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Results for “Cross-Linking Reagents”

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At least 19 recordsLinked to original sources

PubMed1977

A novel approach to the identification of surface receptors. The use of photosensitive hetero-bifunctional cross-linking reagent.

Methyl 4-azidobenzoimidate, a photosensitive hetero-bifunctional cross-linking reagent, was synthesized and characterized. This reagent has an imidoester at one end, which reacts spontaneously with primary amines, and an arylazide at the other end, which reacts with a variety of chemical groups upon photolysis by ultraviolet radiation. The reagent molecules were attached to concanavalin A by reactions between imidoester groups of the reagents and free amino groups of the lectin. These activated lectins were purified on a Sephadex G-25 column and showed the binding affinity to an affinity column, glucosylated Sepharose, and to the human erythrocyte ghost membrane. The activated lectins were incubated with the membranes and then unbound lectins were removed by washing. The lectins bound to receptors in the membranes were irradiated with a shortwave ultraviolet lamp to photolyze arylazides attached to the lectins, thus cross-linking the lectins and receptors together. Then the membranes were solubilized and electrophoresed. On gels, the intensity of the lectin receptor band diminished slightly and concomitantly a new band of a higher molecular weight appeared. When 125I-labeled concanavalin A was used, the new band contained the radioactivity. The extent of the appearance of the new band and the decrease of the receptor band were reduced significantly when the ultraviolet irradiation was omitted or the activated lectins were incubated with the membranes in the presence of the lectin inhibitor, alpha-methylmannoside. The irradiation of nonactivated, receptor-bound concanavalin A did not cause those changes. When the activated lectins alone were irradiated with ultraviolet, the band of the lectin dimer appeared whereas nonirradiated lectins appeared mostly as monomers. It is concluded that a small fraction of the activated lectins were cross-linked to receptors in the membrane upon photolysis. In this study, only 8 reagent molecules were attached to a tetramer of the lectin, compared with the presence of approximately 40 available free amino groups. The efficiency of such cross-links of ligands to receptors may be increased by employing longer versions of the hetero-bifunctional cross-linking reagents and also by attaching more of the reagent molecule to ligands.

Azides↗

PubMed1976

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.

Binding Sites↗

PubMed1978

Chemical modification of the excitable membrane in Paramecium aurelia: effect of a cross-linking reagent.

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Aldehydes↗

PubMed1976

Photoactivated cross-linking of proteins within the erythrocyte membrane core.

We describe the reactions of three lipophilic, photoactivated cross-linking reagents, 1,5-diazidonapthalene, 4,4'-diazidobiphenyl, and the reversible 4,4'-dithiobisphenylazide, with erythrocyte membranes. Cross-linking occurs only upon photoactivation. At pH 7 to 8, only spectrin components are cross-linked by these reagents. At pH 5.0 to 5.5 several additional membrane proteins including the major "integral" membrane proteins are also cross-linked, despite equivalent binding of the cross-linkers at neutral and acid pH. The cross-linking rates of various membrane proteins at pH 5.0 to 5.5 depend distinctly upon duration of photoactivation. Bidimensional electrophoresis of membrane proteins after cross-linking with the reversible cross-linker, 4,4'-dithiobisphenylazide, has allowed for the identification of homopolymeric products of cross-linking (e.g. dimers and tetramers of Band 3) and heterocomplexes (spectrin plus other membrane proteins). The data suggest that at reduced pH, cross-linking can proceed not only at the membrane surface but also in the membrane core.

Azides↗

PubMed1976

The arrangement of subunits in cholera toxin.

Cholera toxin consists of five similar B subunits of apparent molecular weight about 10 600 and one A subunit (29 000) consisting of two peptides (A1 23 000-24 000 and A2 about 5500) linked by a single disulfide bond. Each B subunit also contains one internal disulfide bond which is readily reduced but is protected from carboxymethylation unless the reduced subunits are heated in urea. Tyrosine residues in A1 and in B subunits are readily iodinated, but the intact B assembly does not react with iodine. Upon reaction with the cross-linking reagent dimethyl suberimidate, B subunits may be covalently connected to each other, to A1 and to A2. A1 and A2 may also be cross-linked. The B subunits are probably arranged in a ring with A on the axis. A2 is required for the re-assembly of toxin from its subunits and may serve to hold A1 on the B ring. The maximum activity of cholera toxin in vitro is obtained only when the active peptide, A1, is separated from the rest of the molecule. Such separation, and the insertion of A1 into the cytosol, must follow the binding of the complete toxin, through component B, to the exterior of intact cells. This binding increases the effective concentration of the toxin in the vicinity of the plasma membrane. Possible ways in which A1 then crosses the membrane are considered in the Discussion.

