Conformational studies of the Man(8) oligosaccharide on native ribonuclease B and on the reduced and denatured protein

Site-specific presentation of oligosaccharides in the context of carrier pr oteins can influence markedly their recognition by carbohydrate-binding pro teins. On RNaseB, the Man(5-9) N-glycans at Asn-34 are bound by the serum l ectin conglutinin when the glycoprotein is reduced and denatured, but there is no binding to the N-glycans on the native form of RNaseB. The RNaseB Ma n(8), which is a glycoform preferentially bound by conglutinin, is the subj ect of the present study. The conformational behavior of the protein-linked oligosaccharide Man(8) is investigated on the native and on the reduced an d denatured RNaseB, using a combination of NMR and theoretical calculations . Quantitative data on the NOESY crosspeaks have been obtained, thereby all owing the comparison of mobilities of homologous linkages within the glycan chain, Oligosaccharide conformations compatible with the NMR data have bee n explored by molecular modeling of the free oligosaccharide, using two dif ferent force fields (AMBER and SYBYL). There are some differences between t he results produced by the two force fields, the AMBER simulations providin g a better agreement with the experimental data, The results indicate that both on the native and on the reduced heat-denatured glycoprotein, the RNas e Man(8) oligosaccharide exhibits a conformational behavior very similar to that of the free oligosaccharide. However, this conformational freedom of the N-glcyan does not amount to full availability for carbohydrate-recognit ion proteins and enzymes. (C) 2000 Academic Press.