Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.
Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)
Fa
M
M
Department of Medical Biochemistry, Umeå University, Sweden.
Karolin
J
J
Aleshkov
S
S
Strandberg
L
L
Johansson
L B
LB
Ny
T
T
D001483:Base Sequence; D001896:Boron Compounds; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers; D005029:Ethylenediamines; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006493:Heparin / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019096:Vitronectin / Q000378:metabolism*
D001896:Boron Compounds; D017931:DNA Primers; D005029:Ethylenediamines; D005456:Fluorescent Dyes; C096465:N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacenyl-3-propionyl)-N-(iodoacetyl)ethylenediamine; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D006493:Heparin; D003545:Cysteine