The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin.

Synthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants (K(assoc)), to synthetic LacNAc-Benzene and LacNAc-O(CH(2))(8)CONH(2) glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express alpha bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the alpha bundlin monomer. Collectively, these results suggest that alpha bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of alpha bundlin-expressing EPEC strains to host intestinal epithelial cells.

Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile.

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked alpha Gal(1,3)betaGal(1,4)beta Glc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-alpha Gal(1,3)betaGal(1,4)beta Glc sequences with similar avidities. Additional ES-MS experiments using chemically altered alpha Gal(1,3)betaGal(1,4)beta Glc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.

Blackbody infrared radiative dissociation of nonspecific protein-carbohydrate complexes produced by nanoelectrospray ionization: the nature of the noncovalent interactions.

Gas-phase thermal dissociation experiments, implemented with blackbody infrared radiative dissociation (BIRD) and Fourier transform ion cyclotron resonance mass spectrometry, have been performed on a series of protonated and deprotonated 1:1 and protonated 1:2 protein-carbohydrate complexes formed by nonspecific interactions during the nanoflow electrospray (nanoES) ionization process. Nonspecific interactions between the proteins bovine carbonic anhydrase II (CA), bovine ubiquitin (Ubq), and bovine pancreatic trypsin inhibitor and several carbohydrates, ranging in size from mono- to tetrasaccharides, have been investigated. Over the range of temperatures studied (60-190 degrees C), BIRD of the protonated and deprotonated complexes proceeds exclusively by the loss of the carbohydrate in its neutral form. The rates of dissociation of the 1:1 complexes containing a mono- or disaccharide decrease with reaction time, suggesting the presence of two or more kinetically distinct structures produced during nanoES or by gas-phase processes. In contrast, the 1:1 complexes of the tri- and tetrasaccharides exhibit simple first-order dissociation kinetics, a result that, on its own, is suggestive of a single preferred carbohydrate binding site or multiple equivalent sites in the gas phase. A comparative analysis of the dissociation kinetics measured for protonated 1:1 and 1:2 complexes of Ubq with alphaTal[alphaAbe]alphaMan further supports the presence of a single preferred binding site. However, a similar analysis performed on the complexes of CA and alphaTal[alphaAbe]alphaMan suggests that equivalent but dependent carbohydrate binding sites exist in the gas phase. Analysis of the Arrhenius activation parameters (E(a) and A) determined for the dissociation of 1:1 complexes of CA with structurally related trisaccharides provides evidence that neutral intermolecular hydrogen bonds contribute, at least in part, to the stability of the gaseous complexes. Surprisingly, the E(a) values for the complexes of the same charge state are not sensitive to the structure (primary or higher order) of the protein, suggesting that the carbohydrates are able to form energetically equivalent interactions with the various functional groups presented by the protein. For a given protein-carbohydrate complex, the dissociation E(a) is sensitive to charge state, initially increasing and then decreasing with increasing charge. It is proposed that both ionic and neutral hydrogen bonds stabilize the nonspecific protein-carbohydrate complexes in the gas phase and that the relative contribution of the neutral and ionic interactions is strongly influenced by the charge state of the complex, with neutral interactions dominating at low charge states and ionic interactions dominating at high charge states.

Identifying nonspecific ligand binding in electrospray ionization mass spectrometry using the reporter molecule method.

The application of the reporter molecule (M(rep)) method for identifying nonspecific complexes in the ES-MS analysis of protein-ligand and DNA-ligand interactions in vitro is described. To test the reliability of the method, it was applied to the ES-MS analysis of protein-carbohydrate complexes originating from specific interactions in solution and from nonspecific interactions in the ES process. These control experiments confirm the basic assumptions underlying the M(rep) method, namely that nonspecific ligand binding is a random process, and that the ES droplet histories for specific and nonspecific complexes are distinct. The application of the M(rep) method to the ES-MS analysis of the sequential binding of the ethidium cation, a DNA intercalator, to single and double strand oligodeoxynucleotides is also described, and highlights the general utility of the method.

Method for identifying nonspecific protein-protein interactions in nanoelectrospray ionization mass spectrometry.

The nonspecific self-association of proteins in nanoflow electrospray ionization mass spectrometry (nanoES-MS), and the influence of experimental conditions thereon, are investigated using the protein ubiquitin (Ubq) as a model system. Extents of nonspecific protein association generally increase with protein concentration and, interestingly, with decreasing ES spray potential. The extent of self-association is also sensitive to the duration of the accumulation event in an external rf hexapole. Notably, the relative abundance of metal (Na+ and K+) adducts generally increases with the size of nonspecific Ubq multimer. This result suggests that the gaseous ions of monomeric and nonspecific multimeric Ubq have, on average, different ES droplet histories, with monomer ions originating earlier in the ES process than the nonspecific multimeric complexes. This finding forms the basis for a new method for distinguishing between specific and nonspecific protein complexes in ES-MS. A reporter molecule (Mrep), which does not bind specifically to the proteins and protein complexes of interest, is added to the ES solution at high concentration. The distribution of Mrep bound nonspecifically to gaseous ions of the proteins and protein complexes, as determined from the ES mass spectrum, is used to determine whether a given protein complex originates in solution or whether it forms from nonspecific binding during the ES process. The method is demonstrated in cases where the ions of protein complexes detected by nanoES-MS originate exclusively from nonspecific association, exclusively from specific interactions in solution, or from both specific and nonspecific interactions.

Method for stabilizing protein-ligand complexes in nanoelectrospray ionization mass spectrometry.

The interaction between the bovine pancrease trypsin (Tryp) and its competitive inhibitor benzamidine (1), in solution and the gas phase, is investigated using nanoflow electrospray ionization (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry. In a recent study (Clark, S.M.; Konermann L. Anal. Chem. 2004, 76, 7077-7083), it was reported that the (Tryp + 1) complex could not be detected by ES-MS. Here, it is shown that, with gentle sampling conditions, it is possible to detect gaseous protonated ions of the (Tryp + 1) complex with nanoES-MS. However, the relative abundance of the detected (Tryp + 1)n+ ions is lower than expected, based on solution composition, which suggests that dissociation of (Tryp + 1)n+ ions occurs during MS sampling. The dissociation pathways and corresponding Arrhenius parameters for the protonated (Tryp + 1)n+ ions, at n = 7-9, are determined from time-resolved thermal dissociation experiments, implemented with the blackbody infrared radiative dissociation technique. The gaseous (Tryp + 1)n+ ions are found to have short lifetimes, e.g., <0.6 s, at temperatures of >100 degrees C. The use of solution additives, including polyols, carbohydrates, amino acids, and small organic molecules, to stabilize the (Tryp + 1)n+ ions during nanoES-MS analysis is investigated. Notably, the addition of imidazole to the nanoES solution is shown to preserve the (Tryp + 1)n+ ions. The Kassoc value, (1.9 +/- 0.2) x 104 M-1, determined for the (Tryp + 1) complex by the direct ES-MS method is in agreement with values determined by other analytical methods. The stabilizing effect of imidazole in nanoES-MS is further demonstrated for the interaction between carbonic anhydrase II and 5-(dimethylamino)naphthalene-1-sulfonamide. The stabilizing effect of imidazole may be due to enhanced evaporative cooling achieved by the dissociation of molecules of imidazole, bound nonspecifically, from the protein-ligand complex in the ion source.

Method for distinguishing specific from nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry.

A new methodology for distinguishing between specific and nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry (nanoES-MS) is described. The method involves the addition of an appropriate reference protein (P(ref)), which does not bind specifically to any of the solution components, to the nanoES solution containing the protein(s) and ligand(s) of interest. The occurrence of nonspecific protein-ligand binding is monitored by the appearance of nonspecific (P(ref) + ligand) complexes in the nanoES mass spectrum. Furthermore, the fraction of P(ref) undergoing nonspecific ligand binding provides a quantitative measure of the contribution of nonspecific binding to the measured intensities of protein and specific protein-ligand complexes. As a result, errors introduced into protein-ligand association constants, K(assoc), as determined with nanoES-MS, by nonspecific ligand binding can be corrected. The principal assumptions on which this methodology is based, namely, that the fraction of proteins and protein complexes that engage in nonspecific ligand binding during the nanoES process is determined by the number of free ligand molecules in the offspring droplets leading to gaseous ions and is independent of the size and structure of the protein or protein complex, are shown to be generally valid. The application of the method for the determination of K(assoc) for two protein-carbohydrate complexes, under conditions where nonspecific ligand binding is prevalent, is demonstrated.