title: Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain.
abstract: The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism, and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3-5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately -8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between -9.5 and -12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Vargas-Uribe
authors.forename: Mykola V
authors.initials: MV
authors.lastname: Rodnin
authors.forename: Karin
authors.initials: K
authors.lastname: Öjemalm
authors.forename: Aurora
authors.initials: A
authors.lastname: Holgado
authors.forename: Alexander
authors.initials: A
authors.lastname: Kyrychenko
authors.forename: IngMarie
authors.initials: I
authors.lastname: Nilsson
authors.forename: Yevgen O
authors.initials: YO
authors.lastname: Posokhov
authors.forename: George
authors.initials: G
authors.lastname: Makhatadze
authors.forename: Gunnar
authors.initials: G
authors.lastname: von Heijne
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
mesh_terms: D004167:Diphtheria Toxin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D011485:Protein Binding; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013816:Thermodynamics
chemical_list: D004167:Diphtheria Toxin