tailieunhanh - Báo cáo khoa học: Unfolding and aggregation during the thermal denaturation of streptokinase

The thermal denaturation of streptokinase fromStrepto-coccus equisimilis(SK) together with that of a set of frag-ments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivo-callyunder equilibrium. | Eur. J. Biochem. 269 4121-4133 2002 FEBS 2002 doi Unfolding and aggregation during the thermal denaturation of streptokinase Ana I. Azuaga1 Christopher M. Dobson2 Pedro L. Mateo1 and Francisco Conejero-Lara1 1 Departamento de Quimica Fisica e Instituto de Biotecnologia Facultad de Ciencias Universidad de Granada Granada Spain 2Oxford Centre for Molecular Sciences and New Chemistry Laboratory University of Oxford UK The thermal denaturation of streptokinase from Streptococcus equisimilis SK together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry DSC . Analysis of the effects of pH sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions pH and a sample concentration of less than w mg-mL-1 or pH the heat capacity curves of intact SK can be quantitatively described by three independent two-state transitions each of which compares well with the two-state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH and high sample concentration or at pH at any concentration investigated the thermal unfolding of domain A was accompanied by the time-dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves which become concentration dependent and kinetically controlled and thus precludes their proper analysis by standard deconvolution methods. A simple model involving timedependent high-order aggregation may account for the observed effects. Limited-proteolysis experiments suggest that in the aggregates the N-terminal segment 1-63 and the whole of SK domain C are at least partially structured while domain B is highly unstructured. Unfolding