Thermal Stability and Intersubunit Interactions of Cholera Toxin in Solution and in Association with Its Cell-Surface Receptor Gangliosidegm

The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside G_Mlhas been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside G_Ml, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 °C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 °C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside G_Ml, the B subunit is greatly stabilized as indicated by an increase of 20 °C in the transition temperature. In addition, ganglioside G_Mlgreatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside G_Ml. The exothermic process at the onset of the B subunit pentamer unfolding is not present when the toxin molecule is bound to ganglioside G_Ml. This effect is observed with both micellar and membrane-bound gangliosides but not with pure phospholipid vesicles. Differential solubility thermal gel analysis indicates that under these conditions the A subunit remains associated with the micellar ganglioside G_Mlor ganglioside G_M1-containing membranes.