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 fragment s starting at Ser514 and Thr628, respectively, was investigated in a w ide range of conditions by fluorescence, CD and differential scanning calorimetry (DSC). It was found that the intact protein melts in two d istinct temperature regions reflecting unfolding of different parts of the molecule with different stability The less stable structures unfo ld in a low temperature transition with a t(m) of 69 degrees C or lowe r depending on conditions. Unfolding of the more stable structures was observed at extremely high temperatures, t(m) > 110 degrees C at acid ic pH < 3.5 and t(m) = 90 degrees C at pH 8.6 with 2M GdmCL. Thermodyn amic analysis of the low and high temperature DSC-obtained heat absorp tion peaks indicated unambiguously that the first represents melting o f three thermolabile independently folded domains while two thermostab le domains melt in the second one giving a total of five domains in ea ch a subunit of rFXIII. Both 24 kDa and 12 kDa fragments exhibited a s igmoidal spectral transition at comparatively high temperature where t he thermolabile structures are already denatured, indicating that two thermostable domains are formed by the C-terminal portion of rFXIII an d correspond to the two beta-barrels revealed by crystallography The r emaining 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 mode l of rFXIII confirmed the presence of two structural subdomains in the core region with the boundary at residue 332. The thermolabile domain s appear to interact with each other intra- and/or intermolecularly re sulting in dimerization the a subunits. At acidic pH, where all domain s became destabilized but still remained folded, interdomainial intera ctions seemed to be abolished, resulting in the reversible dissociatio n of the dimer as revealed by ultracentrifugation analysis.