DOMAIN-STRUCTURE, STABILITY AND DOMAIN-DOMAIN INTERACTIONS IN RECOMBINANT FACTOR-XIII

The process of heat denaturation of recombinant factor XIII (rFXIII), as well as its C-terminal 24 kDA and 12 kDa elastase-produced fragments starting at Ser514 and Thr628, respectively, was investigated in a wide range of conditions by fluorescence, CD and differential scanning calorimetry (DSC). It was found that the intact protein melts in two distinct temperature regions reflecting unfolding of different parts of the molecule with different stability The less stable structures unfold in a low temperature transition with a t(m) of 69 degrees C or lower depending on conditions. Unfolding of the more stable structures was observed at extremely high temperatures, t(m) > 110 degrees C at acidic pH < 3.5 and t(m) = 90 degrees C at pH 8.6 with 2M GdmCL. Thermodynamic analysis of the low and high temperature DSC-obtained heat absorption peaks indicated unambiguously that the first represents melting of three thermolabile independently folded domains while two thermostable domains melt in the second one giving a total of five domains in each a subunit of rFXIII. Both 24 kDa and 12 kDa fragments exhibited a sigmoidal spectral transition at comparatively high temperature where the thermolabile structures are already denatured, indicating that two thermostable domains are formed by the C-terminal portion of rFXIII and correspond to the two beta-barrels revealed by crystallography The remaining 56 kDa portion forms three thermolabile domains, one of which corresponds to the N-terminal beta-sandwich and the other two to the catalytic core. Fast accessible surface calculations of the X-ray model of rFXIII confirmed the presence of two structural subdomains in the core region with the boundary at residue 332. The thermolabile domains appear to interact with each other intra- and/or intermolecularly resulting in dimerization the a subunits. At acidic pH, where all domains became destabilized but still remained folded, interdomainial interactions seemed to be abolished, resulting in the reversible dissociation of the dimer as revealed by ultracentrifugation analysis.