Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Structure-function relationships for an alanine-rich, α-helical antifreeze polypeptide (AFP) from winter flounder were studied with the goal of understanding how AFPs depress the freezing point of water. A 37-residue native AFP and 23 analogs with systematic variations in the polypeptide chain were synthesized, and the α-helix content, antifreeze activity, and effect on growth rates of ice crystals along the a and c axes were determined. The results indicate that both the regularly spaced threonine and asparagine (or aspartic acid) residues are critical for maximal activity, and that the asymmetric arrangement of these residues on the helix face causes asymmetric adsorption of AFPs on the ice surface. Charged-residues, except for C-terminal Arg, are not very critical for antifreeze activity. Studies of hydrophobic residue mutants showed that the overall hydrophobicity is not particularly important. However, the Ala residue in position 17 appears to be important, because replacement with a bulky group abolishes antifreeze activity, presumably by interfering with the favorable side-to-side hydrophobic A model for binding of the winter flounder AFP to ice is proposed, whereby the AFP inhibits the growth of ice crystals by hydrogen bonding of Thr, Asn and Asp side chains in a specific pattern to the { 20 21 } hexagonal bipyramidal planes of ice, unidirectionally along the vector <1102>. It is further proposed that ice crystal growth inhibition occurs by a two-step mechanism: first individual AFP molecules hydrogen bond to ice reversibly, allowing slow growth of ice crystal; then at sufficiently high AFP concentrations, the AFP molecules begin to pack together on the binding surface by cooperative, side-to-side, hydrophobic interpeptide interactions, resulting in essentially irreversible binding and arrested ice crystal growth. The D-enantiomer of the AFP was also synthesized. The D and L-enantiomers alone, as well as a 50:50 mixture of D and L, all show identical antifreeze activity. These results indicate that complete coverage of the ice surface is not necessary, and suggest a model whereby AFP molecules bind in patches on the ice surface. / 2999-01-01