APPLICATION OF 2-DIMENSIONAL NMR-SPECTROSCOPY AND MOLECULAR-DYNAMICS SIMULATIONS TO THE CONFORMATIONAL-ANALYSIS OF OLIGOSACCHARIDES CORRESPONDING TO THE CELL-WALL POLYSACCHARIDE OF STREPTOCOCCUS GROUP-A

This paper describes the use of a protocol for conformational analysis of oligosaccharide structures related to the cell-wall polysaccharide of Streptococcus group A. The polysaccharide features a branched stru cture with an L-rhamnopyranose (Rhap) backbone consisting of alternati ng alpha-(1 --> 2) and alpha-(1 --> 3) links and D-N-acetylglucosamine (GlcpNAc) residues beta-(1 --> 3)-connected to alternating rhamnose r ings: [GRAPHICS] Oligomers consisting of three to six residues have be en synthesized and nuclear magnetic resonance (NMR) assignments have b een made. The protocol for conformational analysis of the solution str ucture of these oligosaccharides involves experimental and theoretical methods. Two-dimensional NMR spectroscopy methods (TOCSY, ROESY and N OESY) are utilized to obtain chemical shift data and proton-proton dis tances. These distances are used as constraints in 100 ps molecular dy namics simulations in water using QUANTA and CHARMm. In addition, the dynamics simulations are performed without constraints. ROE build-up c urves are computed from the averaged structures of the molecular dynam ics simulations using the CROSREL program and compared with the experi mental curves. Thus, a refinement of the initial structure may be obta ined. The alpha-(1 --> 2) and the beta-(1 --> 3) links are unambiguous ly defined by the observed ROE cross peaks between the A-B', A'-B and C-B, C'-B' residues, respectively. The branch-point of the trisacchari de CBA' is conformationally well-defined. Assignment of the conformati on of the B-A linkage (alpha-(1 --> 3)) was problematic due to TOCSY r elay, but could be solved by NOESY and T-ROESY techniques. A conformat ional model for the polysaccharide is proposed.