Determination of the Minimal Sequence Required for Antifreeze Activity of Type I Antifreeze Protein (AFP 37)

40 1.07 1.06 0.67 0.60 0.17 20 0.71 0.54 0.33 0.60 0.13 10 0.56 0.51 0.32 0.26 0.08 5 0.52 0.39 0.31 0.17 0.08 2.5 0.32 0.16 0.14 0.14 0.06 1.25 0.19 0.09 0.11 0.11 0.04 0.63 0.07 0.04 0.04 0.03 0.03 Five structurally different types of antifreeze proteins (AFPs) have been found in fish: the antifreeze glycoproteins and four protein types, I, II, III and IV. 1-4 AFPs have in common the ability to lower the freezing point of blood serum, thus allowing fish to survive in subzero ocean temperatures. The type I AFPs, present in the blood of winter flounder, yellowtail flounder, Alaskan plaice and the shorthorn and grubby sculpin are the most widely studied class of fish AFPs. 1 The most well-studied type I AFP is AFP37 isolated from the winter flounder, Pseudopleuronectes americanus. AFP 37 has 37 amino acids arranged in three complete 11-residue repeats Thr-X2-Asx-X7, where Asx is Asp or Asn, and X is generally Ala or another amino acid that favors α-helix formation. 5 NMR study revealed that AFP 37 adopts an α-helical structure with cap structure at the carboxy termini. 6 Analysis of the X-ray structure and ice-binding properties led to the hypothesis that AFP 37 binds to a specific plane of ice through hydrogen bonds from the threonyl hydroxyl (Thr-2, Thr-13, Thr-24, and Thr-35). 7,8 Furthermore, the mutation of the Thr residues to Ser, Val, Ala and allo-Thr have indicated that the hydrophobicity provided by the γ-methyl group of Thr, in addition to hydrogen bonding involving other residues, is a key factor related to the ability to inhibit ice growth.