Cross-linking Sites Research Articles

Targeted Analysis of Mitochondrial Protein Conformations and Interactions by Endogenous ROS-Triggered Cross-Linker Release.

The study of in situ conformations and interactions of mitochondrial proteins plays a crucial role in understanding their biological functions. Current chemical cross-linking mass spectrometry (CX-MS) has difficulty in achieving in-depth analysis of mitochondrial proteins for cells without genetic modification. Herein, this work develops the reactive oxygen species (ROS)-responsive cross-linker delivery nanoparticles (R-CDNP) targeting mitochondria. R-CDNP contains mitochondria-targeting module triphenylphosphine, ROS-responsive module thioketal, loading module poly(lactic-co-glycolic acid) (PLGA), and polyethylene glycol (PEG), and cross-linker module disuccinimidyl suberate (DSS). After targeting mitochondria, ROS-triggered cross-linker release improves the cross-linking coverage of mitochondria in situ. In total, this work identifies 2103 cross-linked sites of 572 mitochondrial proteins in HepG2 cells. 1718 intra-links reveal dynamic conformations involving chaperones with ATP-dependent conformation cycles, and 385 inter-links reveal dynamic interactions involving OXPHOS complexes and 27 pairs of possible potential interactions. These results signify that R-CDNP can achieve dynamic conformation and interaction analysis of mitochondrial proteins in living cells, thereby contributing to a better understanding of their biological functions.

Insight into the Coextrusion Mechanism between Whey Protein Isolate and Cysteine.

The disulfide cross-linking sites of whey protein isolate (WPI) coextruded with dissolved cysteine (Cys) at concentrations of 0, 20, 40, 60, 80, and 100 mM were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software, and the structure and gel water distribution of WPI during coextrusion (≤50 °C) were also investigated. LC/MS/MS demonstrated that α-La (6) and α-La (120) were the most active sites for intermolecular disulfide cross-linking of α-La. Meanwhile, the molecular weight of protein polymers in coextruded WPI-Cys was the largest at 100 mM Cys, and α-lactalbumin was the main reactant for polymerization from the result of SDS-PAGE and size exclusion chromatography. Additionally, the high concentration of Cys caused the secondary structure of WPI to gradually change from a highly ordered to a disordered structure during coextrusion. In addition, with an increasing concentration of Cys, the free sulfhydryl group of proteins and the binding force to immobilized water gradually increased. Therefore, this work revealed the disulfide cross-linking mechanism between WPI and Cys under low-temperature coextrusion at the molecular level, and the obtained coextruded cross-linked WPI could serve as a novel food ingredient with excellent water-holding capacity for the food industry.

Pristine Photopolymerizable Gelatin Hydrogels: A Low-Cost and Easily Modifiable Platform for Biomedical Applications.

The study addresses the challenge of temperature sensitivity in pristine gelatin hydrogels, widely used in biomedical applications due to their biocompatibility, low cost, and cell adhesion properties. Traditional gelatin hydrogels dissolve at physiological temperatures, limiting their utility. Here, we introduce a novel method for creating stable hydrogels at 37 °C using pristine gelatin through photopolymerization without requiring chemical modifications. This approach enhances consistency and simplifies production and functionalization of the gelatin with bioactive molecules. The stabilization mechanism involves the partial retention of the triple-helix structure of gelatin below 25 °C, which provides specific crosslinking sites. Upon activation by visible light, ruthenium (Ru) acts as a photosensitizer that generates sulphate radicals from sodium persulphate (SPS), inducing covalent bonding between tyrosine residues and "locking" the triple-helix conformation. The primary focus of this work is the characterization of the mechanical properties, swelling ratio, and biocompatibility of the photopolymerized gelatin hydrogels. Notably, these hydrogels supported better cell viability and elongation in normal human dermal fibroblasts (NHDFs) compared to GelMA, and similar performance was observed for human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). As a proof of concept for functionalization, gelatin was modified with selenous acid (GelSe), which demonstrated antioxidant and antimicrobial capacities, particularly against E. coli and S. aureus. These results suggest that pristine gelatin hydrogels, enhanced through this new photopolymerization method and functionalized with bioactive molecules, hold potential for advancing regenerative medicine and tissue engineering by providing robust, biocompatible scaffolds for cell culture and therapeutic applications.

Ultra-Tough, yet Rigid and Healable Supramolecular Polymers with Variable Stiffness for Multimodal Actuators.

Variable stiffness materials have shown considerable application in soft robotics. However, previously reported materials often struggle to reconcile high stiffness, stretchability, toughness, and self-healing ability, because of the inherently conflicting requisite of these properties in molecular design. Herein, we propose a novel strategy that involves incorporating acid-base ionic pairs capable of from strong crosslinking sites into a dense and robust hydrogen-bonding network to construct rigid self-healing polymers with tunable stiffness and excellent toughness. To demonstrate these distinct features, the polymer was employed to serve as the strain-regulation layers within a fiber-reinforced pneumatic actuator (FPA). The exceptional synergy between the configuration versatility of FPA and the dynamic molecular behavior of the supramolecular polymers equips the actuator with simultaneous improvement in motion dexterity, multimodality, loading capacity, robustness, and durability. Additionally, the concept of integrating high dexterity at both macro- and micro-scale is prospective to inspire the design of intelligent yet robust devices across various domains.

Equimolar Cross-Coupling Using Reactive Coiled Coils for Covalent Protein Assemblies.

Biocompatible cross-coupling reactions enable the efficient covalent attachment of large biomolecules at near-stoichiometric ratios, ensuring the stability and integrity of the resulting products. We present an affinity-based peptide platform utilizing coiled coils containing reactive side chains for proximity-driven protein cross-coupling in the presence of a cross-linking agent. This platform supports both chemical synthesis and recombinant expression, using canonical amino acids to generate reactive affinity tags. Employing the E3/R3 coiled coil pair as a scaffold, we design four complementary coils with cysteine residues as cross-linking sites, achieving >90% conversion to covalent heterodimeric coupling products using 3,4-dibromomaleimide. Equimolar mixtures of proteins with reactive coils at their termini yield near-quantitative heterodimeric cross-coupling products. The strategic selection of complementary coiled coil pairs and cross-linking agents enables orthogonal assembly of macromolecules with diverse architectures. This method offers a versatile approach for creating covalent fusion proteins, enhancing their stability and functionality for applications in chemical biology, biotechnology, and medicine.

Maleimide-induced crosslinking of polyimide membranes for high gas separation performance and plasticization resistance

Trade-off effect and plasticization are the key issues confronted in the area of membrane separation when dealing with applications involving high-pressure, soluble gases like CO2. In this study, a novel diamine (TAPA-MAA) was synthesized derived from tris(4-aminophenyl)amine (TAPA) and maleic anhydride (MA). This diamine was copolymerized with DAM (2,4,6-trimethylbenzene-1,3-diamine) and 6FDA (4,4′-(hexafluoroisopropylidene)diphthalic anhydride) to form the polyimide (PI) membrane containing maleimide (MI) cross-linking sites via chemical imidization way. Then, under the action of heat treatment, the crosslinked PI (cPI) membrane was constructed since the MI crosslinking initiates a radical cycloaddition reaction to form a succinimide-linked structure, and named as cPSI membrane for short. Further, an increase in the ratio of TAPA-MAA gives rise to the resultant cPSI membrane with elevated cross-linking density. The obtained cPSI membranes showed significantly enhanced gas selectivities and superior anti-plasticization property compared to the un-crosslinked PI membranes due to the MI-induced crosslinking reaction in the PI main chain, resulting in a marked increment (24 %) in ultra-microporosity (<7 Å) and a reduction in the d-spacing value (6.10 Å vs 5.80 Å), generating a strong molecular sieving effect. Meanwhile, the highly interconnected covalent structure formed by the MI-induced crosslinking will suppress the plasticizing phenomenon induced by high-pressure soluble gases. The typical membrane (cPSI-3:7-350) possessed great CO2/CH4 separation performance, CO2 permeability of 334.4barrer and a CO2/CH4 selectivity of 61.9 that is exceeded the 2008Roberson upper bound, along withCO2 plasticization pressures above 44 bar. Besides, its mixed CO2/CH4 separation performance exceeded the 2018 upper gas mixture limit when the upstream pressure changed from 2-20 bar. This study provides valuable insights for the design of PIs containing MI cross-linking sites, which can enhance the performance of membrane-based gas separation.

