title: The use of fluorescent probes to characterize conformational changes in the interaction between vitronectin and plasminogen activator inhibitor-1.
abstract: Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central beta-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1' or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydryl-reactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1' bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an arginine-specific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996, USA.
authors.forename: A
authors.initials: A
authors.lastname: Gibson
authors.forename: K
authors.initials: K
authors.lastname: Baburaj
authors.forename: D E
authors.initials: DE
authors.lastname: Day
authors.forename: I
authors.initials: I
authors.lastname: Verhamme
authors.forename: J D
authors.initials: JD
authors.lastname: Shore
authors.forename: C B
authors.initials: CB
authors.lastname: Peterson
mesh_terms: D005456:Fluorescent Dyes; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D019096:Vitronectin / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin