KINETICALLY CONTROLLED FOLDING OF THE SERPIN PLASMINOGEN-ACTIVATOR INHIBITOR-1

The serpin plasminogen activator inhibitor 1 (PAI-1) folds into an act ive structure and then converts slowly to a more stable, but low-activ ity, ''latent'' conformation [Hekman, C. M., & Loskutoff, D. J. (1985) J. Biol. Chem. 260, [11581-11587]. Thus, the folding of PAI-1 is appa rently under kinetic control. We have determined the urea denaturation and refolding transitions of both latent and active PAI-1 proteins by using intrinsic tryptophan fluorescence. While folding of active PAI- 1 is reversible, the denaturation and refolding of latent PAI-1 are no t. Instead, denatured latent PAI-1 refolds in lower concentrations of urea to give the active protein. Thus, the high-stability latent confo rmation is kinetically inaccessible over a range of urea concentration s. Complete denaturation of latent PAI-1 occurs at 5.5 M urea [Delta G (H2O) similar to 21 kcal] whereas active PAI-1 denatures in only 3.8 M urea [Delta G(H2O) similar to 12 kcal]. The fluorescence emission pro file, as a function of urea of both the active and latent forms of the protein, reveals intermediates with partial structure. Circular dichr oism measurements and limited protease digestion with Lys-C suggest th at the intermediate in the denaturation of latent PAI-1 retains most o f the secondary structure of the fully folded protein, whereas the int ermediate in the denaturation of active PAI-1 exhibits significant los s of secondary structure. The Lys-C digestion patterns show that the a ctive protein is mon susceptible to proteolysis near sheet A than is t he latent form. The studies suggest a model for the kinetically contro lled folding pathway of PAI-1.