title: High-Resolution NMR Studies of Human Tissue Factor.
abstract: In normal hemostasis, the blood clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to the integral membrane protein, human tissue factor (TF). The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the soluble extracellular domain of TF (sTF) exist; however, these structures are missing electron density in functionally relevant regions of the protein. In this context, NMR can provide complementary structural information as well as dynamic insights into enzyme activity. The resolution and sensitivity for NMR studies are greatly enhanced by the ability to prepare multiple milligrams of protein with various isotopic labeling patterns. Here, we demonstrate high-yield production of several isotopically labeled forms of recombinant sTF, allowing for high-resolution NMR studies both in the solid and solution state. We also report solution NMR spectra at sub-mM concentrations of sTF, ensuring the presence of dispersed monomer, as well as the first solid-state NMR spectra of sTF. Our improved sample preparation and precipitation conditions have enabled the acquisition of multidimensional NMR data sets for TF chemical shift assignment and provide a benchmark for TF structure elucidation.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Nuzzio
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Watt
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: John M
authors.initials: JM
authors.lastname: Boettcher
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Gajsiewicz
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: James H
authors.initials: JH
authors.lastname: Morrissey
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.|Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.|Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Chad M
authors.initials: CM
authors.lastname: Rienstra