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Observation of ice-like water layers at an aqueous protein surface

Significance Antifreeze proteins (AFPs) enable the survival of various organisms in freezing or subfreezing habitats by preventing the macroscopic growth of ice crystals. Understanding how AFPs recognize and bind ice crystals is the most important step to unravel their working mechanism. Using surface-specific sum frequency generation spectroscopy, we were able to directly probe the ice-binding site of the protein and discovered that, already at room temperature and in an aqueous solution, the antifreeze proteins arrange water molecules into an ice-like array, which they then use to bind to ice crystals. We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0 °C to −2 °C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice.