Alternative roles for putative ice-binding residues in type I antifreeze protein

Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alph a-helix. Leu and Asn flank the central two of four regularly spaced ice-bin ding Thr in the i-1 and i + 3 positions, respectively. All three residues p roject from the same side of the helix to form the protein's putative ice-a dsorption site and are considered in some models to act together as an "ice -binding motif". Replacement of Asn by residues with shorter side chains re sulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. H owever, substitution of Asn by its slightly larger homologue (Gln) abolishe d thermal hysteresis activity. The Gin-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu w as replaced by Ala, two of the three were more active than their Leu-contai ning counterparts, but all three variants began to precipitate as the pepti de concentration increased. None of the seven variants tested showed dramat ic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventin g peptide aggregation at the antifreeze protein concentrations (10 mg/mL) n ormally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as tra ditionally thought. Nevertheless, the dramatic loss of activity in the Asn- to-Gln replacement demonstrates the steric restriction on residues in or ne ar the ice-binding site of the peptide.