Structural Characterization

Focused ultrasound (FUS) is a noninvasive physical delivery and control modality that penetrates deep biological tissues and induces confined mild hyperthermia to activate heat-sensitive genetic modules. In the cited 2023 study, FUS was coupled to heat-sensitive CRISPR, CRISPRa, and CRISPRi systems to enable remote spatiotemporal regulation of genome and epigenome function in live cells and animals.

As a tool component, CIB1 is most directly supported as the cryptochrome-interacting bHLH partner used with CRY2 to create blue-light-controlled protein association systems. In these systems, CIB1 is typically fused to localization, transcriptional, or enzymatic modules so that blue light drives CRY2–CIB1 binding and light withdrawal reverses the interaction.

Subsequently, we delve into cutting-edge applications of nanoparticles to enhance immune protection, including mosaic and cocktail nanoparticle vaccines, surface-modified targeting strategies, and the integration of mRNA technology with virus-like particles (VLPs).

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.

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.

Prime editing is mentioned in the cited review as part of the broader set of genome-editing approaches considered in bacterial genome engineering. The supplied evidence does not describe its molecular architecture, target scope, or editing outcomes.

Three-dimensional (3D) bioprinting is a rapidly evolving technology that uses complementary biomaterials to emulate native extracellular matrices, enabling the generation of finely patterned, multicellular tissue architectures.

Key ACT modalities include chimeric antigen receptor (CAR) T cells, tumor-infiltrating lymphocytes (TILs), and T cell receptor (TCR)-engineered T cells.

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.

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.

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.

Optical imaging methods covered in this review include... Raman spectroscopy for early-stage cancer detection.

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.

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.

Lipid-polymer hybrid nanoparticles (LPHNPs) are the next-generation nanocarriers that integrate the mechanical strength and sustained-release capacity of polymeric cores with the biocompatibility and high drug-loading efficiency of lipid shells.

Molecular dynamics simulation is a computational method for modeling atomistic conformational dynamics of proteins and analyzing residue fluctuations and vibrational behavior. In the cited studies, it was used as a noninvasive approach to validate dynamic behavior and to compare PAS-domain dynamics across functional groups.

Nanobody-based CAR-T cells further expand design versatility, offering improved stability, tumor penetration, and reduced immunogenicity compared with single-chain variable fragment constructs.

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.

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 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 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.

PpSB2-LOV is a compact "short" light, oxygen, voltage (LOV) photosensory protein from Pseudomonas putida KT2440. It forms a light-induced LOV photoadduct and exhibits rapid dark-state thermal recovery, with a reported recovery time of 3.5 min at 20 °C, making it a candidate building block for genetically encoded photoswitches.

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.

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.

H2B-tKR is a chromatin-targeted phototoxic construct in which histone H2B is fused to tandem KillerRed to control cell division with green light. Upon illumination, it induces nuclear damage-associated mitotic defects, including chromosome nondisjunction during metaphase, and can transiently block proliferation.

their effects on renal functional and structural indices were compared at week 15 with those of diabetic DBA/2J recipients of vehicle alone or of empty vector-transduced DBA/2J ADSCs (EV-ADSCs).

Krokinobacter eikastus rhodopsin 2 (KR2) is the prototypical light-driven sodium pump. Upon illumination, it actively transports small cations across cellular membranes and has been described as a useful optogenetic tool with applications in neuroscience.

CRISPR activation and interference (CRISPRa/CRISPRi) is a CRISPR-based gene regulation method that uses Nab2- or Egr3-targeted single-guide RNAs to modulate transcription. In the cited 2022 study, these tools were used in Neuro2a cells to mimic bidirectional cocaine-associated expression changes in Nab2 and Egr3.

AcrIIC3-LOV2 is an engineered light-switchable anti-CRISPR protein formed by fusing the Neisseria meningitidis Cas9 inhibitor AcrIIC3 to the Avena sativa LOV2 blue-light sensory domain. In mammalian cells, two reported hybrids strongly inhibit NmeCas9 in the dark and allow robust genome editing under blue-light irradiation.

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 resulting aminated-hyaluronic acid (HAA) scaffolds act as rigid structural backbones in virus-inspired polymer-DNA nanoparticles termed as "Skeletoplexes"

Here, we constructed a hybrid gene cluster of the structural gene cluster from Serratia sp. ATCC 39006 and the accessory gene cluster from Bacillus megaterium in Escherichia coli to synthesize a novel gene-encoded gas vesicle ... termed as ARGS1B.

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.

We describe the structural features, including solubility, chemical resistance, and modularity that facilitate the advanced development of bispecific, trivalent, and logic-gated CAR forms.

BROAD is a computational protein design method that combines Rosetta-based structure modeling, machine learning, and integer linear programming to improve design search beyond Rosetta sampling alone. It was demonstrated in antibody design to increase the predicted HIV neutralization breadth of VRC23 across a panel of 180 divergent viral strains.

Caspase-2 active-site mutants are engineered variants of human Caspase-2 designed in silico to broaden substrate recognition at the substrate N-terminal amino acid position. In vitro experiments confirmed that two proposed mutants showed enhanced promiscuity, including increased recognition of branched amino acids relative to unmutated Caspase-2.

The supplied review summary explicitly mentions CLARITY for correspondence between CBV-weighted signal spread and microvascular architecture.

the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS)

Here, we introduce an approach for visualizing individual microrobots in real time with color flow mapping ultrasound imaging based on acoustically induced structural oscillations of the microrobot generating a pseudo-Doppler signal.

Here, we report the X-ray crystal structure of plasmid-encoded Csep1P... Transcriptomic analysis revealed that Csep1P induced a chemokine-dominant inflammatory state in macrophages.

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 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).

The EF-III metal-binding site of calcium- and integrin-binding protein 1 (CIB1) is an EF-hand domain characterized as a mixed Mg2+/Ca2+-binding site. Mutational analysis of its metal-coordinating loop indicates that EF-III modulates CIB1 affinity for the integrin alphaIIb domain and tunes metal sensitivity into a range relevant to intracellular Ca2+ sensing.

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.

Through the integration of computational structural biology and enzyme channel engineering, this study successfully elucidated the key intermediates in the stepwise hydroxylation-cleavage catalytic process of Digitalis purpurea-derived DlCYP87A enzyme. Building on this foundation, we implemented structure-guided rational design to precisely engineer the substrate channel and catalytic pocket.

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.

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.

NoV Virus-like particles (VLPs) composed of the major capsid protein VP1 (~60 kDa) are essential for vaccine development... VP1 from four epidemiologically relevant GI genotypes was expressed using the silkworm-baculovirus system.

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.

We describe the structural features, including solubility, chemical resistance, and modularity that facilitate the advanced development of bispecific, trivalent, and logic-gated CAR forms.

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.

Molecular dynamics is a computational method used to study signaling mechanisms of LOV domains through simulation-based analysis. In the cited literature, it functions as an in silico approach for mechanistic investigation rather than as a biological reagent or genetically encoded tool.

Pseudovirus technology, which uses single-round infectious viral particles lacking replication competence, has thus gained prominence as a safe and versatile tool for antiviral research.

The PAS domain superfamily comprises widespread sensory/regulatory protein domains present in archaea, bacteria, and eukaryota. Available evidence characterizes PAS domains through combined sequence, structure, and dynamics analysis, showing that functional groups can be differentiated by conserved-residue fluctuation patterns and statistically distinct vibrational behaviors.

The same peptide can function as ... a structural component of peptide-drug conjugates (PDCs) ... Additionally, we summarize their application in peptide-drug conjugates (PDCs) ...

Additionally, we summarize their application in ... peptide-guided radionuclides ...

We identified the lipin-like sequence of Nir1 (PILS-Nir1) as a candidate PA biosensor based on structural analysis of Nir1's LNS2 domain.

RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively.

RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively.

The RhoPDE C-terminal phosphodiesterase catalytic domain is the isolated catalytic region of the rhodopsin/phosphodiesterase fusion protein RhoPDE from the choanoflagellate Salpingoeca rosetta. It has been expressed, purified, and structurally characterized by crystallography, while the parent full-length protein was reported to function as a cGMP-selective phosphodiesterase.

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.

The supplied source summary states that the review explicitly covers SMLM, including STORM/dSTORM.

We describe the structural features, including solubility, chemical resistance, and modularity that facilitate the advanced development of bispecific, trivalent, and logic-gated CAR forms.

Codon-optimized full-length Cap and putative NLS-deleted dCap (residues 1-36) were expressed in Escherichia coli. Full-length Cap self-assembled into 13-17 nm icosahedral VLP, structurally consistent with native virions.

Catharanthus roseus Extract-Loaded Zn-Substituted Hydroxyapatite Nanocomposites as a Multifunctional Antioxidant and Anticancer Therapeutic Applications

bioengineered cell membrane-coated nanoparticles (CMNPs) that combine synthetic cores with natural membranes from diverse source cells

Species D adenoviruses, such as human adenovirus type 10 (HAdV-D10), are promising candidates due to low seroprevalence in humans... support the advancement of HAdV-D10 as a next-generation platform for gene delivery and vaccine development.

Here, we consider the potential of macrocyclic peptides to overcome this limitation.

A crystal structure of the tetramer QQPQ with the parallel G4 formed by dTGGGTTGGGTTGGGTTGGGT shows two quinoline subunits interacting with an external G-quartet through c0-stacking, and solution nuclear magnetic resonance (NMR) confirms that the foldamer targets the 3' and 5' ends of this G4.

YF1 is an engineered light-oxygen-voltage (LOV) histidine kinase that acts as a blue-light-regulated signaling switch. Available evidence indicates that blue-light input is transmitted through structural transitions in the photosensor and linker regions that control its signaling state.

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.

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.

These were confirmed by Cryo-EM.

evaluated by structural characterization, DPPH antioxidant assay, antibacterial testing against Staphylococcus aureus and Escherichia coli, and MTT cytotoxicity testing

evaluated by structural characterization, DPPH antioxidant assay, antibacterial testing against Staphylococcus aureus and Escherichia coli, and MTT cytotoxicity testing in MG-63/HOS osteosarcoma cells

Explicitly supported component/tool names from the sources include zinc-substituted hydroxyapatite (Zn-HA), hydroxyapatite (HA/HAp), Catharanthus roseus extract, DPPH assay, MTT assay, MG-63/HOS cells, XRD, and tuna/fish-bone-derived hydroxyapatite.

It reports a 2026 primary study on zinc-substituted hydroxyapatite (Zn-HA) nanoparticles prepared from tuna fish bone waste, then functionalized with Catharanthus roseus flower extract

We propose the concept of the virophore: a genetically or chemically encoded functional unit integrated into the structure of a virus particle that acts as a programmable structural switch, enabling reversible, triggerable reconfiguration in response to defined stimuli.

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.

Biosensing is mentioned only as an emerging assay-related strategy expected to shape future directions in the field. The supplied evidence does not define a specific biosensor modality, analyte, or experimental implementation.

This review is focused on smaller multivalent structures such as glycoclusters emphasizing carbohydrate-centered and heteromultivalent glycoconjugates.

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.

One way to improve these properties is to constrain the secondary structure of linear peptides by cyclisation. Herein we review various classes of cyclic and macrocyclic peptides as chemical probes of protein surfaces and modulators of PPIs.

protons in lipids and proteins have been selectively replaced by deuterons and the resultant deuterium NMR spectrum analyzed to give structural and dynamic information about the molecular associations between a range of membrane components

The supplied source summary states that the review explicitly covers SMLM, including STORM/dSTORM.

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.

From a variety of initial designs two have emerged as promising prototypes for further optimization: FRET (Förster Resonance Energy Transfer)-based sensors and single fluorophore sensors of the GCaMP family.

From a variety of initial designs two have emerged as promising prototypes for further optimization: FRET (Förster Resonance Energy Transfer)-based sensors and single fluorophore sensors of the GCaMP family.

This review is focused on smaller multivalent structures such as glycoclusters emphasizing carbohydrate-centered and heteromultivalent glycoconjugates.

This review is focused on smaller multivalent structures such as glycoclusters emphasizing carbohydrate-centered and heteromultivalent glycoconjugates.

In particular, introduction of isotope labelled carbonyl into a peptide backbone ... have greatly expanded the ability of vibrational spectroscopy to obtain site-specific structural and dynamic information.

Herein we review various classes of cyclic and macrocyclic peptides as chemical probes of protein surfaces and modulators of PPIs.

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...

This review provides a comprehensive overview of recent advances in the supramolecular organisation and hierarchical self-assembly of organo-functionalised hybrid polyoxometalates (hereafter referred to as hybrid POMs), and their emerging role as multi-functional building blocks in the construction of new nanomaterials.

Post-translational covalent modification can provide an additional level of enzyme control.

Using the pressure jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was investigated.

RHEB GTPase refers here to the molecular protein structural elements of the RAS homolog protein enriched in brain, discussed alongside RAS GTPases as components of a signaling switch. The supplied evidence describes a native signaling protein class rather than an engineered biological tool implementation.

Self-healing hydrogels are particularly interesting because of their abilities to repair the structural damages and recover the original functions... self-healing hydrogels with shear-thinning properties can be potentially used as the vehicles for drug/cell delivery or the bioinks for 3D printing by reversible sol-gel transitions.

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

We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing.

Structural studies involving Bcl-xL were also the basis for the discovery of the first small-molecule pro-survival protein inhibitors, leading ultimately to the development of a new class of drugs now successfully used for cancer treatment in the clinic.

Synthetic peptides have proven an excellent type of molecule for the mimicry of protein sites because such peptides can be generated as exact copies of protein fragments, as well as in diverse chemical modifications.

Using the pressure jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was investigated.

In particular, ... incorporation of the extrinsic unnatural amino acids with vibrational moieties on the side chain, have greatly expanded the ability of vibrational spectroscopy to obtain site-specific structural and dynamic information.

Camouflage nanoparticle-based vectors are biomimetic delivery systems developed for in situ bioluminescence-driven optogenetic therapy of retinoblastoma. In the cited 2023 ACS Nano study, this system enabled optogenetic treatment and showed greater tumor inhibition than external blue light irradiation.

NanoLuc-modified macrophage membranes are a membrane-camouflage delivery harness used on nanoparticles for in situ bioluminescence-driven optogenetic therapy of retinoblastoma. The reported construct was combined with folic acid ligands and supported greater tumor inhibition and reduced ocular tumor size relative to external blue light irradiation in the cited study.

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.

B21 is a small-angle X-ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK.

In the present study, transgenic tobacco lines with constitutive SsDHN expression (SsDHN-OE) were employed to examine its influence on seedling development under water-limited conditions.

Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET).

The Escherichia coli YcgF protein contains a C-terminal EAL domain linked to an N-terminal FAD-binding BLUF photosensory domain. In this full-length blue-light-responsive protein context, the EAL domain is predicted to have cyclic-di-GMP phosphodiesterase activity.

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.

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.

In this review, we present key theoretical concepts and recent experimental strategies using femtosecond stimulated Raman spectroscopy (FSRS) to map the structural dynamics of proteins, highlighting the flexible chromophores on ultrafast timescales.

By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed... Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained.

High-resolution LC-MS/MS analysis enabled structural characterization and relative quantification of the lipopeptides.

Light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful, sensitive and informative method for studying protein structural changes in photoreceptive proteins.

