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

J.T Davis,S Hirani,C Bartlett,B.R Reid

doi:10.1016/s0021-9258(17)41867-9

J.T Davis, S Hirani + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)41867-9

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Abstract

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.

Highlights

From the $Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, the YGenzyme Corporation, Cambridge,Massachusetts 02139, and the **Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195
Nonex- tide conformations have beenproposed for the N-glycosylation changeable proton and exchangeable amide (NH)pro- site (Bause andLegler, 1981; Bause, 1983;Abbadi et al, 1986, ton resonances were assigned for the hen ovomucoid Beintema, 1986;Imperiali and Shannon1, 991; Imperiali et al, glycopeptideI, SerIle-Glu-Phe-Gly-Thr"ne-Ser-Lys, with pentasaccharide Manu13 (Manu14)Manpl4GlcNAcpl4GlcNAc~1-NHattached to the Asn7 ycarboxamide
Little is known about intramolecular sugar-peptide interactions and theirole in controlling glycoprotein structure and function

Summary

AND DISCUSSION

Glycopeptide NMR-TheB,-2 glycopeptide, containing the structure, the glycopeptide's GlcNAc-1 Cl-N2 side chain bond Asn53N-glycosylation site, was obtained by proteolysis of hen has a rigid orientation in HzO. This leads to the question of ovomucoid (Yet et al, 1988). EXPERIMENTALPROCEDURES different complex-type oligosaccharides attachedto (Beeley, 1976;Yet et al, 1988), withnumerous p-Gal and p-GlcNAc. Materials and Sample Preparation-The glycopeptide 1, Ser-Ile-Glu- sugars joined to the a1,3 and u1,6 antennae of an invariant.

GLYCOPEPTIDE1

Decapeptide

H2 H3 H4

64 GNC1HN3H2-GN1