Adenylyl Cyclases↗

PubMed1976

Synthesis and properties of carbonylbis(methionyl)insulin, a proinsulin analogue which is convertible to insulin by cyanogen bromide cleavage.

The preparation and use of carbonylbis (L-methionine p-nitrophenyl ester) as a reversible cross-linking reagent for insulin are described. The reaction of 1 equiv of reagent with zinc insulin in dimethylformamide in the presence of triethylamine yields as one of the products NalphaA1, NepsilonB29-carbonylbis(methionyl)insulin, (CBM-insulin). The CBM-insulin was characterized by end group analysis and by the products formed on tryptic and chymotryptic cleavage. It possessed 91% of the immunological and 6.5% of the hormonal activity of insulin. Treatment of CBM-insulin with cyanogen bromide (CNBr) in 70% formic acid for 1 h resulted in nearly complete removal of the methionine bridge to yield insulin. A small amount of a side product was removed on DEAE-cellulose at pH 7.2 to give an overall recovery of insulin of 70-80%. Oxidative sulfitolyses of CBM-insulin gave the hexa(S-sulfonate) which was reduced with dithiothreitol to yield reduced CBM-insulin. The latter compound, containing 6 sulfhydryls, exhibited a pH-dependent circular dichroic spectrum. The form at pH 10 exhibited a spectrum typical of random coil which was converted to a form at pH 7.8 which was characterized by a negative extremum at 213 nm. The change in the spectrum at 213 nm with pH was characterized by an apparent pKa of 8.5. Studies on the reoxidation of reduced CBM-insulin were performed at pH values between 7.8 and 10 and at protein concentrations of 0.01-1 mg/ml. The best yields (ca. 85%) of the correctly paired disulfide bonds were obtained in reoxidations at pH 9.5-10 at protein concentration of 0.01-0.1 mg/ml. CBM-insulin, which had been isolated from reoxidation at high pH of the reduced CBM-insulin, was cleaved by CNBr to yield a fully active insulin in an overall yield of 60% from the reduced CBM-insulin.

Adipose Tissue↗

PubMed2025

Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.

DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.

Arabidopsis↗

PubMed2026

Second-Generation ELZA-sub400 Protocol: Individualized High-Fluence Cross-Linking for Ultra-Thin Keratoconus Corneas.

PURPOSE: To evaluate the safety and efficacy of a second-generation individualized corneal cross-linking (CXL) protocol (ELZA-sub400) using high-fluence UV-A irradiation in ultrathin ectatic corneas. DESIGN: Retrospective, single-center, consecutive interventional case series. METHODS: Twenty-nine eyes of 24 patients with progressive keratoconus or post-LASIK ectasia and a post-soak intraoperative thinnest stromal thickness <400 µm were included. After epithelial removal and riboflavin soaking, continuous UV-A irradiation (365 nm) at 3 or 9 mW/cm² was delivered with total fluence titrated up to 10 J/cm² based on intraoperative ultrasound pachymetry and a previously published nomogram targeting an uncross-linked stromal margin of approximately 70 µm above the endothelium. Outcomes were assessed at baseline and up to 12 months using corrected distance visual acuity (CDVA) and corneal parameters measured using Scheimpflug tomography and anterior segment OCT (AS-OCT) with Placido-based topography. The main outcome measure was the proportion of eyes without progression at 12 months, defined as <1.0 D increase in maximum keratometry (Kmax). Secondary outcomes included changes in CDVA, refraction, Kmax, stromal thickness, demarcation line depth, densitometry, and safety parameters. RESULTS: At 12 months, 22/29 eyes (76%; 95% CI, 57.9%-87.8%) met the nonprogression criterion. Mean change in Kmax was -0.77 ± 5.10 D (95% CI, -2.71 to 1.17; P = .418). Mean demarcation line-to-anterior stroma distance was 205 ± 64 µm (95% CI, 180.7-229.3), and demarcation line-to-endothelium distance was 64 µm (IQR, 49-152). All demarcation lines remained within the stromal layer; 15/29 eyes (51.7%) had a demarcation line located ≤70 µm from the endothelium. Median CDVA changed from 0.10 to 0.32 logMAR (P = .142). Minimum stromal thickness showed a median change of -4.0 µm (P = .309). No significant change was observed in densitometry, and no eye developed deep stromal haze or endothelial decompensation. CONCLUSIONS: Second-generation ELZA-sub400 CXL halted ectasia progression in 76% of ultrathin corneas at 12 months and was associated with an acceptable short-term safety profile, including stromal-confined demarcation line formation and no observed endothelial decompensation. The numerical decline in spectacle CDVA observed in this severely affected cohort did not reach statistical significance but is clinically important and warrants confirmation in larger prospective studies.

Humans↗

PubMed2026

Proteome-wide structural and interaction analysis using cross-linking mass spectrometry and its applications.

Deciphering the mechanisms of protein-protein interactions (PPIs) and protein structural changes within the native cellular environment is crucial for advancing drug discovery. In vivo chemical cross-linking coupled with mass spectrometry (XL-MS) captures weak, transient, and higher-order interactions that are often dysregulated under altered physiological conditions and remain challenging to detect using conventional methods. Applications of in vivo XL-MS range from targeted mapping of PPIs to large-scale identification of interactome networks within the cells. The integration of quantitative approaches further facilitates comparison across different physiological conditions. The recent incorporation of machine learning (ML) tools into XL-MS workflows is transforming the depth and efficiency of this technology. AI-driven algorithms now enable more accurate identification of cross-linked peptides and the mapping of interaction topologies. Furthermore, the synergistic coupling of in vivo XL-MS data with AI-assisted structural modeling platforms such as AlphaFold allows dynamic and high-throughput prediction of protein networks. This review discusses the broader applications of in vivo XL-MS in complex biological samples, ranging from organelles and cells to whole tissues, and highlights how AI integration is expanding structural biology toward a systems-level understanding of proteome architecture.

Mass Spectrometry↗

PubMed2026

Structure-resolved virus-host interactomics by cross-linking mass spectrometry.

Viruses depend on host protein networks to replicate, assemble progeny, and spread between cells and organisms. Defining these virus-host protein interactions is challenging because they are highly dependent on infection stage, cell type, species, and because mechanistic interpretation requires information about structural interfaces and conformational states. Cross-linking mass spectrometry (XL-MS) addresses these challenges by adding a spatial and structural dimension to virus-host interactomics in native systems. In this review, we discuss how XL-MS has advanced from targeted analysis of viral protein complexes to structure-resolved mapping of virion architecture and infected-cell virus-host interactomes. We highlight how XL-MS complements AP-MS, cryo-EM/cryo-ET, quantitative proteomics, genetic perturbation, and structure prediction to connect physical proximity with molecular mechanisms. Finally, we discuss current limitations in sensitivity, chemical coverage, temporal resolution, and model interpretation, and outline how future quantitative and integrative XL-MS workflows may enable systems-level structural virology.

Mass Spectrometry↗

PubMed2026

Generation of Interstrand DNA Cross-Links under Conditions of Acid Stress.

Bacteria encounter acid stress under a variety of circumstances. Acid stress induces DNA damage and genomic instability, most directly via acid-catalyzed depurination reactions that generate apurinic (abasic, AP) sites on the deoxyribose phosphate backbone. DNA damage responses are important in bacterial resistance to acids. A recent report provided evidence that a DNA repair glycosylase, AlkX, which is capable of initiating the repair of interstrand DNA cross-links (ICLs), contributes to acid resistance by the pulmonary pathogen Acinetobacter baumannii (Kunkle et al. Proc. Nat. Acad. Sci. USA, 2024, 121, e2402422121). This suggested the possibility that AP-derived ICLs might contribute to the acid stress in bacteria. This idea is predicated on earlier work showing that AP sites can generate ICLs via reactions of the ring-opened AP aldehyde with the exocyclic amino groups of nucleobases on the opposing strand of duplex DNA (Price, N. E. J. Am. Chem. Soc. 2014, 136, 3483). However, it was not clear from previous work whether AP-derived ICLs could be generated under conditions of acid stress. The results reported here provide evidence for ICL formation under conditions of acid stress via a sequential process involving acid-catalyzed depurination followed by cross-linking of the resulting AP site with an adenine residue on the opposing strand of duplex DNA. This supports the possibility that AP-derived interstrand cross-links could contribute to the effects of acid stress in bacteria, and proteins involved in the repair of these lesions could be involved in resistance to acid stress.

