Author : Monica Gabriela Szczepina
Publisher :
ISBN 13 :
Total Pages : 470 pages
Book Rating : 4.:/5 (112 download)
Book Synopsis Saturation Transfer Difference NMR Studies of Protein-ligand Interactions by : Monica Gabriela Szczepina
Download or read book Saturation Transfer Difference NMR Studies of Protein-ligand Interactions written by Monica Gabriela Szczepina and published by . This book was released on 2011 with total page 470 pages. Available in PDF, EPUB and Kindle. Book excerpt: The mycolyl-arabinogalactan-peptidoglycan complex coats the surface of Mycobacterium tuberculosis. It is a structure composed of galactofuranosyl (Galf) residues attached via alternating -(1→6) and -(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, -D-Galf-(1→6)--D-Galf-(1→5)--D-Galf-O(CH2)7CH3 and -D-Galf-(1→5)--D-Galf-(1→6)--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 -(1→5) and -(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.
