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  1. Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile [Download]

    Title: Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile
    Creator: Murase, Tomohiko
    Description: Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.
    Subjects: Mass Spectrometry (MS), Protein-Protein Interactions, X-ray Crystallography, Antibodies, Bacterial Toxins
    Date Created: 2014
  2. Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein [Download]

    Title: Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein
    Creator: Hug, Isabelle
    Description: Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.
    Subjects: Bacterial Glycoproteins, Glycoengineering, Glycoprotein, Glycoconjugate, Fucosyltransferase, Helicobacter pylori, Glycosyltransferases, Glycosylation, Oligosaccharyltansferase
    Date Created: 2011
  3. Thermal dissociation of streptavidin homotetramer in the gas phase: Subunit loss versus backbone fragmentation [Download]

    Title: Thermal dissociation of streptavidin homotetramer in the gas phase: Subunit loss versus backbone fragmentation
    Creator: Kitova, Elena N.
    Description: The results of time-resolved blackbody infrared radiative dissociation experiments performed on gaseous protonated ions, at charge states +15, +16 and +17, of the homotetramer streptavidin (S4) are reported. Evidence is found for three dissociation pathways involving (i) the loss of a single subunit, (ii) covalent cleavage of the backbone of one of the subunits and ejection of the resultant b212+ ion (followed by loss of complementary y106 ions), and (iii) the direct loss of one or more water molecules. The contribution of the different dissociation channels was found to be dependent on temperature, with the loss of subunit dominating at higher reaction temperatures and backbone fragmentation dominating at lower temperatures, and reaction times, with longer times favoring covalent fragmentation. Analysis of the dissociation kinetics and the influence of reaction time on the relative abundance of product ions indicate that backbone fragmentation and subunit loss are not produced via parallel pathways from a single reactant. Instead, the results suggest the presence of multiple, non-interconverting structures, which contribute differentially to the backbone fragmentation and subunit loss pathways. The results of molecular dynamics simulations performed on S416+ ions with different charge configurations suggest that unfolding of the N-terminus of the subunit may be associated with the backbone fragmentation pathway.
    Subjects: Blackbody infrared radiative dissociation, Dissociation kinetics, Protein complexes, Dissociation pathways
    Date Created: 2013
  4. Energetics of intermolecular hydrogen bonds in a hydrophobic protein cavity [Download]

    Title: Energetics of intermolecular hydrogen bonds in a hydrophobic protein cavity
    Creator: Liu, Lan
    Description: This work explores the energetics of intermolecular H-bonds inside a hydrophobic protein cavity. Kinetic measurements were performed on the gaseous deprotonated ions (at the −7 charge state) of complexes of bovine β-lactoglobulin (Lg) and three monohydroxylated analogs of palmitic acid (PA): 3-hydroxypalmitic acid (3-OHPA), 7-hydroxypalmitic acid (7-OHPA), and 16-hydroxypalmitic acid (16-OHPA). From the increase in the activation energy for the dissociation of the (Lg + X-OHPA)7– ions, compared with that of the (Lg + PA)7– ion, it is concluded that the –OH groups of the X-OHPA ligands participate in strong (5 – 11 kcal mol–1) intermolecular H-bonds in the hydrophobic cavity of Lg. The results of molecular dynamics (MD) simulations suggest that the –OH groups of 3-OHPA and 16-OHPA act as H-bond donors and interact with backbone carbonyl oxygens, whereas the –OH group of 7-OHPA acts as both H-bond donor and acceptor with nearby side chains. The capacity for intermolecular H-bonds within the Lg cavity, as suggested by the gas-phase measurements, does not necessarily lead to enhanced binding in aqueous solution. The association constant (Ka) measured for 7-OHPA [(2.3 ± 0.2) × 105 M–1] is similar to the value for the PA [(3.8 ± 0.1) × 105 M–1]; Ka for 3-OHPA [(1.1 ± 0.3) × 106 M–1] is approximately three-times larger, whereas Ka for 16-OHPA [(2.3 ± 0.2) × 104 M–1] is an order of magnitude smaller. Taken together, the results of this study suggest that the energetic penalty to desolvating the ligand –OH groups, which is necessary for complex formation, is similar in magnitude to the energetic contribution of the intermolecular H-bonds.
    Subjects: Hydrogen bonds, Kinetics energetics, Hydrophobic, Protein–ligand complexes
    Date Created: 2014
  5. Fluorine bonding enhances the energetics of protein-lipid binding in the gas phase [Download]

