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 Man1Glc-NAc2-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 Man5Glc-NAc2-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.
Joao
H C
HC
Department of Biochemistry, University of Oxford, England.
Dwek
R A
RA
D002240:Carbohydrate Sequence; D002942:Circular Dichroism; D004586:Electrophoresis; D006031:Glycosylation; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D012260:Ribonucleases / Q000737:chemistry*
D006639:Histidine; D012260:Ribonucleases; C029600:ribonuclease B