Insight into the Binding of Antifreeze Proteins to Ice Surfaces via 13C Spin Lattice Relaxation Solid-State NMR

The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of α-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP’s ice-binding side, a variable-temperature dependent 13C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain 13C labeled HPLC6 isoforms of the type I AFPs each frozen in H2O and D2O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the α-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T1 data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the α-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface.