New ice-binding face for type I antifreeze protein

Type I antifreeze protein (AFP) from winter flounder is an alanine-rich, 37 amino acid, single alpha-helix that contains three 11 amino acid repeats ( Thr-X-2-Asx-X-7), where X is generally Ala. The regularly spaced Thr, Asx a nd Leu residues lie on one face of the helix and have traditionally been th ought to form hydrogen bonds and van der Waals interactions with the ice su rface. Recently, substitution experiments have called into question the imp ortance of Leu and Asn for ice-binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala resi dues adjacent to the Thr, at right angles to the Leu/Asn-rich face, are com pletely conserved, To investigate the role of these Ala residues, a series of Ala to Leu steric mutations mas made at various points around the helix. All the substituted peptides were full, alpha-helical and remained as mono mers in solution. Wild-type activity was retained in A19L and A20L. A17L, w here the substitution lies adjacent to the Thr-rich face, had no detectable antifreeze activity, The nearby A21L substitution had 10% wildtype activit y and demonstrated weak interactions with the ice surface. We propose a new ice-binding face for type I AFP that encompasses the conserved Ala-rich su rface and adjacent Thr, (C) 1999 Federation of European Biochemical Societi es.