Hydrogen-bonded structure of the complex N-linked fetuin glycopeptide

Abstract

The conformation of the N-linked compex glycopeptide of fetuin was examined with H-exchange techniques. The glycopeptide molecule contains 8 acetamido H stemming from 5 N-acetylglucosamine residues and 3 N-acetylneuraminic acid residues and also 1 from the remaining sugar-peptide linkage. The H-exchange rates of these secondary amides were compared with small molecule model compounds having identical primary structures at their exchangeable H sites. Differences between the model rates and glycopeptide rates therefore cannot be accounted for by primary structure effects but reflect conformational features of the glycopeptide. Two glycopeptide H exhibit significantly hindered exchange; the rest exchange at the model rates. Removal of the 3 N-acetylneuraminic acid residues from terminal positions on the 3 branches of the glycopeptides removes the slowed H. The remaining ones continue to exchange at the model rate. These results indicate that 2 of the 8 sugar acetamido H are involved in intramolecular H-bonds. A likely structure includes 2 H-bonds between the 3 N-acetylneuraminic acid residues. These 2 H slowed to a moderate degree, reflect a preferred conformation stabilized by .apprx. 1 kcal/mol in free energy. The solution conformation of the glycopeptide suggested by these results is one that is partially ordered and can be easily modulated, owing to the relatively small amount of energy stabilizing the preferred conformation.