Lipopolysaccharide solution conformation: antigen shape inferred from high resolution 1H and 13C nuclear magnetic resonance spectroscopy and hard-sphere calculations

Abstract

High resolution ¹H and ¹³C n.m.r. measurements have been used to assess the solution conformations of synthetic oligosaccharides which are related to a Shigella flexneri O-antigen. Unequivocal assignments of all ¹H and ¹³C resonances have been made for the model oligosaccharides and the polysaccharide antigen. N.m.r. parameters that aid the conformational interpretations are: chemical shifts, coupling constants, relaxation measurements, and N.O.E. experiments. The conformation of the repeating unit of the O-antigen has also been estimated by hardsphere calculations, which take into account the exo-anomeric effect. The experimental results are in close agreement with the calculated conformations and permit the extrapolation of the oligosaccharide conformation to the polysaccharide chains, which constitute the O–antigen. N.m.r. measurements of this polysaccharide isolated from bacteria support these conclusions. Ordered conformations, such as those discovered for the Shigella flexneri O-antigen, are important for antibody–polysaccharide interactions; these are discussed with particular reference to the recognition, by antibodies, of internal ohigosaccharide sequences. Space-fitting projections of these conformations are presented and used to facilitate the interpretation of serological data in molecular terms. Limitations of the hapten inhibition technique for mapping antibody combining sites are discussed in stereochemical terms. The possible role of sugar units as ‘spacers’ between contact sugars, defining the polysaccharlde binding surface, is illustrated and the manner in which linear sugar arrays may generate multiple binding surfaces is related to conformation.