title: Engineering plasminogen activator inhibitor 1 mutants with increased functional stability.
abstract: Plasminogen activator inhibitor 1 (PAI-1), a member of the serine protease inhibitor (Serpin) superfamily, is the primary inhibitor of the plasminogen activators tPA and uPA. PAI-1 is produced in an active form but converts to an inactive or latent form with a half-life of approximately 1 h at pH 7.5, 37 degrees C. This study describes the construction, expression, and characterization of PAI-1 mutants with increased functional stability. Three mutations that disrupt an ion pair, present in latent PAI-1, between Arg-30 and Glu-350 (P4'), were introduced into recombinant PAI-1. All three mutant proteins maintained normal functional activity against both uPA and tPA. However, the half-life of each purified PAI-1 mutant was extended compared to the 1.1 h observed for wild-type PAI-1 (wtPAI-1) (1.2 h for Glu-350-->Arg, 2.0 h for Glu-350-->Pro, and 2.1 h for the Arg-30-->Glu mutation). An additional PAI-1 variant containing a second mutation designed to potentially reconstitute the ion pair (Arg-30-->Glu, Glu-350-->Arg) failed to restore the wild-type half-life. Circular dichroism spectra analysis indicated that the active and latent forms of wtPAI-1 and all four mutants contained similar secondary structural elements. Thermal stability determinations showed that latent wtPAI-1 was much more structurally stable than the active conformation. However, the latent form for all four mutants was significantly less stable than the corresponding wtPAI-1 conformer. This is the first report of PAI-1 mutants which have been specifically engineered to produce enhanced functional stability.
authors.affiliation: Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0650.
authors.forename: D A
authors.initials: DA
authors.lastname: Lawrence
authors.forename: S T
authors.initials: ST
authors.lastname: Olson
authors.forename: S
authors.initials: S
authors.lastname: Palaniappan
authors.forename: D
authors.initials: D
authors.lastname: Ginsburg
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator