The serine protease family termed the granzymes has been implicated in the cytotoxic lymphocyte mediated cell death mechanism. Each granzyme is proposed to have an individual primary and extended specificity, like a fingerprint, yet the family has above 50% sequence similarity. How is it that these enzymes have such unique cleavage sites and yet are so similar in their primary sequences? This leads us to hypothesize that the components of extended specificity in this scaffold must come from just a few amino acids, varied through evolution to provide differing sequence specificities. By finding these essential amino acids in granzyme B and defining their interactions with the substrate, the specificities of the granzyme B-like family can be both predicted and engineered. Experiments address these issues through three pathways: solving the structure of granzyme B, using alignments of the family to determine the residues directly involved in specificity, and using site directed mutagenesis to discover the specific differences at these sites. The Computer Graphics Laboratory is invaluable for all three aspects of this project. MidasPlus is used extensively to draw conclusions regarding this family of proteins.
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