Become ice cold without freezing thanks to ice-binding proteins

There is an onion under the microscope in the lab at TU Eindhoven. A wafer-thin slice of a red onion, so that the light passes through and the elongated cells are clearly visible. “At room temperature, they all look like beautifully lit rooms with dark red walls,” says chemistry professor Ilja Voets. “If you now lower the temperature, you will see that the cells become dark one by one.”

She shows a previously recorded red onion video. 15 degrees above zero: nothing to worry about. But as the temperature drops further thanks to liquid nitrogen, to 30 degrees below zero, the cells go black one by one. Ice growth blocks the light transmission. “And now watch what happens when we reheat the onion. The rooms become light, the ice disappears. But the red fluid from the cells flows through the cell membranes and cell walls to the neighboring cells. Something fundamental has changed.”

Studying such freeze-thaw processes, says Voets, is essential for biomedical science and technology. “If you freeze cells, for example for cell cultures or food production, you want them to be exactly the same as before after thawing. But that is not the case. The cell membrane is damaged, irreversible changes occur. Far from ideal. In our lab we want to see if we can do something about this with ice-binding proteins.”

They are also called antifreeze proteins: proteins that slow down or change ice growth. Voets himself prefers the umbrella term ice-binding proteins. “Not all those proteins do the same thing. Some bind to the ice crystals already present and indeed prevent further growth, but there are also proteins that change the growth shape of the ice crystals or ensure that new ice nuclei form more easily.”

Fish blood

Research into ice-binding proteins began in the 1960s, in the icy waters of the Southern Ocean around Antarctica. The young American biologist Arthur DeVries was stationed as a PhD student at the McMurdo research base, on the edge of the Antarctic ice sheet, to investigate what made the region’s fish so unique. It has been known for some time that the freezing point in the ice-cold ocean around Antarctica is exceptionally low due to the high salt content: around -1.5 degrees Celsius. Fish blood normally freezes at around -0.9 degrees Celsius. It was actually not possible at all for those Antarctic fish to swim around unfrozen. But DeVries discovered that they survived thanks to special proteins in their blood.

About fifty different ice-binding proteins are now known in biology. They occur not only in cold-loving fish, but also in insects, fungi, plants and bacteria. “They fulfill different functions there,” says Voets. “In animals they prevent the blood from freezing, in plants they ensure that the ice crystals do not become too large. Those are those antifreeze properties. But you also have proteins that promote the formation of new ice nuclei. And some bacteria cling to floating ice with it. By forming a so-called biofilm with diatoms – single-celled algae that live in the ocean and require sunlight – both stick to the underside of the ice. This way, the algae do not sink into the deep sea, but can capture as much light as possible and the bacteria benefit from the oxygen and other substances that the algae produce through photosynthesis.”

Self-organization

Understanding how the proteins work can ultimately lead to the next step: making synthetic ice-binding proteins. That is what Voets’ research department focuses on, among other things. “We look at self-organization in biological matter. In other words: what structures and conditions do you need to ensure that a material makes itself?”

Anyone who can influence the ice growth process can then use that knowledge widely. “Consider keeping donor organs fresh for longer by freezing them, making crops frost-resistant or improving the quality of frozen food,” says Nuriye van Lamoen, who is a master’s student in Voets’ group.

Van Lamoen studies technical physics. “The physicists and chemists have somewhat taken over the antifreeze research from the biologists. We now want to use artificial intelligence to design new ice-binding proteins that have exactly the right properties for the desired applications.”

Two computer models that won the Nobel Prize for Chemistry in 2024 are crucial for this: Rosetta and AlphaFold2. “With Rosetta you can design a protein and find out from which amino acids – which building blocks – such a protein can be composed,” says Van Lamoen. “With AlphaFold2 you can check whether that design is logical; whether the structure of the designed protein corresponds to a shape required to bind to ice. Because proteins fold themselves in a certain way, and the shape in which they do so determines their function.”

As an example, she uses a 3D-printed protein from the American winter plaice (Pseudopleuronectes americanus), a flatfish found along the west coast of the northern Atlantic Ocean. “This is of course greatly exaggerated – in reality ice-binding proteins are only a few nanometers in size. But here you see that there is a beautiful alpha helix in the structure of the protein, a right-handed spiral that protrudes outwards like a kind of corkscrew. This ensures adhesion to ice crystals.”

Perfect spiral

Voets published together with researchers from Wageningen University and the Institute for Protein Design an article in 2023 in PNAS about this specific flatfish protein. Under normal circumstances, the alpha helix is ​​a perfect spiral, but something remarkable appears to happen in the ice-binding proteins of the American winter plaice: the spiral deforms in such a way that its amino acids fit exactly on the ice crystals. This prevents further ice growth. Van Lamoen: “The next step is now to investigate whether we can also modify this protein in such a way that new ice nuclei are formed, as happens with some bacteria. Then you speak of ice core-forming proteins.”

The research field of ice nucleating materials has grown explosively in recent years, Voets adds. “The process of ice core formation is important, among other things, in climate prediction. Because the more ice crystals there are in clouds, the more precipitation can fall.”

Although much research into ice-binding proteins is currently still at the fundamental stage, practical applications are also known. “In the United States, ice-binding proteins have already been incorporated into packaged ice creams to reduce the sugar content,” says Voets. “The proteins inhibit crystal growth, so that the ice cream remains nice and smooth with less sugar.”

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