W0076

Structure of Eel Pout Type III Antifreeze Protein: The Role of Hydrogen Bonds and Shape Complimentarity in Ice Binding. Zongchao Jia, Steffen P. Graether, Carl I. DeLuca, Jason Baardsnes, Peter L. Davies, Department of Biochemistry, Queen's University, Kingston, Ontario, Canada, K7L 3N6

Fish antifreeze proteins (AFP) inhibit ice growth by absorbing to the surface of ice, altering the curvature of the ice between the bound protein molecules and resulting in a local freezing point depression. The difference between the temperature at which the ice begins to grow quickly again (burst point) and the temperature at which the ice crystal melts is known as thermal hysteresis, and is used as a measure of AFP activity. Even for type I AFP with its linear and repetitive alpha-helical structure, the ice-binding mechanism is still not yet fully understood. Globular AFPs such as type III AFP are more complex. In order to probe the structural features responsible for ice-binding of type III AFP, we have determined the X-ray structure of eel pout type III AFP to 1.25 Å. The structure shows a globular, mostly beta-sheet protein with two flat surfaces. Preliminary point mutations and modelling suggested that surface adsorption may occur through a hydrogen-bond match between the side-chains of Gln9, Asn14, Thr15, Thr18, Gln44 on one flat surface and the ice prism plane {10 x 0} . In total, twenty-one mutations have been made and their structure determined (resolution from 1.43 Å to 2.6 Å) to examine the role of various residues in and near the putative ice-binding surface. An ice-binding model is proposed where: 1) There is an initial contact from Asn14 to an advancing ice layer; 2) Additional contacts are made by the hydrogen-bonding side chains (Gln9, Asn14, Thr15, Thr18, Gln44); 3) Hydrogen bonds are stabilized by surface shape complementarity which also enables non-specific van der Waals interaction to contribute to the binding.

Supported by Medical Research Council of Canada.