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 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal 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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Bhakuni
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Department of Biology and Biocalorimetry Center, Johns Hopkins University, Baltimore, Maryland 21218.
Xie
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Freire
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D002152:Calorimetry, Differential Scanning; D002772:Cholera Toxin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D010449:Peptide Mapping; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014357:Trypsin
D006146:Guanidines; D046911:Macromolecular Substances; D002772:Cholera Toxin; D014357:Trypsin; D019791:Guanidine