Effects on the conformation of FVIIa by sTF and Ca2+ binding: Studies of fluorescence resonance energy transfer and quenching

Carlsson, Karin; Persson, Egon; Lindgren, Mikael; Carlsson, Uno; Svensson, Magdalena

doi:10.1016/j.bbrc.2011.08.135

Cited by 1 publication

(1 citation statement)

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“…The extracellular domain comprises two fibronectin type-III domains, which have primarily β-sheet secondary structure. The isolated extracellular domain of TF (also known as soluble TF, sTF) does not occur naturally, but because it is highly water soluble while retaining the ability to form an active enzyme complex with fVIIa [ 3 ], it has been used in a variety of biophysical studies to investigate TF [ 4 – 9 ]. Apart from blood coagulation, TF has also been implicated in a variety of processes, including inflammation, metastasis, and cell signaling [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

High-Resolution NMR Studies of Human Tissue Factor

et al. 2016

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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.

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Section: Introductionmentioning

confidence: 99%