DNA Damage↗

PubMed2026

Freezing with Light: Photo-Cross-Linking-Assisted Platform Enables GPCR Deorphanization.

Despite their success as drug targets, nearly one hundred G protein-coupled receptors (GPCRs) remain orphan without identified endogenous ligands. Defining these ligand-receptor pairs constitute a fundamental prerequisite for understanding receptor biology and rational drug discovery. However, deorphanization remains inherently challenging due to the transient and interface-specific ligand-GPCR interactions, especially for endogenous ones that are embedded within chemically complex environments. This In Focus article highlights a modularly designed platform that integrates site-specific photo-cross-linking reaction with proteomics to enable ligand-GPCR pairing directly in native biological contexts. Using this strategy, neuropeptide L-LEN was identified as the endogenous ligand for GPR50, forming a regulatory axis that controls energy expenditure and thermogenesis through brain-peripheral interactions.

Receptors, G-Protein-Coupled↗

PubMed2025

Site-Specific Profiling of RNA-Binding Proteins Enabled by Isotopic Signature-Enhanced Mass Spectrometry.

RNA-binding proteins (RBPs) ubiquitously regulate RNA throughout their lifespan, being extensively involved in cellular metabolism and genetic evolution. Therefore, comprehensive identification of the RNA-protein interactions, especially their interfaces with site-specific resolution, is significant to elucidate the intricate biological activities governed by RNA. Nevertheless, it remains challenging for data-dependent acquisition (DDA)-based proteomics to identify the RNA-cross-linked peptides in depth due to the low abundance and negative charge of modified peptides. To address such limitations, we developed an innovative method named "isoRIC" for profiling RNA-binding proteomes with site-specific resolution, which combines the metabolic labeling of isotopic nucleotides for photo-cross-linking of RNA-binding proteins and the real-time targeted LC-MS/MS analysis of RNA-cross-linked peptides. This method shows a dramatic improvement of sensitivity in identifying RNA-cross-linked peptides with low abundance as compared to the DDA-based proteomic approaches, enabling the discovery of novel RNA-binding proteins and precise mapping of RNA-protein binding interfaces at single amino acid resolution. We applied isoRIC in the context of pathogenic mutations and post-translational modifications to highlight the critical role of RNA-binding sites in modulating the RNA-binding ability.

RNA-Binding Proteins↗

PubMed2026

To cleave or not to cleave: a systemic evaluation of DSS versus DSSO for cross-linking mass spectrometry analysis.

Cross-linking mass spectrometry is a powerful method for structural analysis, but choosing between cleavable and non-cleavable cross-linkers remains challenging. We rigorously compared non-cleavable DSS with cleavable DSSO and found that DSS consistently yields more cross-link identifications from isolated protein complexes to bacterial lysates. The advantage of DSS diminishes as sample complexity increases. At the highest complexity tested-human cell lysate-the trend reverses, with DSSO outperforming DSS. The superior performance of DSS in less complex samples is likely explained by its longer and more flexible spacer arm, which interrogates a spatial volume >40% larger than that of DSSO. For both cross-linkers, the number of identified cross-links decreases as the search space expands, but more steeply for DSS. This sharper decline arises from DSS cross-links producing slightly lower fragment ion coverage, not from the absence of signature ions that could reduce search space. Fragment ion coverage is key to interactome mapping: when coverage reaches 85% or above, identification sensitivity hardly decreases as the search space expands, regardless of the cross-linker used. In summary, we recommend DSS for samples no more complex than bacterial lysates. For interactome mapping of mammalian cells, although DSSO outperforms DSS, neither achieves deep interactome coverage.

Cross-Linking Reagents↗

PubMed2026

Library-based, multiplexed strategy for mapping protein interaction networks via crosslinking.

