Serpins are involved in regulation of proteinases of many physiological processes including blood coagulation and fibrinolysis. Serpins utilize a unique mechanism to inhibit proteinases. This mechanism requires a major conformation change in the serpin resulting in a stable and essentially irreversible serpin-proteinase complex. This conformational change is thought to involve the insertion of the reactive center loop into beta-sheet A, possibly separating the P1-P1 residues by as much as 70 Angstrom units, as observed in the crystal structures of cleaved and latent serpins. Three models requiring minimal, partial, or full insertion of the reactive center loop into beta-sheet A have been proposed for the inhibitory mechanism of serpins. Knowledge of the structure of a serpin-proteinase complex would aid in differentiating between the proposed models, but crystallography has yet to elucidated the structure of a serpin-proteinase complex. This proposal seeks to use (1H, 15N)-heteronuclear single quantum correlation NMR to characterize the serpin-proteinase complex in solution. The hypothesis is that formation of a stable serpin-proteinase complex involves translocation of the proteinase from its initial docking position to the opposite pole of the serpin. This translocation requires full insertion of the reactive center loop into beta-sheet A trapping the proteinase as a kinetically stabilized acylenzyme intermediate rigidly held to the serpin. This hypothesis will be tested through the following specific aims. Specific Aim 1: To characterize conformational changes in the kinetically trapped serpin-proteinase complex and distinguish between the proposed models of proteinase inhibition by serpins. Specific Aim 2: To determine the dynamic interactions of the serpin and proteinase in the non-covalent and covalent serpin-proteinase complex. Specific Aim 3: To define the serpin residues which form the serpin-proteinase interface in the covalent serpin-proteinase complex.