The serpin plasminogen activator inhibitor 1 (PAI-1) folds into an active structure and then converts slowly to a more stable, but low-activity, 'latent' conformation [Hekman, C. M., and Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587]. Thus, the folding of PAI-1 is apparently 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 not. Instead, denatured latent PAI-1 refolds in lower concentrations of urea to give the active protein. Thus, the high-stability latent conformation is kinetically inaccessible over a range of urea concentrations. Complete denaturation of latent PAI-1 occurs at 5.5 M urea [ΔG(H2O) ~ 21 kcal] whereas active PAI-1 denatures in only 3.8 M urea [ΔG(HO) ~12 kcal]. The fluorescence emission profile, as a function of urea of both the active and latent forms of the protein, reveals intermediates with partial structure. Circular dichroism measurements and limited protease digestion with Lys-C suggest that the intermediate in the denaturation of latent PAI-1 retains most of the secondary structure of the fully folded protein, whereas the intermediate in the denaturation of active PAI-1 exhibits significant loss of secondary structure. The Lys-C digestion patterns show that the active protein is more susceptible to proteolysis near sheet A than is the latent form. The studies suggest a model for the kinetically controlled folding pathway of PAI-1.
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