BACKGROUND: Protein-protein interactions are fundamental to cellular function, yet resolving their interaction interfaces and dynamic behaviors in native biological contexts remains challenging, particularly for weak or transient interactions. Crosslinking strategies based on noncanonical amino acids offer an effective means to capture such interactions; however, traditional single-site incorporation provides limited coverage and may overlook critical interaction hotspots. RESULTS: By employing a mutagenesis library, multiple interaction partners and cross-linking sites of a target protein can be simultaneously screened in a single experiment, without prior knowledge of its precise structural or functional features, enabling effective and unbiased analysis of its interaction network. In this study, we constructed an amber codon-scanning mutagenesis library of PSMD10, facilitating independent incorporation of the photocrosslinking ncAA p-azido-phenylalanine at multiple distinct residues. This approach allowed us to systematically interrogate and precisely map potential interaction regions across the protein surface. Coupled with crosslinking mass spectrometry, we identified multiple residues involved in intermolecular interactions, as well as previously unreported interaction partners, including T2FA, TBA1C, and ATRIP. CONCLUSIONS: These findings expand our understanding of PSMD10-associated proteasome interactome, demonstrate a multiplexed strategy for in situ mapping of protein interaction interfaces with broad coverage, and offer a valuable platform for developing therapeutics that target protein-protein interactions.

Protein Interaction Mapping↗

PubMed1978

Cross-linking of nucleosomal histones with monofunctional imidoesters.

Cross-linking experiments with the MONOfunctional imidoester methyl-acetimidate, in the pH range 7.0 - 8.0, on rat liver nucleosomes generate a cross-linking pattern almost identical with the one observed for much longer BIfunctional reagents (e.g. dimethylsuberimidate). Combined cross-linking and trypsin digestion experiments suggest that all or at least the great majority of this cross-linking occurs on trypsin digestible segments (or "tails") of the histones. The formation of oligomers over such extremely short distances and especially the observation of an H3 homodimer suggests a very close proximity of half-nucleosomes.

Animals↗

PubMed1976

[Chemical modification of proteins. 2. Blocking of amino groups and basic amino acids and crosslinking of polypeptide chains in casein and field bean globulin by reaction with dialdehyde starch].

The reaction of dialdehyde starch with casein and field-bean globulin leads to a blocking of the protein amino groups and to a decrease of free lysine, arginine and histidine. Maximum values are reached at high protein concentrations and great molar reagent excess. At best 80-93% of the amino groups or of the available lysine may be blocked in this way. For 1% casein and I and 5% globulin solutions, the pH optimum of the reaction lies at approximately 8; for 5% casein solutions, it is shifted towards the neutral to weakly acidic range. The value for the proportion of unblocked lysine is higher (approximately 5%) when determined by amino-acid analysis after acid total hydrolysis than when measured by means of the colorimetric method according to Carpenter (20%). The difference is designed as reversibly blocked lysine proportion. There is a linear correlation between the proportion of blocked lysine and the relative nutritional value as determined by means of the test organism Tetrahymena pyriformis. Dependently on protein concentration and reagent excess, gel chromatographically detectable cross-linking products of higher molecular weight are formed by the reaction of dialdehyde starch with casein. In 5% protein solutions, such products with molecular weights of less than or equal to 900 000 are the sole detectable components.

Amino Acids↗

PubMed1978

Stabilization of the relaxed state of aspartate transcarbamoylase by modification with a bifunctional reagent.

Native aspartate transcarbamoylase from Escherichia coli was modified with the bifunctional reagent tartaryl diazide in the presence of the substrate carbamoyl phosphate and the substrate analog succinate. The product had the same sedimentation coefficient as the native enzyme but showed a marked increase in affinity for the substrate aspartate with a hyperbolic saturation curve. The Michaelis constant for aspartate (7.4 mM) is similar to that estimated for the relaxed state of the enzyme. The high substrate affinity was not produced if modification was conducted in the absence of substrate analogs or with a monofunctional reagent. The modified enzyme was also desensitized towards the allosteric effectors ATP and CTP. It appears to represent a stabilized relaxed state whose conversion to the taut state is presumably prevented by cross-linking.

Aspartate Carbamoyltransferase↗