Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation.
View Article and Find Full Text PDFInteractions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging.
View Article and Find Full Text PDFElectrospray ionization mass spectrometry (ESI-MS) is a powerful label-free assay for detecting noncovalent biomolecular complexes and is increasingly used to quantify binding thermochemistry. A common assumption made in ESI-MS affinity measurements is that the relative ion signals of free and bound species quantitatively reflect their relative concentrations in solution. However, this is valid only when the interacting species and their complexes have similar ESI-MS response factors (RFs).
View Article and Find Full Text PDFMass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured.
View Article and Find Full Text PDFCarbohydrate-active enzymes (CAZymes) play critical roles in diverse physiological and pathophysiological processes and are important for a wide range of biotechnology applications. Kinetic measurements offer insight into the activity and substrate specificity of CAZymes, information that is of fundamental interest and supports diverse applications. However, robust and versatile kinetic assays for monitoring the kinetics of intact glycoprotein and glycolipid substrates are lacking.
View Article and Find Full Text PDFCarbohydrate-Active enZymes (CAZymes) are involved in the synthesis, degradation, and modification of carbohydrates. They play critical roles in diverse physiological and pathophysiological processes, have important industrial and biotechnological applications, are important drug targets, and represent promising biomarkers for the diagnosis of a variety of diseases. Measurements of their activities, catalytic pathway, and substrate specificities are essential to a comprehensive understanding of the biological functions of CAZymes and exploiting these enzymes for industrial and biomedical applications.
View Article and Find Full Text PDFInteractions between glycosphingolipids (GSLs) on the surfaces of cells and glycan-binding proteins (GBPs) mediate a wide variety of essential and pathological processes. Despite the biological importance of these interactions, the GSL ligands of most GBPs remain to be identified and the mechanisms controlling recognition of GSLs are incompletely understood. Recently, it was suggested that, when present together with high affinity ligands, low affinity GSL ligands can contribute significantly to the binding of GBPs with multiple binding sites through a process called heteromultivalent binding.
View Article and Find Full Text PDFGlycan interactions with glycan-binding proteins (GBPs) play essential roles in a wide variety of cellular processes. Currently, the glycan specificities of GBPs are most often inferred from binding data generated using glycan arrays, wherein the GBP is incubated with oligosaccharides immobilized on a glass surface. Detection of glycan-GBP binding is typically fluorescence-based, involving the labeling of the GBP with a fluorophore or with biotin, which binds to fluorophore-labeled streptavidin, or using a fluorophore-labeled antibody that recognizes the GBP.
View Article and Find Full Text PDFGlycan binding by glycan-binding proteins and processing by carbohydrate-active enzymes is implicated in physiological and pathophysiological processes. Comprehensive mapping of glycan interactions is essential to understanding of glycan-mediated biology and can guide the development of new diagnostics and therapeutics. Here, we introduce the competitive universal proxy receptor assay (CUPRA), which combines electrospray ionization mass spectrometry, competitive binding and heterobifunctional glycan-based ligands to give a quantitative high-throughput method for screening glycan libraries against glycan-binding and glycan-processing proteins.
View Article and Find Full Text PDFElectrospray ionization mass spectrometry (ESI-MS) screening of compound libraries against target proteins enables the rapid identification of ligands and measurement of the stoichiometry and affinity of the interactions. However, non-specific association of buffer or salts (added or present as impurities) to the protein ions during gas-phase ion formation can complicate the analysis of ESI-MS data acquired for mixtures of compounds with similar molecular weights. Spectral overlap of ions corresponding to free protein and protein-ligand complexes and their corresponding adducts can hinder the identification of ligands and introduce errors in the measured affinities.
View Article and Find Full Text PDFHuman milk oligosaccharides (HMOs) afford many health benefits to breast-fed infants, such as protection against infection and regulation of the immune system, through the formation of non-covalent interactions with protein receptors. However, the molecular details of these interactions are poorly understood. Here, we describe the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening natural libraries of HMOs against lectins.
