'Antifreeze' developed for biological material

Dutch and American researchers have developed a protein that works like an antifreeze agent and which could be used to freeze and defrost biological material without causing any damage. Their work has been described in the journal PNAS.

Chucking summer fruit like strawberries into the freezer doesn’t work very well. This is partly due to the ice crystals that form inside the strawberry during the freezing and defrosting process and puncture the cell structure from within. The same thing happens to frozen donor organs, sperm and immune cells for immunotherapy.

An international research term, led by Renko de Vries from Wageningen University & Research (WUR) and Ilja Voets from Eindhoven University of Technology (TU/e), has now used computer simulations to develop a protein that combats the formation of ice crystals. They took inspiration from fish in the Arctic Ocean, which swim around freely despite the temperature lying below freezing point.

“They generate antifreeze proteins that prevent the formation of ice in their bodies,” said Rob de Haas, a PhD student at WUR and first author of the team’s study.

Antifreeze proteins are incredibly complex and difficult to recreate, which is why de Haas created a simplistic version — first on the computer and then in the lab. He began with the simplest version known to scientists: an antifreeze protein in the American plaice flatfish. He further simplified the fish’s protein digitally by removing any protrusions.

What remained is what scientists term an alpha helix — a spiral-shaped protein, like the spring in a pen. Just like a spring, an alpha helix has a stable shape, but if you twist it, you can reshape it slightly.

“It occurred to me that the natural antifreeze proteins in fish were reshaped like this,” de Haas said. He built the antifreeze protein on his computer, first in a perfect spiral shape and then gradually twisted. By doing so, he eventually developed four digital variants, which he then recreated in bacteria in the laboratory.

“I tested the functioning of the four antifreeze proteins by adding the bacteria to a shallow pool of water, which I then cooled to almost freezing point,” de Haas said. Studying the ice crystals under the microscope, he found that they appeared slightly smaller and less destructive when the twisted antifreeze proteins were present.

“By twisting the protein, the amino acids that latch on the water crystals align exactly,” de Haas said. In this way, the antifreeze protein fits onto the ice as if in a perfect mould, preventing ice crystals from developing any further beneath it.

Before the artificial protein can be applied to transplant organs, the researchers “first want to test whether our antifreeze agent prevents damage when freezing and defrosting simple cells”, de Haas said. It is the defrosting process that usually causes issues, as it is a gradual process in which sharp ice crystals are given enough time to form and damage biological material.

“The first tests conducted by fellow researchers from the Animal Breeding and Genomics chair group showed a positive effect of our antifreeze protein on the survival of frozen and defrosted pig sperm,” de Vries noted.

The antifreeze protein has been praised for its simplicity, as scientists do not always understand how such proteins work in nature. With his artificial version, de Haas brought the protein back to basics.

“For the first time ever, we can now digitally design antifreeze proteins and measure them solely at the protein level,” Voets said. “Thanks to these two discoveries, we can better study how antifreeze proteins exactly work in the future.”

Image caption: Ice crystals after the freezing and defrosting process using the antifreeze proteins. The proteins prevent the ice crystals from developing further into sharp, pointed shapes. Image credit: Rob de Haas.