Paradoxical effects on ice nucleation are intrinsic to a small winter flounder antifreeze protein.

Antifreeze proteins (AFPs) bind to ice in solutions, resulting in non-colligative freezing point depression; however, their effects on ice nucleation are not well understood. The predominant plasma AFP of winter flounder (Pseudopleuronectes americanus) is AFP6, which is an amphiphilic alpha helix. In this study, AFP6 and modified constructs were produced as fusion proteins in Escherichia coli, subjected to proteolysis when required and purified prior to use. AFP6 and its recombinant fusion precursor generated similar thermal hysteresis and bipyramidal ice crystals, whereas an inactive mutant AFP6 produced hexagonal crystals and no hysteresis. Circular dichroism spectra of the wild-type and mutant AFP6 were consistent with an alpha helix. The effects of these proteins on ice nucleation were investigated alongside non-AFP proteins using a standard droplet freezing assay. In the presence of nucleating AgI, modest reductions in the nucleation temperature occurred with the addition of mutant AFP6, and several non-AFPs, suggesting non-specific inhibition of AgI-induced ice nucleation. In these experiments, both AFP6 and its recombinant precursor resulted in lower nucleation temperatures, consistent with an additional inhibitory effect. Conversely, in the absence of AgI, AFP6 induced ice nucleation, with no other proteins showing this effect. Nucleation by AFP6 was dose-dependent, reaching a maximum at 1.5 mM protein. Nucleation by AFP6 also required an ice-binding site, as the inactive mutant had no effect. Furthermore, the absence of nucleation by the recombinant precursor protein suggested that the fusion moiety was interfering with the formation of a surface capable of nucleating ice.

Interaction of curcumin with a winter flounder alpha-helical antifreeze protein.

The winter flounder, Pseudopleuronectes americanus, synthesizes a variety of alpha-helical antifreeze proteins (AFPs) that adhere to ice and inhibit its growth. The best studied of these is AFP6, which is a 37-residue protein abundant in the flounder blood plasma during winter. Curcumin from the turmeric plant (Curcuma longa) was found to interact with AFP6 in aqueous solutions, resulting in measurable changes in the curcumin, but not in the protein. Specifically, the secondary structure and unfolding of synthetic AFP6, shown by circular dichroism, appeared to be unaffected by curcumin. In contrast, the peak absorbance of curcumin shifted and increased in the presence of AFP6, and the maximum fluorescence emission was greater and blue shifted. These results also suggested the possibility of AFP6 detection by curcumin fluorescence. Synthetic AFP6 did not interact with Coomassie blue, silver or a commercial fluorescent stain following electrophoresis; however, the change in curcumin fluorescence upon binding to electrophoresed AFP6 resulted in a fluorescent signal, which was also detected upon interaction with purified natural AFP and flounder blood plasma containing the protein. Thus, aqueous curcumin can be used for the direct detection of AFP6 and curcumin binding could provide new avenues for the study of this protein.