Two series of mutant polypeptides of the type I, 37-residue winter flounder "antifreeze" protein have been synthesized and analyzed by nanoliter osmometry, the "ice hemisphere" test, measurement of ice growth hysteresis and circular dichroism (CD) spectroscopy. In series 1 peptides the central two threonines and all four threonines of the native protein were mutated to serine. In series 2 peptides two additional salt bridges (K7, E11 and K29, E33) were incorporated, and all four threonine residues in this sequence were mutated simultaneously to each of serine, valine, alanine, and glycine, respectively. The CD studies showed that all mutants are 100% helical in structure at low temperature, except for the glycine derivative which was estimated to be 70% R-helical. Dilute solutions of serine-substituted series 1 peptides showed no detectable, nonbasal faceting, or hysteresis behavior, indicating either no or extremely weak interaction with ice. The analogous serine-substituted mutant in series 2, as well as the glycine derivative, displayed unfaceted growth and showed no hysteresis. Hysteresis values, ice growth patterns, and the helicity measurements showed that the additional salt bridges present in series 2 peptides do not alter significantly the properties of the protein. The valine-substituted mutant gave a distinct etching pattern in which polypeptide accumulates on the {2 0 2 h 1}plane of ice 1h, and exhibited thermal hysteresis comparable to that of the native protein. In the case of the alanine-substituted mutant, reduced hysteresis behavior was measured, together with a distinct etch pattern in the ice hemisphere test. These combined results show that existing hypotheses for the action of native winter flounder peptide are incorrect; these hypotheses include models in which the -OH groups on four threonine side chains, equally spaced 11 residues apart on the 37-residue native polypeptide, are responsible for "binding" of the molecule to the ice/water interface. The antifreeze activity of the valine-and alaninesubstituted mutants indicate a significant contribution to the mechanism of ice growth inhibition by type I antifreeze proteins from the hydrophobic methyl group in threonine and valine. Arguments against the importance of the role of hydrogen-bonding are summarized, and alternate ice growth inhibition mechanisms that include hydrophobic interactions are discussed.

Antifreeze glycoproteins (AFGPs) constitute the major fraction of protein in the blood serum of Antarctic notothenioids and Arctic cod. Each AFGP consists of a varying number of repeating units of (Ala-Ala-Thr) n , with minor sequence variations, and the disaccharide b-D-galactosyl-(1fi3)-a-N-acetyl-D-galactosamine joined as a glycoside to the hydroxyl oxygen of the Thr residues. These compounds allow the fish to survive in subzero ice-laden polar oceans by kinetically depressing the temperature at which ice grows in a noncolligative manner. In contrast to the more widely studied antifreeze proteins, little is known about the mechanism of ice growth inhibition by AFGPs, and there is no definitive model that explains their properties. This review summarizes the structural and physical properties of AFGPs and advances in the last decade that now provide opportunities for further research in this field.High field NMR spectroscopy and molecular dynamics studies have shown that AFGPs are largely unstructured in aqueous solution. While standard carbohydrate degradation studies confirm the requirement of some of the sugar hydroxyls for antifreeze activity, the importance of following structural elements has not been established: (a) the number of hydroxyls required, (b) the stereochemistry of the sugar hydroxyls (i.e. the requirement of galactose as the sugar), (c) the acetamido group on the first galactose sugar, (d) the stereochemistry of the b-glycosidic linkage between the two sugars and the a-glycosidic linkage to Thr, (e) the requirement of a disaccharide for activity, and (f) the Ala and Thr residues in the polypeptide backbone. The recent successful synthesis of small AFGPs using solution methods and solidphase chemistry provides the opportunity to perform key structure-activity studies that would clarify the important residues and functional groups required for activity.Genetic studies have shown that the AFGPs present in the two geographically and phylogenetically distinct Antarctic notothenioids and Arctic cod have evolved independently, in a rare example of convergent molecular evolution. The AFGPs exhibit concentration dependent thermal hysteresis with maximum hysteresis (1.2°C at 40 mgAEmL )1 ) observed with the higher molecular mass glycoproteins. The ability to modify the rate and shape of crystal growth and protect cellular membranes during lipid-phase transitions have resulted in identification of a number of potential applications of AFGPs as food additives, and in the cryopreservation and hypothermal storage of cells and tissues.

The type I`antifreeze' proteins, found in the body fluids of fish inhabiting polar oceans, are alanine-rich a-helical proteins that are able to inhibit the growth of ice. Within this class there are two distinct subclasses of proteins: those related to the winter flounder sequence HPLC6 and which contain 11-residue repeat units commencing with threonine; and those from the sculpins that are unique in the N-terminal region that contains established helix breakers and lacks the 11-residue repeat structure present in the rest of the protein. Although 14 type I proteins have been isolated, almost all research has focused on HPLC6, the 37-residue protein from the winter flounder Pseudopleuronectes americanus. This protein modifies both the rate and shape (or`habit') of ice crystal growth, displays hysteresis and accumulates specifically at the {2 0 2 Å 1} ice plane. Until very recently, all models to explain the mechanism for this specific interaction have relied on the interaction of the four threonine hydroxyls, which are spaced equally apart on one face of the helix, with the ice lattice. In contrast, proteins belonging to the sculpin family accumulate specifically at the {2 1 Å 1 Å 0} plane. The molecular origin of this difference in specificity between the flounder and sculpin proteins is not understood. This review will summarize the structure±activity and molecular modelling and dynamics studies on HPLC6, with an emphasis on recent studies in which the threonine residues have been mutated. These studies have identified important hydrophobic contributions to the ice growth inhibition mechanism. Some 50 mutants of HPLC6 have been reported and the data is consistent with the following requirements for ice growth inhibition: (a) a minimum length of approx. 25 residues; (b) an alanine-rich sequence in order to induce a highly helical conformation; (c) a hydrophobic face; (d) a number of charged/polar residues which are involved in solubility and/or interaction with the ice surface. The emerging picture, that requires further dynamics studies including accurate modelling of the ice/water interface, suggests that a hydrophobic interaction between the surface of the protein and ice is the key to explaining accumulation at specific ice planes, and thus the molecular level mechanism for ice growth inhibition.

Three mutant polypeptides of the type I 37-residue winter flounder`antifreeze' protein have been synthesized. All four threonine residues in the native peptide were been mutated to serine, valine and glycine respectively and two additional salt bridges were incorporated into the sequences in order to improve aqueous solubility. The peptides were analyzed by nanoliter osmometry, the`ice hemisphere' test, the`crystal habit' test, measurement of ice growth hysteresis and CD spectroscopy. While the valine and serine mutants retain the K K-helical structure, only the valine mutant retains`antifreeze' activity similar to that of the native protein. These data show that the threonine hydroxyl groups do not play a crucial role in the accumulation of the native`antifreeze' protein at the ice/water interface and the inhibition of ice growth below the equilibrium melting temperature.z 1998 Federation of European Biochemical Societies.