High-Resolution NMR Studies of Human Tissue Factor

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Hemostasis involves the blood clotting cascade initiated by factor VIIa (fVIIa) binding to human tissue factor (TF).
• Existing X-ray crystal structures of soluble TF (sTF) lack electron density in key functional regions.
• Nuclear Magnetic Resonance (NMR) can offer complementary structural and dynamic information for enzyme activity.

Purpose of the Study:

• To demonstrate high-yield production of isotopically labeled recombinant sTF.
• To enable high-resolution solid-state and solution-state NMR studies of sTF.
• To provide a benchmark for TF structure elucidation.

Main Methods:

• Production of multiple milligrams of isotopically labeled recombinant sTF.
• Acquisition of solution NMR spectra at sub-millimolar concentrations.
• Acquisition of the first solid-state NMR spectra of sTF.
• Development of improved sample preparation and precipitation conditions.

Main Results:

• Successful high-yield production of various isotopically labeled sTF forms.
• Obtained high-resolution solution NMR spectra of monomeric sTF.
• Acquired the first solid-state NMR spectra of sTF.
• Enabled acquisition of multidimensional NMR datasets for TF chemical shift assignment.

Conclusions:

• High-yield production of labeled sTF facilitates advanced NMR studies.
• NMR provides critical structural and dynamic insights into TF.
• This work establishes a foundation for detailed TF structure elucidation.