface on a larger binding partner by applying selective radio-frequency irradiation to a smaller binding partner. In the cited study, irradiation of the αIIb peptide enabled detection of the interaction surface on Ca2+-bound CIB1.

Scintillator-mediated optogenetics is an engineering method in which implanted Ce:GAGG microparticles convert X-ray irradiation into scintillation light that activates red-shifted opsins. In mice, this enabled wireless modulation of neural activity at tissue depth, including bidirectional control of midbrain dopamine neurons and associated place preference behavior.

Serial crystallography at pump-probe delays is a time-resolved structural characterization method that collects crystallographic data at defined times after light excitation. In the cited KR2 study, it was used to follow the light-driven sodium pump photocycle across ten delays spanning femtoseconds to milliseconds and capture structural intermediates over time.

Serial femtosecond crystallography is a time-resolved structural characterization assay used to track light-triggered protein photoreactions from femtoseconds to the microsecond regime. In the cited fluorescent protein study, it resolved chromophore isomerization and twisting and provided structural evidence for a hula twist photoactivation mechanism linked to beta-barrel rearrangements.

Site-directed spin labelling, used with electron-electron double resonance (ELDOR) spectroscopy, is a structural assay method for charting blue-light-induced conformational changes in proteins. In the cited study, it was applied to the engineered LOV histidine kinase YF1 to obtain distance information on light-dependent structural transitions and quaternary rearrangements.

Small-angle X-ray scattering (SAXS) is a structural characterization assay used to directly observe solution-state conformational changes in light-responsive proteins. In the cited phototropin literature, SAXS was used with other biophysical approaches to study multidomain phototropins from Chlamydomonas and Arabidopsis.

SOS-CIS(D) is a quantum-chemical excited-state calculation method used to compute vertical excitation energies. In the cited 2010 BLUF photoreceptor study, it was applied to model flavin-associated structural and spectral changes and to evaluate light-induced states.

Temperature-dependent FTIR spectroscopy is a structural characterization assay used to record conformational heterogeneity and the propagation of structural changes in the LOV2/Jα domain from Avena sativa phototropin 1. In the cited work, it functions as an infrared spectroscopic method for analyzing a light-responsive protein domain.

TiGGER is a 240 GHz time-resolved Gd-Gd electron paramagnetic resonance assay for tracking inter-residue distances during a protein mechanical cycle in the solution state. It was demonstrated on the light-responsive AsLOV2 domain to resolve time-dependent structural separation and relaxation after photoactivation.

Time-resolved infrared spectroscopy, also termed transient infrared spectroscopy, is a light-triggered functional assay that monitors vibrational and structural dynamics of LOV photoreceptors on picosecond-to-microsecond timescales. In the cited studies, it resolved FMN triplet-state progression to cysteinyl-FMN adduct formation and subsequent protein conformational changes, including Jα helix unfolding.

Time-resolved serial oscillation crystallography is a synchrotron-based, room-temperature X-ray diffraction method that collects, processes, and merges monochromatic oscillation data from fewer than 100 crystals. It was used to follow light-driven structural changes in a blue-light photoreceptor domain with 63 ms time resolution and to visualize time-dependent rearrangements of both the protein and its chromophore.

Time-resolved vibrational spectroscopy coupled with isotope labeling is an assay method used to resolve light-triggered structural dynamics in the Avena sativa LOV2 (AsLOV2) photosensory domain. In the cited study, it mapped structural evolution from 100 fs to 1 ms after optical excitation and supported a sequential allosteric model linking the flavin pocket to Jα-helix unfolding.