EFFECTS OF GLYCOSYLATION ON PROTEIN-STRUCTURE AND DYNAMICS IN RIBONUCLEASE-B AND SOME OF ITS INDIVIDUAL GLYCOFORMS

In an attempt to elucidate the role of carbohydrates on protein structure and dynamics in glycoproteins, ribonuclease B (RNase B), containing a single glycosylation site at Asn34, has been investigated and compared with the enzyme in the unglycosylated form (RNase A). RNase B consists of five glycoforms: Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2 and Man9GlcNAc2 (Man5-9GlcNAc2). The individual glycoforms Man1GlcNAc2 (synthetic) and Man5GlcNAc2 of RNase B have been studied to ascertain any specific effects of the different oligosaccharides. NMR measurement of amide-proton/deuterium exchange rates shows that glycosylation of the enzyme leads to the protection of amide-proton resonances from solvent exchange for a large number of residues, both in the vicinity of the glycosylation site (e.g. residues 29-34 and 35) and remote from it (e.g. residues 57-61 and 75-76). In addition, residues 10-13, 32, 34 and 35, which are observed to be protected from exchange as a result of glycosylation in the individual glycoforms Man1GlcNAc2-RNase and Man5GlcNAC2-RNase (when compared with RNase A) are less protected in RNase B. This additional protection in the glycoforms Man1GlcNAc2-RNase and Man5GlcNAc2-RNase may arise from steric hindrance between the oligosaccharide and protein reducing solvent accessibility. The rates of solvent exchange of amide protons for residues 10-13, 32, 34 and 35 are dependent on the oligosaccharide moiety. The average amide-proton/deuterium exchange rate in Man6-9Glc-NAc2-ribonucleases for residues 10-13 and 35 is approximately three times greater than Man5GlcNAc2-ribonuclease while for residues 32 and 34 it is approximately 7-11 times greater. CD analysis of RNase A and RNase B revealed the carbohydrate moiety to have a small stabilizing effect (approximately 5 kJ/mol) on the protein.