Domain structure, stability and domain-domain interactions in recombinant factor XIII

Area of Science:

• Protein biochemistry
• Structural biology
• Biophysics

Background:

• Recombinant factor XIII (rFXIII) is a complex protein with a dimeric structure.
• Understanding the thermal stability and domain organization of rFXIII is crucial for its function and potential therapeutic applications.

Purpose of the Study:

• To investigate the heat denaturation process of intact rFXIII and its C-terminal fragments.
• To determine the number, stability, and organization of domains within the rFXIII subunit.

Main Methods:

• Fluorescence spectroscopy
• Circular dichroism (CD)
• Differential scanning calorimetry (DSC)
• Ultracentrifugation analysis
• Analysis of X-ray crystallographic model

Main Results:

• Intact rFXIII exhibits two distinct thermal transitions, indicating the presence of domains with different stabilities.
• Thermodynamic analysis reveals five domains per subunit: three thermolabile and two thermostable.
• C-terminal fragments (24 kDa and 12 kDa) correspond to the two thermostable beta-barrel domains.
• The N-terminal and catalytic core regions form the three thermolabile domains.
• Thermolabile domains mediate intra- and intermolecular interactions, leading to rFXIII dimerization, which is reversible at acidic pH.

Conclusions:

• rFXIII subunit comprises five distinct domains with varying thermal stabilities.
• The C-terminal region contains two thermostable beta-barrel domains, while the N-terminal and catalytic regions contain three thermolabile domains.
• Interactions between thermolabile domains are essential for rFXIII dimer formation and stability.