| Antifreeze proteins protect many types of organisms from damage caused by freezing. They do this by binding to the ice surface, which causes inhibition of ice crystal growth. However, the molecular mechanism of ice binding leading to growth inhibition is not well understood. In this thesis, NMR investigations of the structure and dynamics of the antifreeze protein from the yellow mealworm beetle Tenebrio molitor are presented. TmAFP is a small, highly disulfide-bonded, right-handed parallel beta-helix consisting of seven tandemly repeated 12 amino acid loops. Analysis of the 15N relaxation parameters shows that TmAFP is a well-defined, rigid structure, and the extracted parameters reveal similar restricted internal mobility throughout the protein backbone at both 30 and 5°C. In TmAFP, an array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. The flexibility of the threonine side chains was investigated using two-dimensional homonuclear NMR spectroscopy and natural abundance 13C relaxation. From measurement of the 3Jalphabeta 1H-1H scalar coupling constants, the chi1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas threonines not on the ice-binding face experience conformational averaging. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. The CalphaH relaxation measurements are compared to the measured 15N backbone parameters and these are found to be in agreement. For analysis of the threonine side chain motions, a model of restricted rotational diffusion about the chi1 dihedral angle was employed. The range of motion experienced by the ice-binding threonine side chains is restricted, corresponding to a range of less than +/-25°. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand. |
