Thermolabile antifreeze protein produced in Escherichia coli for structural analysis.

The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforms; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express® strain of Escherichia coli at 12 °C, just below the thermal denaturation temperature of 16-18 °C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP Ih yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes of the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography.

Hyperactive antifreeze protein from winter flounder is a very long rod-like dimer of alpha-helices.

The winter flounder (Pseudopleuronectes americanus) produces short, monomeric alpha-helical antifreeze proteins (type I AFP), which adsorb to and inhibit the growth of ice crystals. These proteins alone are not sufficiently active to protect this fish against freezing at -1.9 degrees C, the freezing point of seawater. We have recently isolated a hyperactive antifreeze protein from the plasma of the flounder with activity 10-100-fold higher than type I AFP. It is comparable in activity to the AFPs produced by insects, and is capable of conferring freeze resistance to the flounder. This novel AFP has a molecular mass of 16,683 Da and a remarkable amino acid composition that is >60% alanine. CD spectra indicate that the protein is almost entirely alpha-helical at 4 degrees C but partially denatures at 20 degrees C, resulting in a species with a moderately reduced helix content that is stable at up to 50 degrees C. This transformation correlates with irreversible loss of activity. Analytical ultracentrifugation (sedimentation velocity and equilibrium) indicates that the predominant species in solution is dimeric (molecular weight, 32,275). Size-exclusion chromatography reveals a 2-fold higher apparent molecular weight suggesting that this molecule has an unusually large Stokes radius. The axial ratio of the dimer calculated from the sedimentation velocity data is 18:1, confirming that this protein has an extraordinarily long, rod-like structure, consistent with a novel dimeric alpha-helical arrangement. The structural model that best fits these data is one in which the approximately 195 amino acids of each monomer form one approximately 290-A long alpha-helix and associate via a unique dimerization motif that is distinct from that of the leucine zipper and any other coiled-coil.

Hyperactive antifreeze protein in a fish.

Fish that live in the polar oceans survive at low temperatures by virtue of 'antifreeze' plasma proteins in the blood that bind to ice crystals and prevent these from growing. However, the antifreeze proteins isolated so far from the winter flounder (Pleuronectes americanus), a common fish in the Northern Hemisphere, are not sufficiently active to protect it from freezing in icy sea water. Here we describe a previously undiscovered antifreeze protein from this flounder that is extremely active (as effective as those found in insects) and which explains the resistance of this fish to freezing in polar and subpolar waters.

Isolation and characterization of type I antifreeze proteins from Atlantic snailfish (Liparis atlanticus) and dusky snailfish (Liparis gibbus).

Antifreeze proteins (AFPs) were isolated from the blood plasma of Atlantic snailfish Liparis atlanticus and dusky snailfish Liparis gibbus, which belong to the Teleost family Cyclopteridae, a close relative of sculpins. Using a combination of gel filtration chromatography and reversed-phase HPLC, proteins were purified to individual peaks. Atlantic snailfish plasma contained two different proteins (MW=9344, 9415) while dusky snailfish plasma contained five protein isoforms (MW=9514-9814), as determined by mass spectrometry. Further characterization revealed that these proteins are rich in alanine (>50 mol%), and have alpha-helical secondary structure that can undergo reversible thermal denaturation. Thermal hysteresis activities of these proteins were similar to each other but lower than the major type I AFPs from winter flounder. Results of this study have indicated that although the AFPs from snailfish are significantly larger than previously described type I AFPs, they share enough characteristics to be classified in this group.