Researchers Engineer Cold-Tolerant Proteins to Give U.S. an Arctic Edge

Newswise — As the Arctic region becomes increasingly contested, the U.S. military faces a new era of challenges in one of the world’s most inhospitable environments. Researchers at the Johns Hopkins Applied Physics Laboratory (APL) in Laurel, Maryland, are tapping into the unique properties of ice-affecting biomolecules to develop novel materials and technologies that could give the nation a critical strategic and operational edge.

The effort is part of the Defense Advanced Research Projects Agency’s (DARPA) Ice Control for cold Environments (ICE) program, which seeks to harness nature’s cold-weather adaptations to develop solutions to the operational challenges of extreme cold.

APL plays a key role in the program through a project called BOREAS, for Bio-Optimized Regulation of Environmental Ice for Arctic Supremacy. The project team’s goal is to build a toolkit of ice-manipulating technologies and protein formulations to help the Department of War operate in the harshest cold environments.

APL started developing ice-manipulating technologies in 2020 through internally funded opportunities, and subsequent demonstration of those capabilities helped secure a role in the DARPA program. “It’s a great story of APL investing resources to build capabilities in anticipation of a sponsor need,” said Joel Sarapas, a polymer chemist in APL’s Research and Exploratory Development Department.

Libraries of Molecules

Sarapas and his team have built a physical library containing hundreds of bioinspired synthetic polymers — chain-like molecules with branches containing chemical side groups. Those branches can alter the behavior of the polymer and how the system interacts with the surrounding environment.

The team has uncovered dozens of molecules that alter ice formation, including a surprising find: a derivative of a sugar additive commonly used in cosmetics. When attached to a polymer, the compound proved unusually effective at encouraging ice to grow.

“That was a big surprise for us,” Sarapas said. “Based on its structure, we assumed it would inhibit ice, like similar molecules do. But when we applied it to a chilled surface and passed humid air over it, ice appeared exactly where the polymers were placed.”

To illustrate the effect, the team used a formulation with these molecules to paint letters on a surface and placed it within a cold, humid environment. Within minutes, “APL” appeared in ice.

Working in parallel, a team led by APL molecular biologist Will Stone developed a second library of proteins that affect ice formation, including ice-nucleating proteins, which help ice form, and antifreeze proteins, which prevent ice growth. Counterintuitively, by combining these protein types, the team expects it can create stronger ice crystals.

Both antifreeze and ice-nucleating proteins work by interacting with individual water molecules to control how ice takes shape. Ice-nucleating proteins provide a scaffold for water molecules to form crystals, while antifreeze proteins cling to existing ice crystals to prevent them from growing, which many polar fish use to stop their blood from freezing. By combining these proteins, these tethered water molecules could come closer together to create bigger, more resilient ice seed crystals.

“By exploring unexpected synergies like these, we’re uncovering new molecular motifs that don’t exist in nature but could unlock stronger, more robust ice,” Stone said.

To create these novel combinations, the team identified 36 well-studied antifreeze and ice-nucleating proteins and used machine learning to screen massive databases for other sequences with similar molecular features. From 14,000 candidates, they selected just 108 proteins, most of them uncharacterized, and randomly linked them to create a library of 11,664 unique protein pairs, which they produced and tested.

“Now, a large component of the process is designing techniques to measure the activity of all of these molecules,” Stone said. “To do that we had to come up with new, high-throughput lab assays to find those ‘needle in the haystack’ combinations that work.”

Finding a Needle

One key approach is the use of standardized microwell plate assays, which can process 384 samples in just under an hour. The team is also developing a cutting-edge mechanics assay that can test the adhesion energy of hundreds of samples per hour — a staggering pace considering that traditional adhesion tests can take 5-10 minutes per sample.

What has been truly revolutionary, however, is the team’s use of droplet microfluidics. These assays encapsulate single cells or molecules individually into tiny droplets and then expose them to freezing conditions. This allows the team to study the ice-affecting actions of thousands of individual molecules within a short period. “This is a very advanced, cutting-edge assay, and one we’re still perfecting,” Sarapas said. “But it’s going to be a game changer.”

As BOREAS enters its next phase, the team hopes to apply the insights and discoveries of the first phase to the development of real-world materials. “It’s cool to have a molecule that does something in a tube, but it’s entirely different to have it do something useful in the field,” Sarapas noted.

The possibilities include creating stronger or more robust ice for construction purposes, or preventing ice formation on surfaces like aircraft wings. The team is also investigating medical applications, such as frostbite prevention and the development of freeze-tolerant medicines. One day this technology could even be used to enable the freezing of donated transfusion blood for storage, enabling it to travel farther to places where it’s needed.

“It’s very early days on all of this,” Sarapas said. “But the project is so exciting, galvanizing, and such a natural motivator for people to do things that will be useful in so many ways.”