Saturation transfer difference NMR studies of protein-ligand interactions

The mycolyl–arabinogalactan–peptidoglycan complex coats the surface of Mycobacterium tuberculosis. It is a structure composed of galactofuranosyl (Galf) residues attached via alternating β-(1→6) and β-(1→5) linkages synthesized by bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We have used Saturation Transfer Difference (STD) NMR spectroscopy to examine the active site architecture of GlfT2 using trisaccharide acceptor substrates, β-D-Galf-(1→6)-β-D-Galf-(1→5)-β-D-Galf-O(CH2)7CH3 and β-D-Galf-(1→5)-β-D-Galf-(1→6)-β-D-Galf-O(CH2)7CH3. The STD NMR epitope maps demonstrated a greater enhancement toward the “reducing” ends of both trisaccharides, and that UDP-galactofuranose (UDP-Galf) made more intimate contacts through its nucleotide moiety. This observation is consistent with the greater flexibility required within the active site of the reaction between the growing polymer acceptor and the UDP-Galf donor. Competition STD NMR titration experiments with the trisaccharide acceptor substrates demonstrated that they bind competitively at the same site, suggesting that GlfT2 has one active site pocket capable of catalyzing both β-(1→5) and β-(1→6)-galactofuranosyl transfer reactions. STD NMR spectroscopy was also used to probe the bioactive conformation of the carbohydrate mimic MDWNMHAA of the O-polysaccharide of the Shigella flexneri Y bacterium when bound to its complementary antibody, mAb SYA/J6. The dynamic ligand epitope was mapped with the CORCEMA-ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) program that calculates STD-NMR intensities. Comparison of these predicted STD enhancements with experimental data was used to select a representative binding mode. The bound conformation was further refined with a simulated annealing refinement protocol known as STD-NMR Intensity-restrained CORCEMA Optimization (SICO) to give a more accurate representation of the bound peptide epitope. X-ray crystallographic data of MDWNMHAA when bound to mAb SYA/J6 indicated the immobilization of water molecules in the combining site. Water Ligand Observed via Gradient Spectroscopy (WaterLOGSY) was used in conjunction with STD NMR spectroscopy to provide insight into the presence of water molecules that exist at the interstitial sites between the peptide and the antibody. Molecular dynamics calculations have also provided a more accurate picture of the possibilities for bound-ligand conformations, and water molecules involved in providing complementarity between the peptide and SYA-J6

Devaney's definition of chaos in topological spaces

Głównym celem pracy jest pokazanie, że w przestrzeniach metrycznych wrażliwość na warunki początkowe wynika z pozostałych dwóch warunków definicji chaosu Devaneya. Omówiono także jakie implikacje zachodzą między warunkami występującymi w definicji Devaneya w ogólnych przestrzeniach topologicznych.The primary purpose of the present thesis is to prove that, for topological dynamical systems on infinite metric spaces, the sensitive dependence on initial conditions follows from the other two conditions appearing in Devaney's definition of chaos. Possible implications between these conditions in general topological spaces are also discussed

Ether formation on the tridentate Schiff base ligands of copper(II) complexes

A series of copper(II) complexes, CuL·imidazole, where L2− are tridentate Schiff base ligands formed by condensation of salicylaldehyde with a series of amino acids, have been synthesized. Visible spectral data indicate that copper(II) in these complexes are five coordinate in the solid state and in solution. Electrospray mass spectrometry has been used to show how these complexes react in alcohol/NaOH solutions with and without the presence of d-galactose. In the absence of d-galactose where the amino acid in the ligand is serine, the alcohol group on the ligand is converted to its alkyl ether after sonication of the solution for up to 4 h. In the presence of d-galactose, an alkoxy group is added to the ligands except for the ligand containing serine after sonication of the solutions for up to 4 h. At the same time, d-galactose is oxidized to its aldehyde. Where the ligand contains methionine, oxygen is also added to the ligand, most likely to the thioether sulfur

Ligand-Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

The direct evaluation of dissociation constants (KD) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor–ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of KD, as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor–ligand dissociation constants (KD) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein–ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein–ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein–ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein–ligand systems: the binding of the saccharides GlcNAc and GlcNAcß1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental KD measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein–ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available

Synthesis and Immunological Characterization of Modified Hyaluronic Acid Hexasaccharide Conjugates

The synthesis of a tetanus toxoid (TT)-conjugate of a hyaluronic acid (HA) hexasaccharide is described. The compound was intended for use in monitoring HA levels as a disease marker and as a potential vaccine against Group A <i>Streptococcus</i> (GAS) infections. We also report the synthesis of a chemically modified HA-hexasaccharide-TT conjugate in which the <i>N</i>-acetyl moiety of the <i>N</i>-acetyl-d-glucosamine residue is replaced with an <i>N</i>-propionyl unit in order to enhance immunogenicity. The oligosaccharides are synthesized in a convergent manner. The TT-conjugate syntheses rely on the reaction of the amines on the 6-aminohexyl aglycon of the hexasaccharides with diethyl squarate to give the monoethyl squarate adducts. Subsequent reactions with lysine ε-amino groups on TT then give the glycoconjugates containing an average of 8 hexasaccharide haptens per TT molecule. Immunological studies in mice show very similar antibody responses with both conjugates, suggesting that the <i>N</i>-acetyl groups of the glucosaminyl residues of the HA-hexasaccharide are not a critical part of the epitope recognized by the anti-HA polyclonal immune response. Furthermore, it would appear that the <i>N</i>-acyl moieties are not in close contact with the amino acid residues of the antibody combining sites