Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewisx bound to E-selectin |
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Authors: | Karoline Scheffler Jean-Robert Brisson Rüdiger Weisemann John L Magnani Wei Tong Wong Beat Ernst Thomas Peters |
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Institution: | (1) Institute for Chemistry, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany;(2) National Research Council, Institute for Biological Sciences, Ottawa, ON, Canada, K1A 0R6;(3) Bruker Analytik GmbH, Silberstreifen, D-76287 Rheinstetten, Germany;(4) Glyco Tech Corporation, 14915 Broschart Road, Rockville, MD, 20850, U.S.A;(5) Zentrale Forschungslaboratorien, Rosental, Ciba-Geigy AG, CH-4002 Basel, Switzerland |
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Abstract: | The conformation of the sialyl Lewis x tetrasaccharidebound to E-selectin was previously determined from transfer NOE (trNOE)experiments in conjunction with a distance-geometry analysis. However, theorientation of the tetrasaccharide ligand in the binding site of E-selectinis still unknown. It can be predicted that the accurate quantitativeanalysis of all trNOEs, including those originating from spin diffusion, isone key to analyze the orientation of sialyl Lewisx in thebinding pocket of E-selectin. Therefore, we applied homonuclear 3D NMRexperiments and 1D analogs to obtain trNOEs that could not unambiguously beassigned from previous 2D trNOESY spectra, due to severe resonance-signaloverlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a1D trNOESY-TOCSY experiment of the sialyl Lewisx/E-selectincomplex furnished new interglycosidic trNOEs and provided additionalinformation for the interpretation of trNOEs that have been describedbefore. A 2D trROESY spectrum of the sialyl Lewisx/E-selectincomplex allowed one to identify the amount of spin-diffusion contributionsto trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysisof spin-diffusion pathways, was obtained, creating a basis for aquantitative analysis of trNOEs in the sialylLewisx/E-selectin complex. |
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Keywords: | 3D TOCSY-trNOESY 1D TOCSY-trNOESY 1D trNOESY-TOCSY trNOE trROE E-selectin sialyl Lewisx |
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