title: Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition.
abstract: The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
authors.forename: G
authors.initials: G
authors.lastname: Ramsay
authors.forename: D
authors.initials: D
authors.lastname: Montgomery
authors.forename: D
authors.initials: D
authors.lastname: Berger
authors.forename: E
authors.initials: E
authors.lastname: Freire
mesh_terms: D002152:Calorimetry, Differential Scanning; D004167:Diphtheria Toxin; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments; D011487:Protein Conformation
chemical_list: D004167:Diphtheria Toxin; D010446:Peptide Fragments