LOV2 is a blue-light sensory domain from Avena sativa that was fused into the anti-CRISPR protein AcrIIC3 to create light-switchable inhibitors of Neisseria meningitidis Cas9. In this engineered context, AcrIIC3-LOV2 hybrids inhibited Nme Cas9 in the dark and permitted genome editing under blue light in mammalian cells.

The Avena sativa LOV2 blue light sensory domain is a photosensory protein module used in engineered AcrIIC3-LOV2 fusion proteins to confer blue-light control over Neisseria meningitidis Cas9 activity. In this reported context, LOV2 enabled dark-state inhibition and light-permissive genome editing in mammalian cells.

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.

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.

Combining microscopy with other quantitative measurement techniques such as optical tweezers and scissors, gives a very powerful tool for unraveling the intricacies of mechanobiology enabling measurement of forces, torques and displacements at play.

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.

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.

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.

YcgF (Blrp) is an Escherichia coli full-length blue-light sensing protein that contains an N-terminal FAD-binding BLUF domain and a C-terminal EAL domain. It functions as a BLUF-family photoreceptor in which the FAD chromophore undergoes light-responsive changes, and spectroscopy indicates coupling between the BLUF photochemistry and structural responses in the full-length protein.

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.

Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++)

In this study, we probed the effects of a few key mutations on the coupled binding and folding of α-synuclein by using a combination of single-molecule (smFRET) and ensemble (far-UV CD) measurements.

Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography

Methods like FRET, chemical probing and even the humble polyacrylamide gel can be rapid and very powerful, allowing the examination of a large number of sequence variants relatively quickly.

Methods like FRET, chemical probing and even the humble polyacrylamide gel can be rapid and very powerful, allowing the examination of a large number of sequence variants relatively quickly.

Methods like FRET, chemical probing and even the humble polyacrylamide gel can be rapid and very powerful, allowing the examination of a large number of sequence variants relatively quickly.

The most significant new data have come from X-ray crystallography of four-way DNA junctions... Ultimately crystallography provides the gold standard for structural analysis.

The YcgF BLUF domain is the N-terminal FAD-binding blue-light sensor from Escherichia coli YcgF. In isolation, it forms a light-induced signaling state with flavin FTIR and UV-visible properties, and dark-state recovery kinetics, that match those of full-length YcgF.

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.

Through mechanistic decipherment of the photophysical processes, structural design strategies for generating new AIE luminogens are developed.

Computational methods for LOV-based optogenetic tool development are design-enabling approaches used in the ongoing development of Light-Oxygen-Voltage domain-based optogenetic systems. The cited evidence supports a role for computational methods as one of several factors advancing LOV-based tools for light-controlled biological regulation.

Bridging this divide, covalent adaptable networks (CANs) structurally resemble thermosets with permanent covalent crosslinks but are able to flow in a manner that resembles thermoplastic behavior only when a dynamic chemical reaction is active.

Cr_ChR2 is a light-gated cation channel from Chlamydomonas reinhardtii used in optogenetics to activate neuronal excitability. The supplied evidence describes it as the first and most widely applied channelrhodopsin and as a benchmark comparator for newer light-gated cation channels such as Gt_CCR4.

Gt_CCR4 is a Guillardia theta-derived light-gated cation channel proposed as an optogenetic actuator. Reported evidence indicates that it has significantly higher light sensitivity than Cr_ChR2 while maintaining a channel open lifetime in the same range.

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.

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.

MicroRNAs are small non-coding RNAs that participate in light-regulated biological processes. The supplied evidence supports a role for miRNAs in mediating light-dependent pathways, but does not define a specific engineered construct or application format.

RAS GTPases are small signaling proteins that function as GTP-dependent molecular switches, with activation into the GTP-bound ON state described as a key regulator of brain functions. The supplied evidence discusses endogenous neuronal H-RAS signaling and does not describe a specific engineered light-responsive or optogenetic RAS tool.

The better understanding of the dynamic features of this membrane system requires the use of non-invasive techniques, such as small angle neutron scattering (SANS), which is capable of providing accurate, statistically and spatially averaged information on the repeat distances of periodically organized thylakoid membranes under physiologically relevant conditions with time resolutions of seconds and minutes.

