1H NMR studies on an Asn-linked glycopeptide. GlcNAc-1 C2-N2 bond is rigid in H2O

The conformation of an Asn-linked glycopeptide in H2O was studied by two-dimensional 1H NMR. Nonexchangeable proton and exchangeable amide (NH) proton resonances were assigned for the hen ovomucoid glycopeptide 1, Ser-Ile-Glu-Phe-Gly-Thr-Asn Ile-Ser-Lys, with pentasaccharide Man alpha 1-3 (Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc beta 1-NH attached to the Asn7 gamma-carboxamide. The pentasaccharide increases the local correlation times of amino acid residues near the N-glycosylation site. Nuclear Overhauser effect (NOE) measurements on 1 and the corresponding Man3-GlcNAc2 pentasaccharide 3 show that the attached peptide does not perturb O-glycoside conformation. Sequential dNN (i, i + 1) NOEs in the Thr6-Ser9 region indicate populations of folded structure near the N-glycosylation site of both glycopeptide 1 and aglycosyl peptide 2. However, the Man3GlcNAc2 pentasaccharide does not dramatically affect the average conformation of either the peptide backbone or the Asn7 side chain. GlcNAc NH protons were studied at pH 3.0; and NOE and 3JNH data were used to constrain the glycopeptide's GlcNAc-1 side chain dihedral angle (tau) (C1-C2-N2-C7(Ac)). The glycopeptide's core GlcNAc-1 C2-N2 side chain bond is not flexible in H2O. A strong GlcNAc-1 NH2-H3 NOE, a medium strength NH2-H1 NOE, and a weak NH2-H2 interaction suggest that GlcNAc-1 has a rigid C2-N2 bond, with tau between 95 and 115 degrees. No evidence was found for intramolecular hydrogen bonds restricting this C2 side chain torsion. It may be that GlcNAc-1's rigid planar N-glycosidic linkage limits the conformational space available to the adjacent C2 acetamido side chain.