title: Human factor VIIa and its complex with soluble tissue factor: evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods.
abstract: Ultracentrifugation and fluorescence anisotropy decay measurements were used to evaluate the asymmetry and conformational dynamics of human blood clotting enzyme VIIa (VIIa) and the complex it forms with a soluble truncation mutant of human tissue factor (sTF) which acts as an essential cofactor for VIIa. Sedimentation velocity experiments showed that both VIIa and the sTF.VIIa complex are highly asymmetric. In each case, the friction ratio f/fsphere, is consistent with a family of general elliposids ranging from prolate to oblate. Fluorescence anisotropy decay experiments were used to limit the family of elliposids which can describe the hydrodynamic behavior of VIIa and sTF.VIIa. For both VIIa and the sTF.VIIa complex, the oblate ellipsoid of revolution was eliminated. In addition, the fluorescence anisotropy decay data clearly show that upon binding sTF.VIIa loses a segmental motion involving a domain containing the active site of the enzyme. This suggests that sTF causes a stabilization of a limited range of VIIa conformations. This stabilization may be important for proper recognition of the TF.VIIa substrate, factor X.
authors.affiliation: Department of Biochemistry, Mount Sinai School of Medicine, City University of New York, New York 10029.
authors.forename: E
authors.initials: E
authors.lastname: Waxman
authors.forename: W R
authors.initials: WR
authors.lastname: Laws
authors.forename: T M
authors.initials: TM
authors.lastname: Laue
authors.forename: Y
authors.initials: Y
authors.lastname: Nemerson
authors.forename: J B
authors.initials: JB
authors.lastname: Ross
mesh_terms: D055598:Chemical Phenomena; D002627:Chemistry, Physical; D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D014461:Ultracentrifugation; D014783:Viscosity
chemical_list: D015942:Factor VIIa