Winter flounder Z

The major antifreeze polypeptide (AFP) from winter flounder (37 amino acid residues) is a single alpha-helix. Aspartic acid and arginine are found, respectively, at the amino and carboxyl-termini. These charged amino acids are ideally located for stabilizing the alpha-helical conformation of this AFP by means of charge-dipole interaction (Shoemaker, K. R., Kim, P.S., York, E.J., Stewart, J. M., and Baldwin, R. L. (1987) Nature 326, 563-567). In order to understand these and other molecular interactions that maintain the AFP structure, we have carried out the chemical synthesis of AFP analogs and evaluated their conformations by circular dichroism spectroscopy. We synthesized the entire AFP molecule (37-mer) and six COOH-terminal peptide fragments (36-, 33-, 27-, 26-, 16-, and 15-mers). Peptides containing acidic NH2-terminal residues displayed greater helix formation and thermal stability compared to those peptides of similar size, but with neutral NH2-terminal residues. Helix formation was maximum above pH 9.2. The peptide conformations also displayed a pH-dependent sensitivity to changes in ionic strength. Helix formation was reduced in the presence of acetonitrile. We conclude that the AFP helix is most likely stabilized by: charge-dipole interactions between charged terminal amino acids and the helix dipole, a charge interaction between Lys18 and Glu22 (either a salt bridge or a hydrogen bond), and hydrophobic interactions.

The antifreeze polypeptide (AFP) from the winter flounder displays partial alpha-helix formation at lower temperatures. To investigate the relationship between antifreeze activity and alpha-helical structure, we designed and then chemically synthesized an AFP analog with enhanced alpha-helicity, and compared its conformation and antifreeze properties with those of the native AFP. The synthetic analog was more helical than the native AFP; however, the antifreeze activity of both peptides were identical. The antifreeze activity of the peptides displayed a strong pH dependence, which paralleled pH-induced changes in helix content. At pH 8.5, the antifreeze activity of both peptides displayed identical concentration dependences. In addition to antifreeze activity measurements, the effects of the peptides on the rate of ice crystal growth were also measured. While both peptides affected the a- and c-axis growth rates of ice crystals, the highly helical analog was able to exert its effect on ice crystal growth rates at 7-8-fold lower concentrations than the native AFP. These data indicate that there is a direct but complex relationship between alpha-helicity and antifreeze activity.

An alanine-rich, alpha-helical antifreeze polypeptide (AFP) from the winter flounder and seven analogs with variations in the arrangement of neutral, polar amino acids were synthesized. Circular dichroism studies determined that all of the peptides, except for one containing a proline residue, were essentially 100% alpha-helical. Freezing point depression data, analyzed by three methods, showed that rearrangement of polar residues resulted in moderate to complete loss of anti-freeze activity. It was observed that ice crystals grow as hexagonal bipyramids in dilute solutions, with a constant c to alpha axis ratio of about 3.3. Above a critical threshold concentration, which may depend on the AFP to ice binding constant and reflect the onset of cooperative interactions, growth ceases until the temperature is lowered to the freezing point. We conclude that a specific arrangement of both threonine and asparagine (or aspartic acid) residues is critical for maximal activity and that the AFPs probably bind to the pyramidal faces of ice with a specific orientation. These conclusions are consistent with a recent report (Knight, C. A., Cheng, C. C., and DeVries, A. L. (1991) Biophys. J. 59, 409-418) that a similar AFP adsorbs to the [2021] pyramidal planes of ice in dilute solution.