The serpins (an acronym for serine protease inhibitors) comprise a superfamily of about forty homologous proteins which are implicated in various regulatory processes in humans, including the inflammatory cascades of blood coagulation, complement activation, and matrix proteolysis. The "warhead" of the serpin is the so-called reactive loop, containing ca. 20 amino acids, which is primarily responsible for protease recognition and specificity. Although the structures of several inactive serpins have been determined by X-ray crystallographic methods, no three-dimensional structures has yet been reported of an uncleaved, active (i.e., inhibitory) serpin. Given the biological and pharmaceutical importance of serpin structure-function relationships, it is unfortunate that these circumstances have hindered the field of serpin research. In this proposal we describe the X-ray crystallographic structure determination of the uncleaved serpin antichymotrypsin. In particular, the structure of an antichymotrypsin variant, engineered to be an inhibitor of human neutrophil elastase, has been determined to resolution of 2.5 A; refinement is currently in progress (R=0.195). We propose to continue the refinement of the antichymotrypsin structure, and we additionally propose to determine the structures of the wild-type serpin as well as variants which exhibit novel properties. Given that plasma antichymotrypsin concentrations rise dramatically in response to inflammatory episodes resulting from neutrophil degranulation, we expect that the structural work described herein will set a powerful foundation for the protein engineering of therapeutic agents useful in the treatment of inflammation, e.g., as inhibitors of neutrophil granule enzymes cathepsin G and elastase. Three-dimensional structures of antichymotrypsin and its variants will provide required information for the rational modification of serpin specificity, activity, and stability in vitro as well as in vivo.