Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy

The three-dimensional structures of eel calcitonin (CT) and two glycosylate d CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man(6)-GlcNAc(2))3 ]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-he lix consisting of residues Ser5 through Leu19 followed by an unstructured r egion at the C-terminus, The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relativ e exchange rates of the Leu12 amide proton might suggest the possibility th at fluctuations of the alpha-helix are reduced by glycosylation. The presen ce of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc a nd CT-M6 and the amide proton chemical shift data suggested that the carboh ydrate interacted with the peptide, and this might account for the conforma tional stabilization of the alpha-helix. Both the unmodified CT and the gly cosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.