The high resolution structure of type III antifreeze protein and its implications for ice binding

Determining the modus operandi of an antifreeze protein (AFP) is particularly difficult because of the lability of its ligand, ice. It is currently not possible to observe ice in contact with an AFP at the molecular level without perturbing that interaction, and therefore, indirect methods must be used. In one study the unique ice-crystal morphologies observed on dilution and mutation of type III antifreeze protein helped explain the binding preference of this globular antifreeze for the prism surfaces of ice. The high resolution crystal structure was determined in order to gain a better understanding of the ice-binding process. The protein fold is unique and contains a number of imperfect {dollar}beta{dollar} structural elements. One of the most interesting structural features is the planarity of the ice-binding surface and of the surfaces adjacent to that surface. All of the ice-binding residues appear to be restrained either by hydrogen bonds or by van der Waals interactions with other residues. Using this structure a docking of the protein to ice was modelled which predicted the role of an additional ice-binding residue (Thr15). The distinctive planarity of the ice-binding surface of the protein implies that this feature plays a role in the protein's function possible through a surface match between the protein and ice. The structure was also determined for mutants of type III antifreeze protein that disrupted this planarity. These structures confirmed the need for a planar protein face but also demonstrated that the mutations had secondary effects on the ice-binding residues because of their tight packing on the ice-surface. Additional residues (Arg47, Asp58, Lys61) on the protein were identified that on mutation showed no loss in thermal hysteresis activity but affected the protein's ability to stop ice crystal growth. These mutations are clustered together on a planar surface orthogonal to the main ice-binding surface, which may be involved in shielding the basal plane from water.; The creation of fusions of type III AFP to other proteins demonstrated that this antifreeze does not need cooperative interactions to bind tightly to ice. This indicates that the entire binding energy exists within one molecule of the protein. The fusion proteins also displayed higher activity than type III antifreeze protein alone due to better surface coverage or increased height of the antifreeze protein above the surface. These more active antifreezes may have potential biotechnological applications.