    Title: Fluorine bonding enhances the energetics of protein-lipid binding in the gas phase
    Creator: Liu, Lan
    Description: This paper reports on the first experimental study of the energies of noncovalent fluorine bonding in a protein-ligand complex in the absence of solvent. Arrhenius parameters were measured for the dissociation of gaseous deprotonated ions of complexes of bovine β-lactoglobulin (Lg), a model lipid-binding protein, and four fluorinated analogs of stearic acid (SA), which contained (X =) 13, 15, 17, or 21 fluorine atoms. In all cases, the activation energies (Ea) measured for the loss of neutral XF-SA from the (Lg + XF-SA)7– ions are larger than for SA. From the kinetic data, the average contribution of each > CF2 group to Ea was found to be ~1.1 kcal mol–1, which is larger than the ~0.8 kcal mol–1 value reported for > CH2 groups. Based on these results, it is proposed that fluorocarbon–protein interactions are inherently stronger (enthalpically) than the corresponding hydrocarbon interactions.
    Subjects: Protein-ligand complexes, Energetics, Fluorine bonding, Molecular recognition, Hydrophobic interactions
    Date Created: 2014
  6. Dissociation of multisubunit protein-ligand complexes in the gas phase: Evidence for ligand migration [Download]

    Title: Dissociation of multisubunit protein-ligand complexes in the gas phase: Evidence for ligand migration
    Creator: Zhang, Yixuan
    Description: The results of collision-induced dissociation (CID) experiments performed on gaseous protonated and deprotonated ions of complexes of cholera toxin B subunit homopentamer (CTB5) with the pentasaccharide (β-D-Galp-(1→3)-β-D-GalpNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Galp-(1→4)-β-D-Glcp (GM1)) and corresponding glycosphingolipid (β-D-Galp-(1→3)-β-D-GalpNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Galp-(1→4)-β-D-Glcp-Cer (GM1-Cer)) ligands, and the homotetramer streptavidin (S4) with biotin (B) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Btl), are reported. The protonated (CTB5 + 5GM1)n+ ions dissociated predominantly by the loss of a single subunit, with the concomitant migration of ligand to another subunit. The simultaneous loss of ligand and subunit was observed as a minor pathway. In contrast, the deprotonated (CTB5 + 5GM1)n- ions dissociated preferentially by the loss of deprotonated ligand; the loss of ligand-bound and ligand-free subunit were minor pathways. The presence of ceramide (Cer) promoted ligand migration and the loss of subunit. The main dissociation pathway for the protonated and deprotonated (S4 + 4B)n+/– ions, as well as for deprotonated (S4 + 4Btl)n– ions, was loss of the ligand. However, subunit loss from the (S4 + 4B)n+ ions was observed as a minor pathway. The (S4 + 4Btl)n+ ions dissociated predominantly by the loss of free and ligand-bound subunit. The charge state of the complex and the collision energy were found to have little effect on the relative contribution of the different dissociation channels. Thermally-driven ligand migration between subunits was captured in the results of molecular dynamics simulations performed on protonated (CTB5 + 5GM1)15+ ions (with a range of charge configurations) at 800 K. Notably, the migration pathway was found to be highly dependent on the charge configuration of the ion. The main conclusion of this study is that the dissociation pathways of multisubunit protein–ligand complexes in the gas phase depend, not only on the native topology of the complex, but also on structural changes that occur upon collisional activation.
    Subjects: Collision-induced dissociation, Dissociation mechanism, Multisubunit protein complex, Ligand migration
    Date Created: 2013
  7. Dissociation kinetics of the streptavidin-biotin interaction measured using direct electrospray ionization mass spectrometry analysis [Download]