Stimuli-sensitive genetic circuits are genetic constructs that incorporate natural or engineered modules whose activity changes in response to external stimuli to control gene expression. In the supplied evidence, light is an input modality, and these circuits are described in bacterial hosts for conditional induction or repression of genes of interest.

Thermosensors are stimuli-sensitive gene expression modules used in bacterial hosts for conditional expression of genes of interest. The supplied evidence describes them at a review level as systems in which temperature, and in some contexts light, regulates gene expression through structural changes in responsive biomolecules.

AlphaFold3 is a computational structure-prediction method used in the cited study to model the MagMboI–DNA complex. In that work, it was applied to infer interactions with the 5'-GATC-3' recognition sequence and to guide optimization of the photoactivatable endonuclease variant MagMboI-plus for top-down genome engineering.

BNp-Red-1.2 is a 6 kDa protein domain from the UNICYCL system that binds the cyanobacteriochrome GAF domain NpF2164g6 to form a 1:1 complex in the dark. It serves as a compact red-light-responsive protein interaction module whose association state is coupled to the photostate of NpF2164g6.

NpF2164g6 is a 17 kDa cyanobacteriochrome GAF domain used in the UNICYCL red-light-responsive protein interaction system. In this system, it forms a 1:1 complex with the 6 kDa binder BNp-Red-1.2 in the dark with an approximately 1–5 μM dissociation constant.

MagMboI is a photoactivatable split version of the type II restriction endonuclease MboI configured as a multi-component switch for light-controlled nuclease function. The supplied evidence indicates that an optimized variant, MagMboI-plus, produced stronger genome rearrangement activity than the original MagMboI in Saccharomyces cerevisiae.

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.

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.

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.

LOV photoreceptors are modular light-sensing protein domains that have been broadly used as sensor domains in optogenetic tools. Evidence indicates that mutations in a conserved hydrophobic pocket can alter the lifetime of the photo-adduct signaling state, thereby tuning sensor kinetics and steady-state on/off equilibria.

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.

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.

The Avena sativa LOV2 domain is a light-sensing LOV photoreceptor domain whose dark-state recovery kinetics can be altered by homologous mutations in a conserved hydrophobic pocket. The available evidence supports its use as a tunable photosensory module in LOV-based and optogenetic tool design.

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.

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.

Lamprey parapinopsin (UVLamP) is a Japanese lamprey (Lethenteron camtschaticum) parapinopsin used as a bistable, narrow-bandwidth optogenetic probe for control of the Gi/o pathway. It is activated by a millisecond UV light pulse and switched off by a millisecond blue light pulse, enabling reversible optical control of GPCR signaling.

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 photo-activatable Akt probe (PA-Akt) is a light-controlled multi-component Akt activation system built from the plant CRY2-CIB1 light-inducible interaction module. Upon illumination, it activates Akt signaling with sequential phosphorylation of Akt at S473 and T308 and engages downstream GSK-3β signaling in a light-intensity-dependent manner.

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.

LOV-based optogenetic tools are multi-component light-responsive systems built from Light-Oxygen-Voltage sensing domains. These domains function as versatile photoreceptors involved in cellular signaling and environmental adaptation across kingdoms of life, and their continued development is intended to expand optical control of biological systems.

UVR8 is an Arabidopsis thaliana UV-B photoreceptor that senses solar UV-B light in the 280-315 nm range. It functions as a light-responsive multi-component switch through UV-B-induced dissociation of a UVR8 dimer.

UNICYCL is a red-light-regulated multi-component protein interaction switch built from the cyanobacteriochrome NpF2164g6 and the binder BNp-Red-1.2. In the dark, BNp-Red-1.2 forms a 1:1 complex with NpF2164g6 with an approximately 1–5 μM dissociation constant, enabling reversible light-controlled association.

