title: Contribution of hydrophobic residues to ice binding by fish type III antifreeze protein.
abstract: Type III antifreeze protein (AFP) is a 7-kDa globular protein with a flat ice-binding face centered on Ala 16. Neighboring hydrophilic residues Gln 9, Asn 14, Thr 15, Thr 18 and Gln 44 have been implicated by site-directed mutagenesis in binding to ice. These residues have the potential to form hydrogen bonds with ice, but the tight packing of side chains on the ice-binding face limits the number and strength of possible hydrogen bond interactions. Recent work with alpha-helical AFPs has emphasized the hydrophobicity of their ice-binding sites and suggests that hydrophobic interactions are important for antifreeze activity. To investigate the contribution of hydrophobic interactions between type III AFP and ice, Leu, Ile and Val residues on the rim of the ice-binding face were changed to alanine. Mutant AFPs with single alanine substitutions, L19A, V20A, and V41A, showed a 20% loss in activity. Doubly substituted mutants, L19A/V41A and L10A/I13A, had less than 50% of the activity of the wild type. Thus, side chain substitutions that leave a cavity or undercut the contact surface are almost as deleterious to antifreeze activity as those that lengthen the side chain. These mutations emphasize the importance of maintaining a specific surface contour on the ice-binding face for docking to ice.
authors.affiliation: Department of Biochemistry, Queen's University, Stuart St., Kingston, ON, Canada K7L 3N6.
authors.forename: Jason
authors.initials: J
authors.lastname: Baardsnes
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D021343:Antifreeze Proteins, Type III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005399:Fishes; D007053:Ice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D021343:Antifreeze Proteins, Type III; D017931:DNA Primers; D007053:Ice; D011994:Recombinant Proteins