We study the ice-binding site (IBS) of a hyperactive antifreeze protein from the beetle Dendroides canadensis (DAFP-1) using vibrational sum-frequency generation spectroscopy. We find that DAFP-1 accumulates at the air–water interface due to the hydrophobic character of its threonine-rich IBS while retaining its highly regular β-helical fold. We observe a narrow band at 3485 cm–1 that we assign to the O–H stretch vibration of threonine hydroxyl groups of the IBS. The narrow character of the 3485 cm–1 band suggests that the hydrogen bonds between the threonine residues at the IBS and adjacent water molecules are quite similar in strength, indicating that the IBS of DAFP-1 is extremely well-ordered, with the threonine side chains showing identical rotameric confirmations. The hydrogen-bonded water molecules do not form an ordered ice-like layer, as was recently observed for the moderate antifreeze protein type III. It thus appears that the antifreeze action of DAFP-1 does not require the presence of ordered water but likely results from the direct binding of its highly ordered array of threonine residues to the ice surface.

J. Phys. Chem. Lett.
Ultrafast Spectroscopy

Meister, K., Lotze, S., Olijve, L. L. C., DeVries, A., Duman, J. G., Voets, I., & Bakker, H. (2015). Investigation of the Ice-Binding Site of an Insect Antifreeze Protein Using Sum-Frequency Generation Spectroscopy. J. Phys. Chem. Lett., 6(7), 1162–1167. doi:10.1021/acs.jpclett.5b00281