The synthesis, solution conformation and ice-growth inhibition properties of four new analogues of the type I 37-residue winter flounder 'antifreeze' protein are reported. All four analogues contain two extra salt bridges to facilitate comparison of results with previously published data. In two analogues, all four threonine residues in the native polypeptide were mutated to 2-amino butyric acid (an unnatural amino acid) and isoleucine, respectively. The butyric acid analogue was approximately 85% helical at 3 degrees C, modified the shape of ice growth, and exhibited reduced hysteresis compared to the native protein (9% at 4 mM). These results show that the gamma-methyl group of threonine, which is present in the sidechain of 2-amino butyric acid, is not sufficient for activity. The isoleucine analogue, in which the threonine hydroxyl group is replaced by an ethyl group, was 100% helical at 3 degrees C, showed no hysteresis but was able to modify the shape of ice crystal growth. In the third and fourth analogues, mutations of the aspartic acids 1 and 5 to alanine, and asparagines 16 and 27 to leucine in the threonine- and valine-substituted analogues did not affect the helicity of the polypeptides, but removed the ability to inhibit ice growth.