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Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

Chemical cross-linking would conceivably cause structural disruption of a protein, but few cross-linkers have been fully evaluated in this aspect. Furthermore, integral membrane proteins may differ from soluble proteins in the selection of suitable cross-linkers, which has never been investigated. In this study, we systematically evaluated the impact of five conventional cross-linkers targeting Lys, Asp and Glu, and two Arg-reactive cross-linkers on the structural and functional integrity of two G protein-coupled receptors (GPCRs). Perturbation of the receptor structure and ligand-binding activity was observed, depending on the receptor and cross-linking conditions. In particular, our study demonstrated that the concentrations of PDH and KArGO need to be fine-tuned in order to minimize the structural and functional disturbance of specific GPCRs. A set of amenable cross-linkers was selected to acquire the most comprehensive cross-link maps for two GPCRs. Our in-depth cross-linking mass spectrometry (CXMS) analysis has revealed dynamic features of structural regions in GPCRs that are not observable in the crystal structures. Thus, CXMS analysis of GPCRs using the expanded toolkit would facilitate structural modeling of uncharacterized receptors and gain new insights into receptor-ligand interactions. Image 1 • Assess the impact of cross-linkers on GPCR structural integrity with three orthogonal assays. • Evaluate and optimize seven cross-linkers for CXMS analysis of GPCRs. • Arg-reactive cross-linkers are particularly useful for increasing GPCR cross-linking coverage. • Acquire the most comprehensive cross-link maps for two GPCRs to reveal structural dynamics. [ABSTRACT FROM AUTHOR]

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions., (© 2023 Wiley‐VCH GmbH.)

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidlywith surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximityenhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical crosslinking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes. [ABSTRACT FROM AUTHOR]

Nile tilapia skin collagen sponges were fabricated by freeze-drying technology and modified with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of N-hydroxysuccinimide (EDC/NHS), genipin + PBS, genipin + ethanol, tea polyphenol (TP), nordihydroguaiaretic acid (NDGA) and diphenyl phosphoryl azide (DPPA). Physicochemical and biological properties, micromorphology and compatibility before and after modification were investigated to evaluate collagen sponge as a hemostatic biomedical material. The mechanical property of collagen sponges strengthened after cross-linking. The elongation at break of cross-linked collagen sponges decreased except for EDC/NHS, which was close to that of non-crosslinked. The collagen sponge cross-linked with EDC/NHS exhibited the highest hygroscopicity in comparison with other cross-linkers. The resistance to collagenase biodegradation of collagen sponges after cross-linking strengthened significantly except for NDGA. Collagen sponges cross-linked with EDC/NHS, TP and NDGA maintained high porosity (97–98%), similar to non-crosslinked (98.42%). Collagen sponges could shorten the blood coagulation time. From the variations of the FTIR spectrum pattern and SEM, DPPA could change the secondary structure of collagen and destroy the spongy structure of collagen sponge, which was not suitable for the cross-linking of collagen sponge. Whereas, EDC/NHS was recognized as a perfect cross-linker owing to its excellent properties and porous microstructure. All fabricated collagen sponges were recognized to be biocompatible by the hemolysis assay in vitro . Therefore, collagen sponge modified with EDC/NHS could be used as a perfect biomedical hemostatic material. [ABSTRACT FROM AUTHOR]

Gram-positive thermophilic Bacilli contain quinol-cytochrome c reductase and cyto-chrome c oxidase as two major respiratory complexes of the electron transfer chain, and these enzymes can be extracted with mild detergents as an associated quinol oxidase super-complex.The reductase is composed of three subunits; cytochrome b8 cytochrome c1, and FeS protein, whereas cytochrome c oxidase consists of four subunits numbered 1 through 4. In order to clarify the interactions between the subunits, the super-complex isolated from Bacillus PS3 was cross-linked with three bifunctional cross-linkers; disuccinimidyl tartrate, 3,3′-dithiobis(succinimidylpropionate), and ethylene glycolbis(sulfosuccinimi-dylsuccinate). The most prominent cross-linking was observed for the combination of subunit 1 plus 2 in cytochrome c oxidase, and for that of cytochrome b8 plus cytochrome C1 in the reductase. In addition to these intra-complex cross-linkings, inter-complex linking was observed for the combination of cytochrome b8 plus subunit 1 with ethylene glycolbis-(sulfosuccinimidylsuccinate), and for the combinations of cytochrome b8 plus subunit 1 and cytochrome fit, plus subunit 2 with 3,3 -dithiobis(succinimidylpropionate). Incubation in the presence of Triton X-100, which was confirmed to cleave the two enzyme complexes, selectively reduced the inter-complex cross-linking, suggesting that the chemical cross-linking reflect the spatial arrangement of subunits in the super-complex. [ABSTRACT FROM AUTHOR]

Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)