Matthew Blakeley from ILL and his colleagues from ESRF and elsewhere have discovered how antifreeze in Arctic fish blood keeps them alive in sub-zero conditions. He and Eleanor Hayes explain.

When we think of the Arctic, many of us think of icebergs, polar bears and seals. Freezing temperatures, icy winds and desolate snowy wastes – a challenge to any animal that lives there.

We are familiar with many of the ways in which Arctic animals have adapted to their environment: the deep fur of polar bears, the thick blubber layer of seals, the migratory habits of many birds. These animals, however, are all endotherms – they maintain a constant body temperature, well above that of their icy surroundings.

For fish, the situation is very different – they are mostly exotherms, which means that their bodies are the same temperature as their surroundings. And their surroundings are very cold indeed: the Arctic Ocean frequently reaches temperatures as low as -1 °C, when it is only prevented from freezing by the high salt content of the water.

AFPs have a complex task. To prevent the fish from freezing to death, they must bind to ice-crystal nuclei forming in the fish’s body, preventing the nuclei from growing into ice crystals. How, though, do they distinguish ice from liquid water?

AFPs have been extensively studied using many techniques, including X-ray diffraction, revealing the existence of a specialised ice-binding surface.

This is where a second technique, neutron diffraction, is beneficial. Neutrons are scattered by atomic nuclei rather than electrons, and the strength of the scattering depends on the specific nuclear forces. These forces in turn depend not only on the elements but also on the elements’ isotopes, which differ in the number of neutrons in their nucleus (Figure 1b, above). Fortunately for us, hydrogen atoms can be easily detected by neutron scattering and the hydrogen isotope deuterium (heavy hydrogen, which has an extra neutron) can be detected even more reliably. Thus the signal for water is significantly different from that of oxygen.

Part of the answer to how type-III AFPs distinguish ice from liquid water lies in how the structure of the AFPs differs from that of typical proteins, which normally have hydrophobic amino acids in the core (away from water molecules in their surroundings) and hydrophilic amino acids on the surface. As has been shown by previous researchers, AFPs are unusual in having many hydrophobic amino acids on their surface, preventing the protein binding to liquid water via hydrogen bonds. These hydrophobic amino acids form part of the ice-binding surface, which binds to ice nuclei but not liquid water. How does this work?

Herein lies the second part of the answer: how the structure of ice differs from that of liquid water. Using neutron diffraction, we were able to locate the positions of water molecules on the ice-binding surface. We identified a tetrahedral cluster of water molecules bound to the protein’s ice-binding surface. This tetrahedral cluster is found in liquid water (as in our experiment) but is also typical of ice; this gave us the starting point for modelling the rest of the ice crystal and deducing how it would bind to the AFP. The resulting model is composed of six-membered water rings in what is known as a boat configuration: six water molecules in a hexagonal arrangement, leaving a hole in the middle (see Figure 2, above). It is this hole that enables the type-III AFPs to distinguish the structure of ice-crystal nuclei from that of water: the hydrophobic regions of the ice-binding site fit into the holes, binding with the ice via Van der Waals forces (Figure 3, below). In contrast, liquid water has no hole into which hydrophobic regions such as methyl groups can fit. This prevents a large contact surface between liquid water and the ice-binding site, which would be necessary for a tight interaction.

In itself, it is interesting to learn more about how Arctic fish survive in their environment, but this research also has potential industrial applications. Already, type-III AFPs are incorporated into some ice cream to minimise the build-up of large ice crystals and thus improve the consistency. In future, genes that code for AFPs may be incorporated into crops to allow them to be grown in sub-zero environments.