|We are studying the relationship between structure and function in two different classes of proteins: antifreeze proteins and calcium-dependent cysteine proteinases (calpains). Antifreeze proteins are found in some fish, insects and plants. They bind to ice crystals and prevent them from growing to a size where they would damage the host. Our research involves the isolation and characterization of antifreeze proteins from different sources, and the cloning and expression of their genes to produce recombinant proteins for 3-D structural analysis by NMR and/or X-ray crystallography. They are proving to have remarkably diverse structures.
We are trying to identify their ice-binding sites/residues using site-directed mutagenesis in order to learn more about their mechanism(s) of action and what constitutes an ice-binding motif. Eventually, we would like to design new AFPs based on this information. place davies6gif.gif here Graphic to be inserted
A model of spruce budworm AFP (sbwAFP) showing its surface complementarity with the prism and basal planes of ice. N and C represent the N-terminal and C-terminal ends of this threonine-rich left-handed beta-helical protein. Threonine side chains are displayed. The left panel shows an end-on view of sbwAFP aligned above an ice prism plane. Circles depict thelattice positions of water oxygen atoms in ice. The centre panel shows a perpendicular view to that of the left panel. This view down the c-axis illustrates the match of adjacent loops to the ice surface. Since oxygen atoms have the same interatomic distance along the a-axis in both basal and prism planes, the match to the protein is the same for both planes. The right panel shows the sbwAFP aligned above the basal plane, depicting the analogous surface match to that of the prism plane.
Calcium-dependent cysteine proteinases. Our work on mammalian calpains is done in collaboration with Dr. John Elce, of this department, and is funded by the Protein Engineering Network of Centres of Excellence. Some of the issues we are investigating in this two-subunit, multi-domain enzyme are the functions of each domain, their interactions within the enzyme, and how calpains are regulated by autoproteolysis, calcium levels and phospholipid. Our approach is through the production of recombinant calpain in E. coli by co-expression of cloned sequences for the large and small enzyme subunits, in conjunction with mutagenesis and domain swap experiments