Structural studies of the human α1 glycine receptor via site-specific chemical cross-linking coupled with mass spectrometry

By identifying distance constraints, chemical cross-linking coupled with mass spectrometry (CX-MS) can be a powerful complementary technique to other structural methods by interrogating macromolecular protein complexes under native-like conditions. In this study, we developed a CX-MS approach to identify the sites of chemical cross-linking from a single targeted location within the human α1 glycine receptor (α1 GlyR) in its apo state. The human α1 GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neurotransmission. A single chemically reactive cysteine was reintroduced into a Cys null α1 GlyR construct at position 41 within the extracellular domain of human α1 homomeric GlyR overexpressed in a baculoviral system. After purification and reconstitution into vesicles, methanethiosulfonate-benzophenone-alkyne, a heterotrifunctional cross-linker, was site specifically attached to Cys41 via disulfide bond formation. The resting receptor was then subjected to UV photocross-linking. Afterward, monomeric and oligomeric α1 GlyR bands from SDS-PAGE gels were trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intrasubunit and intersubunit sites of α1 GlyR cross-linking were differentiated and identified from single gel bands of purified protein, showing the utility of this experimental approach to identify a diverse array of distance constraints of the α1 GlyR in its resting state. These studies highlight CX-MS as an experimental approach to identify chemical cross-links within full-length integral membrane protein assemblies in a native-like lipid environment.

Solvent-free Acrylate/BCB drop-on-demand (DOD) inkjet dielectric ink for 3D printing

Currently, drop-on-demand (DOD) inkjet-printed dielectric inks used to produce next-generation high-frequency electronic devices fail to meet the requirements of circuit board substrates in terms of thermal, mechanical, and dielectric properties. To address this gap, we synthesized a low-viscosity acrylate monomer featuring an intrinsically low dielectric structure (hexafluorobisphenol A) and multiple cross-linking sites, and a benzocyclobutene monomer with low curing shrinkage and excellent thermomechanical properties. By mixing these monomers with other reactive diluents and verifying them through theoretical calculations and printing, we have developed a range of dielectric inks suitable for inkjet 3D printing. To enhance the overall performance of the cured inks, a sequential UV/thermal curing strategy was employed to form interpenetrating networks (IPNs). The optimal ink formulations met the requirements of circuit board substrates, demonstrating excellent heat resistance (Td5% = 334 °C, Tg = 181 °C), mechanical properties (tensile strength = 85 MPa, elongation at break = 13.5 %), and dielectric properties (Dk = 2.526 and Df = 0.113 at 15 GHz). These formulations meet the performance requirements for circuit board substrates and lead the industry in mechanical and dielectric properties. Furthermore, the feasibility of inkjet printing heterogeneous integrated electronic devices was verified, showing excellent print resolution and continuity of the conductive network. This work provides new insights into the future development of dielectric materials for inkjet 3D printing.

Covalent Probes To Capture Legionella pneumophila Dup Effector Enzymes.

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires' disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and X-ray crystallography approaches were used to identify the site of covalent cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

Ultra‐Tough, yet Rigid and Healable Supramolecular Polymers with Variable Stiffness for Multimodal Actuators

AbstractVariable stiffness materials have shown considerable application in soft robotics. However, previously reported materials often struggle to reconcile high stiffness, stretchability, toughness, and self‐healing ability, because of the inherently conflicting requisite of these properties in molecular design. Herein, we propose a novel strategy that involves incorporating acid‐base ionic pairs capable of from strong crosslinking sites into a dense and robust hydrogen‐bonding network to construct rigid self‐healing polymers with tunable stiffness and excellent toughness. To demonstrate these distinct features, the polymer was employed to serve as the strain‐regulation layers within a fiber‐reinforced pneumatic actuator (FPA). The exceptional synergy between the configuration versatility of FPA and the dynamic molecular behavior of the supramolecular polymers equips the actuator with simultaneous improvement in motion dexterity, multimodality, loading capacity, robustness, and durability. Additionally, the concept of integrating high dexterity at both macro‐ and micro‐scale is prospective to inspire the design of intelligent yet robust devices across various domains.

