Structure and dynamic behavior of the oligosaccharide side chain of bovine pancreatic ribonuclease B. Application of carbon 13 nuclear magnetic resonance spectroscopy.

Abstract

Natural abundance 13C NMR (at 67.9 MHz) is used to study the primary structure and dynamic behavior of the carbohydrate side chain [(Man)6(GlcNAc)2-Asn] of ribonuclease B and of the shorter carbohydrate side chain [Man(GlcNAc)2-Asn] of a modified ribonuclease B (ribonuclease Bm). A comparison of the 13C NMR spectra of ribonuclease B and of the model compounds Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6 (Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound A) and Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound B) indicates that the (Man)5(GlcNAc)2 configuration of Compound A is present as a core structure in ribonuclease B and that only up to about 30% of our sample of ribonuclease B has the (Man)6(GlcNAc)2 structure of Compound B. Spin-lattice relaxation times, nuclear Overhauser enhancements, and linewidths of the carbohydrate carbon resonances of ribonuclease Bm indicate that the mannose residue and the N-acetylglucosamine linked to mannose are undergoing fast internal rotation (at least as fast as the rate of overall molecular tumbling). The terminal mannose residues of ribonuclease B also exhibit fast internal rotation. A comparison of the chemical shifts of the nonprotonated aromatic carbons of ribonuclease B and ribonuclease A strongly suggests that the carbohydrate side chain of ribonuclease B has a negligible effect (overall or localized) on the conformation of bovine pancreatic ribonuclease.