    Title: Dissociation kinetics of the streptavidin-biotin interaction measured using direct electrospray ionization mass spectrometry analysis
    Creator: Deng, Lu
    Description: Dissociation rate constants (koff) for the model high affinity interaction between biotin (B) and the homotetramer of natural core streptavidin (S4) were measured at pH 7 and temperatures ranging from 15 to 45 °C using electrospray ionization mass spectrometry (ESI-MS). Two different approaches to data analysis were employed, one based on the initial rate of dissociation of the (S4 + 4B) complex, the other involving nonlinear fitting of the time-dependent relative abundances of the (S4 + iB) species. The two methods were found to yield koff values that are in good agreement, within a factor of two. The Arrhenius parameters for the dissociation of the biotin–streptavidin interaction in solution were established from the koff values determined by ESI-MS and compared with values measured using a radiolabeled biotin assay. Importantly, the dissociation activation energies determined by ESI-MS agree, within 1 kcal mol–1, with the reported value. In addition to providing a quantitative measure of koff, the results of the ESI-MS measurements revealed that the apparent cooperative distribution of (S4 + iB) species observed at short reaction times is of kinetic origin and that sequential binding of B to S4 occurs in a noncooperative fashion with the four ligand binding sites being kinetically and thermodynamically equivalent and independent.
    Subjects: Protein–ligand complexes, Electrospray ionization mass spectrometry, Rate constants
    Date Created: 2013
  8. Reliable determinations of protein-ligand interactions by direct ESI-MS measurements: Are we there yet? [Download]

    Title: Reliable determinations of protein-ligand interactions by direct ESI-MS measurements: Are we there yet?
    Creator: Kitova, Elena N.
    Description: The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein–ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein–ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.
    Subjects: Electrospray ionization mass spectrometry, Binding assay, Library screening, Protein-ligand interaction, Association constant
    Date Created: 2012
  9. Nonspecific interactions between proteins and charged biomolecules in electrospray ionization mass spectrometry [Download]

    Title: Nonspecific interactions between proteins and charged biomolecules in electrospray ionization mass spectrometry
    Creator: Sun, Nian
    Description: An investigation of the nonspecific association of small charged biomolecules and proteins in electrospray ionization mass spectrometry (ES-MS) is described. Aqueous solutions containing pairs of proteins and a small acidic or basic biomolecule that does not interact specifically with either of the proteins were analyzed by ES-MS and the distributions of the biomolecules bound nonspecifically to each pair of proteins compared. For the basic amino acid arginine and the peptide RGVFRR, nonequivalent distributions were measured in positive ion mode, but equivalent distributions were measured in negative ion mode. In the case of uridine 5′-diphosphate, nonequivalent distributions were measured in negative ion mode, but equivalent distributions observed in positive ion mode. The results of dissociation experiments performed on the gaseous ions of the nonspecific complexes suggest that the nonequivalent distributions result from differences in the extent to which the nonspecific complexes undergo in-source dissociation. To test this hypothesis, the distributions of nonspecifically bound basic molecules measured in the presence of imidazole, which protects complexes from in-source dissociation, were compared. In all cases, equivalent distributions were obtained. The results indicate that nonspecific binding of charged molecules to proteins during ES is a statistical process, independent of protein structure and size. However, the kinetic stabilities of the nonspecific interactions are sensitive to the nature of the protein ions. It is concluded that the reference protein method for correcting ES mass spectra for nonspecific ligand-protein binding can be applied to the analysis of ionic ligands, provided that in-source dissociation of the nonspecific interactions is minimized.
    Subjects: Affinities, Binding, Noncovalent complexes, Ligand complexes, Constants, Stability, Analogs, Nanoelectrospray ionization, Infrared radiative dissociation
    Date Created: 2010
  10. Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry [Download]

    Title: Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry
    Creator: El-Hawiet, Amr
    Description: A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (Lref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to Lref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.
    Subjects: Monoclonal-antibody, Complexes, Substrate, Concanavalin-A, Blood-group glycosyltransferases, Recognition, Dissociation-constants, Binding affinities
    Date Created: 2010