Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone

The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a β-domain and an α-helical lid, showed two transitions centered at 56.2 and 76.0 °C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the α-helical lid, exhibited a single transition at 57.0 °C. Therefore, the transition of DnaK384-638 at 56.2 °C is mainly attributed to the unfolding of the β-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the β-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 °C, indicating that the substrate binding shifts the β-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the β-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the β-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the β-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the β-domain.