Role of terminal beads in fracture of topological gels: A coarse-grained molecular dynamics study

Topological gels feature movable cross-linking sites which yield great deformation capability, but we show that their long-term performance would be undesirable. Because a long backbone chain has only 2 terminal beads to restrict sliding of rings, the entire chain would fail by just a small debonding. Chain segmentation by adding terminal beads is a promising strategy to improve durability. In this work, we develop a coarse-grained (CG) model of topological gels based on MARTINI force field, to simulate tensile behaviors of the gel. The developed CG model accurately predicts elastic modulus of typical topological gels with different crosslinking densities. Based on the model, we show that adding terminal beads to the chain can effectively improve mechanical properties of topological gels after initial debonding. With analyses on distribution of ring molecules along the chain and effective load-bearing chains during tensile deformation, mechanism of the improvement on durability is discussed.

Synthesis and characterization of novel water hyacinth (Eichhornia Crassipes) biodegradable mulch films

In this paper, carboxymethyl cellulose (CMC) and hemicellulose derived from water hyacinth were used to prepare hemicellulose-based biodegradable mulch film by covalent cross-linking and ionic cross-linking in order to expand its application in agricultural production practice. The esterification reaction between hemicellulose, CMC and citric acid resulted in an increase in tensile strength and elongation at break of the membranes. When citric acid was not used as cross-linking agent and the pH was lowered, the sodium carboxylate group was protonated into carboxylic acid group, which provided abundant active sites for chemical cross-linking of hydroxyl group on hemicellulose and hydroxyl group on CMC. Furthermore Zn2+ could cross-link with carboxylic acid group through hydrogen bonding, and when the DS of carboxymethyl group was high, the cross-linking of Zn2+ with Zn2+ was higher, and the conversion into nano ZnO was lower, which was conducive to the uniform distribution and reduction of agglomeration phenomenon in the films. It is favorable for its uniform distribution in the film and reduces the agglomeration phenomenon. The mulch films made from water hyacinth has excellent mechanical properties, light transmittance, water absorption, soil moisture retention and heat preservation, and is biodegradable. This study will provide new ideas for water pollution control and farmland pollution for sustainable agricultural production.

CROSSLINK DENSITY AND ITS DISTRIBUTION IN HEAT AND OIL RESISTANT ELASTOMERS BY DOUBLE QUANTUM NUCLEAR MAGNETIC RESONANCE

ABSTRACT Double quantum (DQ) nuclear magnetic resonance (NMR) was used to characterize the crosslink density, crosslink density distribution, and defect level in a series of heat and oil resistant elastomers. A wide range of defect levels, crosslink densities, and crosslink density distributions was measured, and results depended on elastomer type and compound formulations, including the vulcanization system. The sol fraction defect level generally correlated with the concentration of added plasticizer in the formulation. The presence of polar side chains appeared to cause additional dynamic contributions to the dangling chain end fraction. The large differences in elastomer composition and rubber formulations prevented meaningful correlation of the measured crosslink densities with the low strain modulus. Fast Tikhonov regularization and log normalization fitting of the corrected DQ build-up curve was extremely useful to provide insight into the modality and widths of the crosslink density distributions. A high degree of heterogeneity of the crosslink network of heat and oil resistant elastomers was found. Crosslink density distributions were explained in terms of the polymer chain structure comprised of monomer sequencing coupled with the position of the crosslinking sites. The type of vulcanization system had a lesser effect of the nature of the crosslink density distribution. The primary polymer chain crosslinking sites may become segregated from the continuous phase due to polarity differences seen in the microstructure of oil and heat resistance elastomers. The development of such micromorphologies can favor curative partitioning. The sole use of DQ NMR can provide valuable insight into the nature of the polymer chain structure and crosslink network in rubber.

