Insect antifreeze proteins (AFP) are considerably more active at inhibiting ice crystal growth than AFP from fish or plants. Several insect AFPs, also known as thermal hysteresis proteins, have been cloned and expressed. Their maximum activity is 3-4 times that of fish AFPs and they are 10-100 times more effective at micromolar concentrations. Here we report the solution structure of spruce budworm (Choristoneura fumiferana) AFP and characterize its ice-binding properties. The 9-kDa AFP is a beta-helix with a triangular cross-section and rectangular sides that form stacked parallel beta-sheets; a fold which is distinct from the three known fish AFP structures. The ice-binding side contains 9 of the 14 surface-accessible threonines organized in a regular array of TXT motifs that match the ice lattice on both prism and basal planes. In support of this model, ice crystal morphology and ice-etching experiments are consistent with AFP binding to both of these planes and thus may explain the greater activity of the spruce budworm antifreeze.

High-resolution three-dimensional structures are now available for four of seven non-homologous sh and insect antifreeze proteins (AFPs). For each of these structures, the ice-binding site of the AFP has been de ned by site-directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice-binding proteins shows that they have the following attributes in common. The binding sites are relatively at and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic-more so than that portion of the protein exposed to the solvent. Surface-surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts.

BackgroundIce nucleation proteins (INPs) allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa), membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown.ResultsHere we have predicted a novel β-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which PbINP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the PbINP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins.ConclusionsDimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As PbINP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.

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The inhibition activities of two antifreeze proteins (AFPs) on the formation of tetrahydrofuran (THF) clathrate hydrate have been tested. AFPs from fish (wfAFP) and insect (CfAFP) changed the morphology of growing THF hydrate crystals. Also, both AFPs showed higher activities in inhibiting the formation THF hydrate than a commercial kinetic inhibitor, poly(vinylpyrrolidone) (PVP). Strikingly, both AFPs also showed the ability to eliminate the "memory effect" in which the crystallization of hydrate occurs more quickly after the initial formation. This is the first report of molecules that can inhibit the memory effect. Since the homogeneous nucleation temperature for THF hydrate was measured to be 237 K, close to that observed for ice itself, the action of kinetic inhibitors must involve heterogeneous nucleation. On the basis of our results, we postulate a mechanism for heterogeneous nucleation, the memory effect and its elimination by antifreeze proteins.