Structure-function studies of hyperactive antifreeze proteins from insects and fish

Many organisms that are exposed to freezing temperatures are protected by antifreeze proteins (AFPs), which bind to and inhibit the growth of ice crystals. There are several non-homologous types of AFPs. Those from insects have been called 'hyperactive' because they are 10--100 fold more potent than fish AFPs. The beetle Tenebrio molitor (Tm) produces a repetitive beta-helical AFP comprised of coils that present a regular array of threonine residues on a flat, parallel beta-sheet. Site-directed mutagenesis was used to determine that Tm AFP binds ice through this face. The repetitive nature of Tm AFP was exploited to engineer a protein series of variable length by inserting or excising repeats, each of which forms one coil. These constructs demonstrated that antifreeze activity is highly dependent on the size of the ice-binding site and can be enhanced substantially by adding coils. Determining specific activity required the removal of misfolded protein, which was not possible by conventional chromatography, but was accomplished by selecting well-folded protein using 'ice affinity purification' (IAP). IAP was then adapted as an analytical technique to investigate the mechanistic basis for the superior activity of insect AFPs. One possibility is that they bind more tightly to ice, although that is at odds with the hypothesis that AFPs must bind irreversibly to exert antifreeze activity. An alternative explanation is that insect AFPs bind key crystallographic planes of ice that better contain ice growth. By comparing the extent to which they bind and become included into growing ice, the affinities of insect and fish AFPs for ice were compared indirectly. They partitioned similarly into ice, suggesting that hyperactivity is not a function of the tightness of binding, but of the plane(s) bound. The ability of IAP to isolate any protein that binds ice led to the discovery of a novel hyperactive AFP in winter flounder. This long overlooked, thermolabile protein is the only known fish AFP with activity comparable to those from insects. The biophysical characterization of this protein indicated that it is dimeric, entirely helical, and rod shaped, a structure that differs from all known AFPs.