Why Fish Don't Freeze When Water Turns to Ice

But just how these mysterious proteins perform this miraculous feat remained elusive until Ilja Voets and colleagues started looking a bit closer at the way they act under different temperatures and in different states. They wanted to learn more about how these so-called anti-freeze proteins (AFPs) bind ice crystals in the circulatory system, reduce the freezing point of bodily fluids and prevent ice recrystallisation.

Credit: © photografspb

The PRICE fellowship grant gave them the opportunity to explore how AFPs actually do their magic through a series of biophysical experiments on bioengineered anti-freeze proteins.

Knowing what is happening at a molecular level is fascinating science in itself, but there are also potential practical uses for this knowledge, according to the team. A synthetic analogue or ‘biomimetic’ version of these cryoprotective AFPs could be applied to medicine and food production, for example preserving organs for transplant and longer-lasting frozen food products.
Ice and proteins

PRICE used Escherichia coli bacteria to make type III AFPs and solid-phase peptide synthesis to produce type I AFP in the laboratory. The researchers used X-ray scattering to study a hyperactive type I AFP and moderately active type III AFP in two fish species: winter flounder and ocean pout. And together with researchers at AMOLF in Amsterdam, advanced spectroscopic tools were used to study the hydration layer of the type III AFP, revealing how these proteins bind ice.

“We carried out a thorough study on many antifreeze proteins with three independent activity assays or tests, to have a broader scope than any previous studies under uniform conditions,” says Voets, who is now with the Department of Chemical Engineering and Chemistry at Eindhoven University of Technology.

Thanks to an EU Marie Skłodowska Curie fellowship, she was able to perform detailed biophysical experiments on key AFP functions. Based on these results, the team was able to establish the absence of a distinct correlation between ice recrystallisation inhibition and thermal hysteresis activity.

PRICE researchers established that the moderately active type III AFP from ocean pout, despite previous reports, effectively and quickly blocks ice crystal growth and is a powerful inhibitor of ice recrystallisation. “That was a big discovery, which we published in the Proceedings of the National Academy of Sciences, because it offers a knowledge base to direct efforts to engineer potent bio-inspired anti-freezes,” stresses Voets.

“It means we now understand better why and when anti-freeze proteins effectively depress the hysteresis freezing point and inhibit ice recrystallisation, so the process of developing biomimetic analogues for biological applications or anti-icing solutions to prevent ice-adhesion in machinery can be fast-tracked,” she adds.

Instead of commonly-used anti-freezes, such as salts and alcohols, the advantage of using AFPs as an additive is that minute amounts are effective, and they do not alter the physico-chemical properties of the water-based material, which makes them a greener alternative.

While it is too early to talk about commercial interests, the worldwide anti-freeze and coolant market, including industrial and automotive applications, is predicted by Markets and Markets to continue growing from USD 4.52 billion (€4.18 billion) in 2016 to reach USD 6.62 billion (€6.13 billion) by 2021.

A true innovation like a bio-inspired anti-freeze, which has a lower environmental impact, could be game-changer for the sector, according to the team.

“The EU funding helped to kick-start my own group which focused on this poorly understood area. Like others who have benefited from these fellowship grants, it really helped us to get up to speed through the exchange of experience between teams in the Netherlands and abroad,” concludes Voets.

Sagitta Peters
PRICE