Hyperactive antifreeze protein from winter flounder is a very long rod-like dimer of α-helices

Abstract

The winter flounder (Pseudopleuronectes americanus) produces short, monomeric α-helical antifreeze proteins (type I AFP), which adsorb to and inhibit the growth of ice crystals. These proteins alone are not sufficiently active to protect this fish against freezing at -1.9 °C, the freezing point of seawater. We have recently isolated a hyperactive antifreeze protein from the plasma of the flounder with activity 10-100-fold higher than type I AFP. It is comparable in activity to the AFPs produced by insects, and is capable of conferring freeze resistance to the flounder. This novel AFP has a molecular mass of 16,683 Da and a remarkable amino acid composition that is >60% alanine. CD spectra indicate that the protein is almost entirely α-helical at 4 °C but partially denatures at 20 °C, resulting in a species with a moderately reduced helix content that is stable at up to 50 °C. This transformation correlates with irreversible loss of activity. Analytical ultracentrifugation (sedimentation velocity and equilibrium) indicates that the predominant species in solution is dimeric (molecular weight, 32,275). Size-exclusion chromatography reveals a 2-fold higher apparent molecular weight suggesting that this molecule has an unusually large Stokes radius. The axial ratio of the dimer calculated from the sedimentation velocity data is 18:1, confirming that this protein has an extraordinarily long, rod-like structure, consistent with a novel dimeric α-helical arrangement. The structural model that best fits these data is one in which the ∼195 amino acids of each monomer form one ∼290-Å long α-helix and associate via a unique dimerization motif that is distinct from that of the leucine zipper and any other coiled-coil. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.