Antifreeze proteins (AFPs) have the unique ability to adsorb to ice and inhibit its growth. Many organisms ranging from fish to bacteria use AFPs to retard freezing or lessen the damage incurred upon freezing and thawing. The ice-binding mechanism of the long linear alpha-helical type I AFPs has been attributed to their regularly spaced polar residues matching the ice lattice along a pyramidal plane. In contrast, it is not known how globular antifreeze proteins such as type III AFP that lack repeating ice-binding residues bind to ice. Here we report the 1.25 A crystal structure of recombinant type III AFP (QAE isoform) from eel pout (Macrozoarces americanus), which reveals a remarkably flat amphipathic ice-binding site where five hydrogen-bonding atoms match two ranks of oxygens on the [1010] ice prism plane in the <0001> direction, giving high ice-binding affinity and specificity. This binding site, substantiated by the structures and properties of several ice-binding site mutants, suggests that the AFP occupies a niche in the ice surface in which it covers the basal plane while binding to the prism face.

Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.

In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type 111 AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 P-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal &sandwich (Sonnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 2591154-1157). Based on its structure and an alignment of Type 111 AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, 444, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type 111 AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50'70, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal @-sheet of Type 111 AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and 444 as key residues for the AFP-ice interaction.Keywords: NMR; site-directed mutagenesis; thermal hysteresis Some cold water marine fishes produce proteins or glycoproteins that lower the freezing point of their blood without significantly increasing its osmolarity (DeVries, 1983;Davies & Hew, 1990). These antifreeze proteins or glycoproteins bind to and halt the growth of seed ice crystals that form in solution at or below Reprint requests to: Peter L. Davies, Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.Abbreviations: AFGP, antifreeze glycoprotein; AFP, antifreeze protein; FPLC, fast protein liquid chromatography; H bond, hydrogen bond; NOESY, nuclear Overhauser effect spectroscopy; QAE, quaternary aminoethyVAFP isoform that binds QAE-Sephadex; TOCSY, total correlation spectroscopy; lD, one-dimensional; 2D, two-dimensional; 3D, three-dimensional.