title: 19F NMR studies of plasminogen activator inhibitor-1.
abstract: Plasminogen activator inhibitor-1 (PAI-1) is a 43 kDa protein involved in the regulation of fibrinolysis. PAI-1 is the principal inhibitor of tissue-type plasminogen activator (t-PA), trapping the proteinase as an acyl-enzyme covalent complex (approximately 105 kDa). Four single tryptophan mutants of PAI-1 have been constructed in which three of the four tryptophan residues (Trp86, Trp139, Trp175, and Trp262) were replaced with phenylalanine. Biosynthetic incorporation of 5-fluorotryptophan (5F-Trp) into wild-type PAI-1 (5FW wtPAI-1) and the single tryptophan mutants (5FW86, 5FW139, 5FW175, and 5FW262) was achieved, allowing a (19)F NMR spectroscopic study of PAI-1 in its active and cleaved forms and in complex with t-PA. The (19)F NMR spectrum of active 5FW wtPAI-1 shows four clearly resolved peaks at -39.20, -49.26, -50.74, and -52.57 ppm relative to trifluoroacetic acid at 0 ppm. Unequivocal assignments of these four resonances in the spectrum of 5FW wtPAI-1 to specific tryptophan residues were accomplished by measuring the chemical shifts of the (19)F resonances of the single tryptophan mutants. There was close agreement between the resonances observed in 5FW wtPAI-1 and of those in the mutants for all three protein forms. This would imply little structural perturbation in the local structures of the tryptophan residues resulting from substitution by phenylalanine. The 5FW wtPAI-1 was observed to have lower second-order rate constant (k(app)) for the inhibition of t-PA than the natural tryptophan wtPAI-1, suggesting that the decreased activity may result from a small structural effect of the fluorine substituent of the indole ring. Further alterations in the k(app) and the stoichiometry of inhibition (SI) were observed in each of the mutants indicating an effect of the three tryptophan to phenylalanine mutations. Detailed interpretation of the (19)F NMR spectra of the PAI-1 mutants provides insights into the local segmental structure of the active form of the proteins and the structural changes that occur in the cleaved and t-PA complexed forms.
authors.affiliation: Department of Chemistry, University of Waterloo, Ontario, Canada.
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Abbott
authors.forename: Grant E
authors.initials: GE
authors.lastname: Blouse
authors.forename: Michel J
authors.initials: MJ
authors.lastname: Perron
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Shore
authors.forename: Linda A
authors.initials: LA
authors.lastname: Luck
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Szabo
mesh_terms: D005461:Fluorine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D005461:Fluorine; C026927:5-fluorotryptophan; D014364:Tryptophan; D010959:Tissue Plasminogen Activator