Energetics of Diphtheria Toxin Membrane Insertion and Translocation: Calorimetric Characterization of the Acid pH Induced Transition

Glen Ramsay, Diana Montgomery, Dolores Berger, Ernesto Freire

Research output: Contribution to journalArticlepeer-review

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 °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 °C whereas that of the nucleotide-free toxin is centered at 51.8 °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 °C) and the B fragment (Tm = 58.4 °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 °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.

Original languageEnglish (US)
Pages (from-to)529-533
Number of pages5
JournalBiochemistry
Volume28
Issue number2
DOIs
StatePublished - 1989

ASJC Scopus subject areas

  • Biochemistry

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