High-Resolution NMR Studies of Human Tissue Factor

• Published in: PloS one Vol. 11; no. 9; p. e0163206
• Main Authors: Nuzzio, Kristin M, Watt, Eric D, Boettcher, John M, Gajsiewicz, Joshua M, Morrissey, James H, Rienstra, Chad M
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01-09-2016; Public Library of Science (PLoS)
• Subjects: Biochemistry; Blood coagulation; Chemical precipitation; Chemistry; Clotting; Coagulation factor VIIa; Crystal structure; Crystallography; E coli; Electron density; Enzymatic activity; Enzyme activity; Glycerol; Hemostasis; Hemostatics; High resolution; Hydrologic data; Integral membrane proteins; Isotopic labeling; Laboratories; Membrane proteins; Molecular weight; NMR; Nuclear magnetic resonance; Physical sciences; Proteins; R&D; Radioactive labeling; Research & development; Research and analysis methods; Sample preparation; Studies; Tissue factor; United States > US; Illinois
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0163206

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.