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

¹ California State University, Los Angeles

^* To whom correspondence should be addressed. E-mail: yba{at}calstatela.edu.

Submitted on July 25, 2005
Revised on September 2, 2005
Accepted on 10 April 2006

	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 -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, 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 17^th and 21^st Ala side chains (i+4, 8), and the second one on the equivalent 8^th, 19^th and 30^th Ala side chains (i+6). The two kinds of labels are on the opposite sides of the -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 while the surrounding water molecules to the i+6 methyl groups could be looser. This study suggests that the side of the -helical AFP comprising the i+4 and i+8 Ala methyl groups could interact with the ice surface in the ice/water interface.

Key Words: ¹³C Spin Latice Relaxation, Antifreeze proteins, Binding to ice surface, Solid state NMR