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Abstract

  1. Top of page
  2. Abstract
  3. REFERENCES

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 Man6–9GlcNAc2-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.

Abbreviations
CE

capillary electrophoresis

NOESY

nuclear Overhauser spectroscopy

COSY

correlation spectroscopy

HO-HAHA

homonuclear Hartman-Hahn spectroscopy

RNase

ribonuclease

RNase Man5

Man5GlcNAc2-ribonuclease

RNase Man1

Man1GlcNAc2-ribonuclease

Enzyme
 

Bovine pancreatic ribonuclease A (EC 3.1.27.5)

REFERENCES

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  2. Abstract
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