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

Vinod Bhakuni, Dong Xie, Ernesto Freire

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28 Scopus citations


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 ΔHcal of 328 kcal/mol of B-subunit pentamer and ΔHvh/ΔHcal 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.

Original languageEnglish (US)
Pages (from-to)5055-5060
Number of pages6
Issue number20
StatePublished - May 1 1991

ASJC Scopus subject areas

  • Biochemistry


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