A natural variant of type I antifreeze protein with four ice‐binding repeats is a particularly potent antifreeze

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Abstract
A 4.3‐kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed‐phase HPLC. By the criteria of mass, amino acid composition, and N‐terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11‐amino acid repeats, one more than the major serum AFP components. The larger protein is completely α‐helical at 0 °C, with a melting temperature of 18 °C. It is considerably more active as an antifreeze than the three‐repeat winter flounder AFP and the four‐repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four‐repeat winter flounder AFP, including its larger size, additional ice‐binding residues, and differences in ice‐binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.
Funding Information
  • Medical Research Council of Canada
  • Protein Engineering Network of Centres of Excellence