Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker.
Ontology highlight
ABSTRACT:
SUBMITTER: Vila-Perello M
PROVIDER: S-EPMC3242410 | biostudies-literature | 2007 Jul
REPOSITORIES: biostudies-literature
Publications
Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker.
Vila-Perelló Miquel M Pratt Matthew R MR Tulin Frej F Muir Tom W TW
Journal of the American Chemical Society 20070613 26
Similar Datasets
Project description:Minimalist photo-cross-linker 1 and its derivatives are extensively utilized in photoaffinity labeling studies. However, obtaining compound 1 in high yield has traditionally required a lengthy synthetic process. In this paper, we present a concise and efficient method to synthesize 1 in just four steps, leveraging the "Normant reagent" as a pivotal component in our strategy. Additionally, we extended our synthetic approach to generate new derivatives of the fully functionalized diazirine tag, providing versatile handles for coupling with other small molecules. This work provides a quick and high-yielding approach to access photo-cross-linker 1 compared to previously reported approaches.
| S-EPMC11800027 | biostudies-literature
Project description:A significant challenge for solid tumor treatment is ensuring that a sufficient concentration of therapeutic agent is delivered to the tumor site at doses that can be tolerated by the patient. Biomolecular targeting can bias accumulation in tumors by taking advantage of specific interactions with receptors overexpressed on cancerous cells. However, while antibody-based immunoconjugates show high binding to specific cells, their low dissociation constants ( KD) and large Stokes radii hinder their ability to penetrate deep into tumor tissue, leading to incomplete cell killing and tumor recurrence. To address this, we demonstrate the design and production of a photo-cross-linkable affibody that can form a covalent bond to epidermal growth factor receptor (EGFR) under near UV irradiation. Twelve cysteine mutations were created of an EGFR affibody and conjugated with maleimide-benzophenone. Of these only one exhibited photoconjugation to EGFR, as demonstrated by SDS-PAGE and Western blot. Next this modified affibody was shown to not only bind EGFR expressing cells but also show enhanced retention in a 3D tumor spheroid model, with minimal loss up to 24 h as compared to either unmodified EGFR-binding affibodies or nonbinding, photo-cross-linkable affibodies. Finally, in order to show utility of photo-cross-linking at clinically relevant wavelengths, upconverting nanoparticles (UCNPs) were synthesized that could convert 980 nm light to UV and blue light. In the presence of UCNPs, both direct photoconjugation to EGFR and enhanced retention in tumor spheroids could be obtained using near-infrared illumination. Thus, the photoactive affibodies developed here may be utilized as a platform technology for engineering new therapy conjugates that can penetrate deep into tumor tissue and be retained long enough for effective tumor therapy.
| S-EPMC6689236 | biostudies-literature
Project description:Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid instrument with improved fragmentation capacities in a systematic comparison to identify which fragmentation method would be best for the identification of cross-linked peptides. Specifically, we explored three fragmentation methods and two combinations: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human serum albumin (HSA) served as a test sample, yielding over all methods and in triplicate analysis in total 2602 matched PSMs and 1390 linked residue pairs at 5% false discovery rate, as confirmed by the crystal structure. HCD wins in number of matched peptide-spectrum-matches (958 PSMs) and identified links (446). CID is most complementary, increasing the number of identified links by 13% (58 links). HCD wins together with EThcD in cross-link site calling precision, with approximately 62% of sites having adjacent backbone cleavages that unambiguously locate the link in both peptides, without assuming any cross-linker preference for amino acids. Overall quality of spectra, as judged by sequence coverage of both peptides, is best for EThcD for the majority of peptides. Sequence coverage might be of particular importance for complex samples, for which we propose a data dependent decision tree, else HCD is the method of choice. The mass spectrometric raw data has been deposited in PRIDE (PXD003737).
| S-EPMC5047652 | biostudies-literature
Project description:Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.
| S-EPMC3178686 | biostudies-literature
Project description:Ubiquitin-mediated signaling pathways regulate essentially every aspect of cell biology in eukaryotes. Ubiquitin receptors typically contain ubiquitin-binding domains (UBDs) that have the ability to recognize monomeric ubiquitin (Ub) and polymeric Ub (polyUb) chains. However, how signaling specificity is achieved remains poorly understood, and many of the UBDs that selectively recognize polyUb chains of particular linkages still need to be identified and characterized. Here we report the incorporation of a genetically encoded photo-cross-linker, p-benzoyl-l-phenylalanine (Bpa), into recombinant Ub and enzymatically synthesized polyUb chains. This allows photo-cross-linking (covalent bond formation) of monoUb and K48- and K63-linked diUb chains to UBDs. This approach provides a framework for understanding Ub cellular signaling through the capture and identification of (poly)Ub-binding proteins.
