Comparison by 1 H‐nmr spectroscopy of the conformation of the 2600 dalton antifreeze glycopeptide of polar cod with that of the high molecular weight antifreeze glycoprotein

The antifreeze glycopeptide (AFGP‐8) from polar cod, B. saida , is a 14‐amino acid polypeptide having alternating glycotripeptide sequences of Ala‐[Gal(β1 → 3)GalNAc(β1 → O)]‐Thr‐Pro and Ala‐[Gal(β1 → 3)GalNAc(β1 → O)]‐Thr‐Ala, with alanyl residues at amino and carboxy terminals. Conformational studies of AFGP‐8 have been carried out by 1 H‐nmr and empirical energy calculations to investigate the difference in its antifreeze behavior from that of the more active high‐molecular weight AFGP 1‐4 of P. borchgrevinki . The 1 H‐nmr spectra, including the resonances of the exchangeable amide protons, were assigned by two‐dimensional correlated spectroscopy (COSY), one‐dimensional difference decoupling, and nuclear Overhauser effect (NOE) measurements. For the four threonyl residues, the amide proton coupling constants and the small coupling constants between H α and H β indicate similar conformations, despite significant chemical shift differences. The strong NOE between the α protons and the amide protons of the residue following together with large temperature coefficients of chemical shifts, indicate an extended conformation not consisting of α‐helix, turns or bends. Energy computations indicate several low‐energy conformations consistent with the observed coupling constants for ϕ. Among these, a left‐handed helical conformation with three repeating residues per turn has been proposed, which is in accordance with the observed NOE between the methyl group of the α‐GalNAc and Ala H β s. While the observed Overhauser effects in the threonyl side chain suggest a certain amount of conformational averaging, the effect involving the acetmido methyl of α‐GalNAc and H β s of Ala indicate that it as is a major conformer. In view of the close similarity between the conformations of AFGP‐8 and the more active antifreeze polymer, AFGP 1‐4, we propose that the difference in their activities is due to the length of the regular repeating structure with glycosylation at every third amino acid residue, and not due to any fundamental difference in their conformations.