Self-healing cellulose nanocrystals/fluorinated polyacrylate with double dynamic interactions of coumarin groups and multiple hydrogen bonds

In this work, self-healing cellulose nanocrystals/fluorinated polyacrylate with dual dynamic networks of photoreversible crosslinking network and high-density hydrogen bonds was prepared by Pickering emulsion polymerization. The main work was to study the effects of 7-(2-methacryloyloxy)-4-methylcoumarin (CMA) and 2-ureido-4[1H]-pyrimidinone methyl methacrylate (UPyMA) monomer dosage on emulsion polymerization and latex film properties. The monomer conversion increased first and then decreased as the CMA and UPyMA monomer dosage increased, while a reverse trend was noted for the particle size and particle size distribution. Incorporating UPyMA allowed the rapid formation of hydrogen bonds at the crosslinking sites, which increased the interaction force between the healing surfaces. Besides, reversible photocrosslinking reaction of coumarin groups provided another support for self-healing performance. Moreover, the influence of self-healing temperature, self-healing time and UV irradiation on the self-healing ability was also systematically investigated The tensile strength of the prepared cellulose nanocrystals/fluorinated polyacrylate latex film exhibited a self-healing efficiency of 91.4 % under 365 nm UV irradiation and 80 °C for 12 h. The latex film had excellent thermal stability as was shown by TG and DTG analyses. The outstanding self-healing capability of latex film was attributed to the reversible photodimerization of coumarin groups and multiple hydrogen bonds. In addition, the water-oil repellent and mechanical properties of the latex films were improved as the CMA and UPyMA monomer dosage increased.

Chemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid-protein interaction sites by mass spectrometry.

UV (ultra-violet) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA-and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo . We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains. In vivo UV and chemical XL-MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid-protein crosslink inventory with crosslink sites at amino acid level for more than 1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.

Mussel-inspired tough ionogel with robust adhesion and mechanical adaptivity for impact resistance

Intrinsic adhesiveness and mechanical adaptivity allowing spontaneous anchoring and impact-stiffening, which facilitate safer impact protection, remain challenges for the design and synthesis of advanced tough impact-resistant materials. Herein, a novel biomimetic mechanically robust impact-resistant ionogel with the mussel-inspired diphasic structure was elaborately developed. The salt-bridge hydrogen bonds between carboxyl and imidazole groups inside rigid clusters serve as reversible crosslinking sites and prime energy-dissipation motifs, leading to strong noncovalent cohesion. Furthermore, the hydrophobic ionic liquid enriches surface interactions, providing extensive adhesive performance for in-situ attachment under external impact. Benefitting from the dynamic nature of the salt-bridge hydrogen bonds, the ionogel possesses impressive energy dissipation, self-healing, and recyclable properties. Notably, the prepared ionogel exhibits rate-dependent mechanical adaptivity and outstanding impact-stiffening performance with extraordinary initial modulus (∼6.3 GPa), impact strength (∼178.5 MPa), and impact toughness (∼89.6 MJ m−3) under high-speed impact (∼5200 s−1). This work offers promising biomimetic design inspirations for the future development of advanced, flexible, sustainable, and protective materials.

Effects of quaternization sites and crossing methods on the slow-release and antibacterial effects of hydroxypropyltrimethyl ammonium chloride chitosan/dialdehyde chitosan-based film