View Article and Find Full Text PDFUsing amphiphilic cyclodextrin as a scaffold, the first class of P-glycoconjugates capable of high avidity binding to both Stx1 and Stx2 toxins in solid-phase assay formats is reported. The generated glycomicroarray effectively mimics the plasma membrane surface while discriminating binding of the two Stx toxins, with unprecedented affinity to Stx2.
View Article and Find Full Text PDFThe affinities of thirty-two free human milk oligosaccharides (HMOs) for four human galectin proteins, a stable mutant of hGal1 (hGal-1), a C-terminal fragment of hGal-3 (hGal-3C), hGal-7, and an N-terminal fragment of hGal-9 (hGal-9N), were measured using electrospray ionization mass spectrometry (ESI-MS). The binding data show that each of the four galectins recognize the majority of the HMOs tested (hGal-1 binds thirty-two HMOs, hGal-3C binds twenty-six, hGal-7 binds thirty-one, and hGal-9N binds twenty-six). Twenty-five of the HMOs tested bind all four galectins, with affinities ranging from 10 to 10 M.
View Article and Find Full Text PDFIn vitro selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with "silent barcodes" to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]4-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]4-GGAGGAGGA, AGT-[X]4-GGTGGTGGT, etc.
View Article and Find Full Text PDFWe describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD.
View Article and Find Full Text PDFA focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction.
View Article and Find Full Text PDFWe describe the rapid reaction of 2-amino benzamidoxime (ABAO) derivatives with aldehydes in water. The ABAO combines an aniline moiety for iminium-based activation of the aldehyde and a nucleophilic group (Nu:) ortho to the amine for intramolecular ring closure. The reaction between ABAO and aldehydes is kinetically similar to oxime formations performed under stoichiometric aniline catalysis.
View Article and Find Full Text PDFCopovidone, a copolymer of vinyl acetate and N-vinyl-2-pyrrolidone, was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and after deacetylation the polymer was functionalized by introduction of amino, azide, and alkyne pendant groups to allow attachment of glycans and peptide. Candida albicans β-mannan trisaccharides 1 and 2 and M. tuberculosis arabinan hexasaccharide 3 with appropriate tethers were conjugated to the polymers by squarate or click chemistry.
View Article and Find Full Text PDFA new microtiter-plate-based method for the rapid generation and evaluation of focused compound libraries was developed and applied to screening ligand analogues for the E. coli Shiga-like toxin Stx2a. The method is general, it mitigates the masking of intrinsic affinity gains by multivalency and enables the discovery of potential hits when starting from ligands that exhibit extremely low affinity with proteins that depend on multivalency for their function.
View Article and Find Full Text PDFShiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany.
View Article and Find Full Text PDFLight-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library.
View Article and Find Full Text PDFA convenient scaffold based on poly(N-vinyl-2-pyrrolidone-co-vinyl alcohol) is proposed for presenting ligands in multivalent format. This amphiphilic polymer supports synthesis of conjugates in both organic and aqueous media, permits enzymatic processing of the ligand precursor, and, finally, offers a choice of formats for evaluation of biological activity either as a soluble inhibitor or as a capture reagent after deposition on a hydrophobic surface or standard microtiter plates.
View Article and Find Full Text PDFHemolytic uremic syndrome is a potentially fatal complication of food poisoning caused by Escherichia coli O157:H7, especially those strains that produce the Stx2 Shiga toxin. Multivalent inhibitors based on the P(k) trisaccharide are most effective against Stx1 the less dangerous of the two Shiga toxins. Inhibitors containing a terminal 2-acetamido-2-deoxy-α-d-galactopyranosyl residue in place of the terminal α-d-galactopyranosyl residue of P(k) trisaccharide have been shown to exhibit preferential binding to Stx2.
View Article and Find Full Text PDFNoroviruses (NoVs), the major cause of viral acute gastroenteritis, recognize histo-blood group antigens (HBGAs) as receptors or attachment factors. To gain a deeper understanding of the interplay between NoVs and their hosts, the affinities of recombinant P dimers (P₂'s) of a GII.4 NoV (VA387) to a library of 41 soluble analogs of HBGAs were measured using the direct electrospray ionization mass spectrometry assay.
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