Moving along the Free Energy Landscape of Membrane Insertion of the Diphtheria Toxin Translocation Domain

Abstract

The pH-triggered membrane insertion of the translocation (T) domain is critical for the entry of the diphtheria toxin into the target cell. Previously we characterized the kinetic pathway of membrane insertion of the T domain, which consists of a sequence of conformational changes that convert a water-soluble state into a transmembrane state. Here we utilize various thermodynamic approaches to determine the changes in the Gibbs free energy associated with these conformational changes. The initial conformational change, which occurs in solution, was studied by thermal and chemical denaturation using differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. We found that acidification of solution, which results in the formation of the membrane-competent form, reduces the thermodynamic stability of the T-domain by about 3-5 kcal/mol, depending on the experimental conditions. For thermodynamic studies of membrane insertion we applied a novel approach that combines fluorescence correlation spectroscopy with the use of fluorinated surfactants as chemical chaperones. We estimated that the free energy values for the transition from a membrane-competent state in solution to the interfacial intermediate, and to the final transmembrane state are about −8.2 ± 0.2 kcal/mol and −12.0 ± 0.2 kcal/mol, respectively. The free energy barrier between the two states is modulated by the presence of anionic lipids. We summarize our findings in a proposed free energy landscape for the refolding and bilayer insertion of the T domain. Supported by NIH GM-069783 (A.S.L.), and Fulbright-CONICYT and BRTP (M.V.U.)