In this study, the active food packaging film were prepared using hydroxypropyltrimethyl ammonium chloride chitosan with different substitution sites (O-HACC & N-HACC) and dialdehyde chitosan (DCS) grafted with protocatechuic acid (PA). To explore the effect of chitosan quaternization positions and crosslinking approaches on the slow-release and antibacterial properties, the double-crosslinked film were fabricated through the self-coupling reaction of PA and Schiff base reaction between amino groups on HACC and aldehyde groups on DCS. The HACC/DCS-based film exhibited stable porous three-dimensional networks with high nisin loading ratios (>90 %). With the participation of the catechol-catechol structure, the dense double-crosslinked film effectively restricted the diffusion of the water molecules, resulting in excellent slow-release properties fitting with the Korsmeyer-Peppas kinetic model. Especially, O-HACC/PA-g-DCS film, which had more reaction sites for Schiff base crosslinking than N-HACC, exhibited the equilibrium swelling ratio of 800 % at 60 h and could sustainably release nisin via non-Fickian diffusion behavior until 48 h. Moreover, the HACC/DCS-based double-crosslinked film performed good long-time antibacterial activity and preservation effects on salmon. On the 10th day of storage, the TVBN of N-HACC/PA-g-DCS and O-HACC/PA-g-DCS groups were only 28.26 ± 1.93 and 29.06 ± 1.68 mg/100 g and still lower than the thresholds.

Dual Photoreactive Ternary Ruthenium(II) Terpyridyl Complexes: A Comparative Study on Visible-Light-Induced Single-Step Dissociation of Bidentate Ligands and Generation of Singlet Oxygen.

The versatile and tunable ligand-exchange dynamics in ruthenium(II)-polypyridyl complexes imposed by the modulation of the steric and electronic effects of the coordinated ligands provide an unlimited scope for developing phototherapeutic agents. The photorelease of a bidentate ligand from the Ru-center is better suited for potent Ru(II)-based photocytotoxic agents with two available labile sites for cross-linking with biological targets augmented with possible phototriggered 1O2 generation. Herein, we introduced a phenyl-terpyridine (ptpy) ligand in the octahedral Ru(II) core of [Ru(ptpy)(L-L)Cl]+ to induce structural distortion for the possible photorelease of electronically distinct bidentate ligands (L-L). For a systematic study, we designed four Ru(II) polypyridyl complexes: [Ru(ptpy)(L-L)Cl](PF6), ([1]-[4]), where L-L = 1,2-bis(phenylthio)ethane (SPH) [1], N,N,N',N'-tetramethylethylenediamine (TMEN) [2], N1,N2-diphenylethane-1,2-diimine (BPEDI) [3], and bis[2-(diphenylphosphino)phenyl]ether (DPE-Phos) [4]. The detailed photochemical studies suggest a single-step dissociation of L-L from the bis-thioether (SPH) complex [1] and diamine (TMEN) complex [2], while no photosubstitution was observed for [3] and [4]. Complex [1] and [2] demonstrated a dual role, involving both photosubstitution and 1O2 generation, while [3] and [4] solely exhibited poor to moderate 1O2 production. The interplay of excited states leading to these behaviors was rationalized from the lifetimes of the 3MLCT excited states by using transient absorption spectroscopy, suggesting intricate relaxation dynamics and 1O2 generation upon excitation. Therefore, the photolabile complexes [1] and [2] could potentially act as dual photoreactive agents via the phototriggered release of L-L (PACT) and/or 1O2-mediated PDT mechanisms, while [4] primarily can be utilized as a PDT agent.

Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.

Simultaneously Strengthening and Toughening All‐Natural Structural Materials via 3D Nanofiber Network Interfacial Design

AbstractConstructing structural materials from sustainable raw materials is considered an efficient way to reduce the potential threat posed by plastics. Nevertheless, challenges remain regarding combining excellent mechanical and thermal properties, especially the balance of strength and toughness. Here, we report a 3D nanofiber network interfacial design strategy to strengthen and toughen all‐natural structural materials simultaneously. The introduced protonated chitosan at the interface between the surface oxidized 3D nanonetwork of bacterial cellulose forms the interfacial interlocking structure of nanonetworks, achieving a robust physical connection and providing enough physical contact sites for chemical crosslinking. The obtained sustainable structural material successfully integrates excellent mechanical and thermal properties on the nanoscale of cellulose nanofibers, such as light weight, high strength, and superior thermal expansion coefficient. The relationship between structural design and comprehensive mechanical property improvement is analyzed in detail, providing a universal perspective to design sustainable high‐performance structural materials from nanoscale building blocks.