Current clinical LVV delivery systems do not include HIV-1 major accessory genes; however, critical structural and non-structural HIV-1 proteins are encoded by the 4-plasmid combination that composes the 3rd generation LVV transduction systems.

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.

Recent breakthroughs in cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based structure prediction have revolutionized protein modeling by enabling highly accurate computational predictions from amino acid sequences.

AlphaFold-a deep learning-based protein structure prediction system-has transformed structural biology by providing near-experimental accuracy models directly from amino acid sequences.

We discuss the complementary roles of cryo-EM and AI, including developments in direct electron detectors, advanced image processing, and deep learning algorithms exemplified by AlphaFold 2 and the emerging AlphaFold 3.

Using approximate analytical approach, the mechanical properties of structures can be predicted quickly and efficiently.

We designed and synthesized a novel LNP by integrating the pro-inflammatory fatty acid, arachidonic acid (ARA), as a functional structural component (ARA-LNP).

Much of our knowledge of the molecular details of these interactions has come from biochemical and structural studies on the pro-survival protein Bcl-xL. The first high-resolution structure of any Bcl-2 family member was of Bcl-xL, which revealed the conserved topology amongst all family members.

plant virus-like particles (VLPs), derived from Brome Mosaic Virus (BMV), as a biodegradable and biocompatible nanocarrier for small interfering RNA (siRNA) delivery aimed at modulating immune checkpoints in melanoma

CAR-NK cell therapy, as an emerging immunotherapeutic approach, has demonstrated significant potential. CAR-NK cells recognize and eliminate tumor cells through chimeric antigen receptors (CARs).

(b) cryptophyte cation channelrhodopsins (CCRs), structurally distinct from the green algae CCRs used extensively for neural activation and from cryptophyte ACRs

Cell-free protein synthesis (CFPS) has been used as a transformative technology in synthetic biology, providing a programmable, scalable, and automation-compatible platform for biological engineering.

Circular permutation (CP) is an invaluable approach to redesigning a protein by rearranging the amino acid sequence, where the connectivity of the secondary structural elements is altered without changing the overall structure of the protein.

Here we report an in vitro study to demonstrate the on-demand release of azidothymidine 5'-triphosphate ... from 30 nm CoFe2O4@BaTiO3 magneto-electric nanoparticles by applying a low alternating current magnetic field.

Recent breakthroughs in cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based structure prediction have revolutionized protein modeling by enabling near-atomic resolution visualization.

Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET).

we developed a druggable ligand-dependent IL-21 secretion system using destabilization domains (DDs).

we reprogram a classically unfavorable EDAC-mediated rearrangement into a productive synthetic route, enabling direct cationization of hyaluronic acid (HA) through spontaneous O-acylisourea rearrangement. This water-based, catalyst-free process achieves up to 70 % substitution

Advanced tissue engineering approaches (3D bioprinting, exosome therapy, and genetic engineering) directly target inflammation, enhance vascular and neuromuscular regeneration, and restore structural integrity of injured muscle.

very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions

Explicitly named in the supplied web research summary as a reference structural model for radial microgel morphology discussed by the review.

Graph neural networks (GNNs) are one of the fastest growing classes of machine learning models. They are of particular relevance for chemistry and materials science, as they directly work on a graph or structural representation of molecules and materials and therefore have full access to all relevant information required to characterize materials.

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs).

computational methods (such as homology modeling, molecular docking, molecular dynamics and enhanced sampling techniques) can provide structural insights to guide photoswitch design and to understand the observed light-regulated effects.

IMP305 was developed as an ultrasound-sensitive liposome for tumor-localized drug release. In particular, IMP305 is dominantly capable of release by ultrasound-mediated cavitation.

Here, we present IsoDiffR, a robust tool designed to identify RNA isoforms with expression patterns that differ from their corresponding genes or major isoforms across cell types, enabling both pairwise and multicell-type comparisons.

