Thermodynamic Identification of Stable Folding Intermediates in the B-Subunit of Cholera Toxin

The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 °C and characterized by a ΔH_cal of 328 kcal/mol of B-subunit pentamer and ΔH_vh/ΔH_cal of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van’t Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 °C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer. Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl. Trypsin digestion studies show that at pH 5.0 the B-subunit is 4 times more susceptible to digestion than at pH 7.0 and that at pH 5.0 limited proteolysis results in two fragments of ~7 and ~5 kDa. These studies provide strong evidence that the B-subunits of cholera toxin are composed of two folding/unfolding domains and that the interactions between the two domains within the same subunit and between subunits are able to account for the cooperative behavior of the entire pentameric ring.