Hydrophobic analogues of the winter flounder 'antifreeze' protein

The synthesis, solution conformation and ice-growth inhibition properties o f 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 ana logues, all four threonine residues in the native polypeptide were mutated to 2-amino butyric acid (an unnatural amino acid) and isoleucine, respectiv ely, The butyric acid analogue was similar to 85% helical at 3 degreesC, mo dified the shape of ice growth, and exhibited reduced hysteresis compared t o the native protein (9% at 4 mM). These results show that the gamma -methy l 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, mas 100% helical a t 3 degreesC, showed no hysteresis but was able to modify the shape of ice crystal growth, In the third and fourth analogues, mutations of the asparti c acids 1 and 5 to alanine, and asparagines 16 and 27 to leucine in the thr eonine- and valine-substituted analogues did not affect the helicity of the polypeptides, but removed the ability to inhibit ice growth, (C) 2001 Fede ration of European Biochemical Societies. Published by Elsevier Science B.V . All rights reserved.