Purification and structural analysis of a type III antifreeze protein from the European eelpout Zoarces viviparus

It has recently been reported that the eelpout Zoarces viviparus synthesizes a family of antifreeze proteins (AFP) similar in sequence to type III AFPs. A method has been set up to separate these antifreeze proteins from blood serum of this teleost species. A total of nine proteins with antifreeze activity have been isolated, several to a purity suited for NMR experiments. One of the proteins, Zvafp13, has been subject to partial structure determination by NMR. 1D- and 2D-¹H NMR analyses were carried out. In the 1D-experiments it was observed that the protein contained 28 slow-exchanging amides, suggesting a compact structure. The 2D-experiments were utilized to assign observed signals to specific amino acids. From TOCSY- and NOESY-experiments 35 out of a total of 66 amino acids were assigned. The amide exchange pattern, protein primary sequence, chemical shifts and NOE-cross-peaks between amides and α-protons in the β-sheets suggest that Zvafp13 structurally resembles the recombinant type III AFP rQAE m1.1.