Lentiviral vectors (LVVs) are used as a viral gene therapeutic and were derived from human immunodeficiency virus subtype 1 (HIV-1). LVVs are used to deliver and induce the stable expression of transgenes through genome integration.

This review discusses the roles of different cellular and structural tumor components in regulating the metastatic process, targeting approaches using small molecule inhibitors, nanoparticles, manipulated exosomes, and miRNAs to inhibit tumor invasion...

we developed the molecular glue and ternary binding (MGTbind) database, providing comprehensive resources about ternary structures and experimental data for the coverage of MG-engaged interactome

Microfluidics has emerged as a set of powerful tools that have greatly advanced some areas of biological research, including research using C. elegans... provide guides for the design, fabrication, and use of microfluidic devices for C. elegans research studies.

Molecular docking and knowledge of bioinformatics are also being used to predict potential applications and manufacturing by industry.

Molecular modelling in conjunction with experiments is also a very important component of the general approach.

The coat protein of the MS2 self-assembles into virus-like particles (VLPs) with a diameter of 26 nm. These VLPs are devoid of the phage genome yet are efficiently recognized by the immune system, eliciting robust humoral and cellular immune responses. The structural characteristics of VLPs position them as a promising platform for the development of vaccines and diagnostic tools.

This review synthesizes genome-guided interventions (CRISPRa and mtDNA editing)... combined with mtDNA base editors...

The supplied web research summary lists nanoparticles as an explicit delivery component supported by therapeutic miRNA and siRNA studies or reviews in TNBC ncRNA therapy.

Nanoparticle-assisted targeted protein degraders (NanoTACs) offer a compelling solution by coupling the catalytic efficiency of TPD with the spatial precision and tunability of nanotechnology.

Among these systems, organic mechanoluminescent nanoparticles have garnered attention for their biocompatibility, structural flexibility, light weight properties, and biodegradability, positioning them as ideal candidates for next-generation biomedical devices.

Photoswitchable peptides have been recognized for many years; however, their functional implementation in biological systems has only recently been achieved... These exciting proof-of-principle studies highlight the tremendous potential of photocontrollable peptides as optochemical tools for chemical biology and biomedicine.

Here we review recent progress in combining time-resolved crystallography at X-ray free electron lasers and quantum chemical calculations to study structural changes in photoenzymes, photosynthetic proteins, photoreceptors, and photoswitchable fluorescent proteins following photoexcitation.

Two RNA secondary structure prediction tools (RNAstructure and ViennaRNA) ... were adopted to assess structural changes induced by these modifications.

VP1 from four epidemiologically relevant GI genotypes was expressed using the silkworm-baculovirus system.

virus-inspired polymer-DNA nanoparticles termed as "Skeletoplexes"

This review discusses the roles of different cellular and structural tumor components in regulating the metastatic process, targeting approaches using small molecule inhibitors, nanoparticles, manipulated exosomes, and miRNAs to inhibit tumor invasion...

In the past few years, spiropyran has emerged as the molecule-of-choice for the construction of novel dynamic materials. This unique molecular switch undergoes structural isomerisation in response to a variety of orthogonal stimuli, e.g. light, temperature, metal ions, redox potential, and mechanical stress.

In this review, we have chosen to present an overview on different mechanisms to impart responsiveness to dendritic polymers, with the particular aim of delivery and release of bioactive molecules.

In this study, we introduce StructRMDB, the first database designed to characterize the impact of chemical modifications on RNA secondary structure.

We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing.

Such compounds are designed in a modular fashion, combining a known ligand of the target protein and a photochromic group, as well as an additional electrophilic group for tethered ligands.

High-signal enrichment leads include earlier domain reviews that frame psychiatric LIFU/tFUS and summarize human parameterization. Explicitly supported related component/tool names in discovered sources include transcranial focused ultrasound/transcranial ultrasound stimulation (tFUS/TUS).

Two RNA secondary structure prediction tools (RNAstructure and ViennaRNA) ... were adopted to assess structural changes induced by these modifications.

Potential impact of an X-ray free electron laser on structural biology