TRANSFERRED NUCLEAR OVERHAUSER ENHANCEMENT (NOE) AND ROTATING-FRAME NOE EXPERIMENTS REFLECT THE SIZE OF THE BOUND SEGMENT OF THE FORSSMAN PENTASACCHARIDE IN THE BINDING-SITE OF DOLICHOS-BIFLORUS LECTIN

A complex between the Forssman pentasaccharide -GalNAc-(1-->3)-beta-D- GalNAc-(1-->3)-alpha-D-Gal- (1-->4)-beta-D-Gal-(1-->4)-D-Glc and the s eed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evo lution of transferred NOEs and ROEs as a function of the pentasacchari de/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc compared to the rest of the mole cule, which reflects distinct relaxation properties and effects of exc hange broadening of the corresponding ligand resonances. Significantly , several intermolecular transferred NOEs were observed between proton s of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D -GalNAc and aliphatic as well as aromatic amino acid side chain proton s in the binding pocket of the lectin. It is concluded that the non-re ducing disaccharide fragment is buried in the lectin-binding pocket, w hereas the reducing trisaccharide portion alpha-D-Gal(1-->4)-beta-D-Ga l-(1-->4)-D-Glc has no immediate contacts with the protein. The experi mental transfer NOE data were qualitatively compared to theoretical pr oton-proton distances from a model that was based on a previous homolo gy modeling study of a complex between the disaccharide fragment alpha -D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found t hat all intermolecular transferred NOEs matched short interatomic dist ances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.