| S-EPMC6699165 | biostudies-literature
Project description:While photo-cross-linking (PXL) with alkyl diazirines can provide stringent distance restraints and offer insights into protein structures, unambiguous identification of cross-linked residues hinders data interpretation to the same level that has been achieved with chemical cross-linking (CXL). We address this challenge by developing an in-line system with systematic modulation of light intensity and irradiation time, which allows for a quantitative evaluation of diazirine photolysis and photo-reaction mechanism. Our results reveal a two-step pathway with mainly sequential generation of diazo and carbene intermediates. Diazo intermediate preferentially targets buried polar residues, many of which are inaccessible with known CXL probes for their limited reactivity. Moreover, we demonstrate that tuning light intensity and duration enhances selectivity towards polar residues by biasing diazo-mediated cross-linking reactions over carbene ones. This mechanistic dissection unlocks the full potential of PXL, paving the way for accurate distance mapping against protein structures and ultimately, unveiling protein dynamic behaviors.
| S-EPMC11258254 | biostudies-literature
Project description:N-glycans are displayed on cell-surface proteins and can engage in direct binding interactions with membrane-bound and secreted glycan-binding proteins (GBPs). Biochemical identification and characterization of glycan-mediated interactions is often made difficult by low binding affinities. Here we describe the metabolic introduction of a diazirine photo-cross-linker onto N-acetylglucosamine (GlcNAc) residues of N-linked glycoproteins on cell surfaces. We characterize sites at which diazirine-modified GlcNAc is incorporated, as well as modest perturbations to glycan structure. We show that diazirine-modified GlcNAc can be used to covalently cross-link two extracellular GBPs, galectin-1 and cholera toxin subunit B, to cell-surface N-linked glycoproteins. The extent of cross-linking correlates with display of the preferred glycan ligands for the GBPs. In addition, covalently cross-linked complexes could be isolated, and protein components of cross-linked N-linked glycoproteins were identified by proteomics analysis. This method may be useful in the discovery and characterization of binding interactions that depend on N-glycans.
| S-EPMC8792112 | biostudies-literature
Project description:Cross-linking mass spectrometry (XL-MS) has seen significant improvements which have enhanced its utility for studying protein-protein interactions (PPIs), primarily due to the emergence of novel crosslinkers and the development of streamlined analysis workflows. Nevertheless, poor membrane permeability and side reactions with water limit the extent of productive intracellular crosslinking events that can be achieved with current crosslinkers. To address these problems, we have synthesized a novel crosslinker with o-nitrobenzyl-based photoresponsive groups. These o-nitrobenzyl ester (o-NBE) groups enhance the stability and hydrophobic properties of the crosslinker and add the potential for temporal resolution, i. e. the ability to control the initiation of the crosslinking reaction. Upon exposure to UV light the resulting aldehyde product reacts with adjacent amino groups and subsequent reductive amination of the formed Schiff-bases yields stable secondary amine linkages. This controlled activation mechanism enables precise UV-triggered protein crosslinking. We demonstrate proof-of principle of our o-NBE cross-linker to reliably detect PPIs by XL-MS using a recombinant model protein. We also demonstrate its ability to enter intact Hela cells, thereby indicating its future potential as a useful tool to study PPIs within the cellular environment.
| S-EPMC11664911 | biostudies-literature
Project description:Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA-protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA-protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA-protein interactions.
| S-EPMC7003851 | biostudies-literature
Project description:Peptides are important species for a variety of biological functions. Detection and analysis of these molecules can be complicated by the presence of background matrix or contaminants. Therefore, a selective method to capture peptides could provide researchers with an option to isolate these remarkable species. Our goal was to perform a set of experiments that would validate the concept of a novel, selective peptide capture, whereby peptides are isolated on functionalized magnetic beads through the use of the heterobifunctional cross-linker, Sulfo-LC-SPDP. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to monitor mass changes associated with the cross-linking reaction. MALDI-TOF MS was then used to monitor conjugation between the cross-linked peptides and sulfhydryl magnetic beads by analyzing supernatant solutions for the presence or absence of cross-linked peptide. Through these experiments, we have proof of concept data confirming that peptides can be isolated on sulfhydryl magnetic beads by using Sulfo-LC-SPDP. This method is a suitable selective global peptide isolation strategy to separate the molecules from contaminating species or sample matrix. This novel method has a variety of potential applications and detection methods.
| S-EPMC4118834 | biostudies-literature