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

Abstract

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 H 2O and D 2O, 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 T 1 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.