The Journal of Membrane Biology

, Volume 248, Issue 3, pp 383–394 | Cite as

Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain

  • Mauricio Vargas-Uribe
  • Mykola V. Rodnin
  • Karin Öjemalm
  • Aurora Holgado
  • Alexander Kyrychenko
  • IngMarie Nilsson
  • Yevgen O. Posokhov
  • George Makhatadze
  • Gunnar von Heijne
  • Alexey S. Ladokhin


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.


Bacterial toxins Membrane protein folding pH-triggered insertion Free energy Conformational switching 



Diphtheria toxin T-domain


State membrane-incompetent state populated at neutral pH


State membrane-competent (protonated) state populated at acidic pH


State interfacial state


State(s) transmembrane state(s)


Large unilamellar vesicles






Fluorescence correlation spectroscopy


Circular dichroism


Differential scanning calorimetry


Enthalpy change


Gibbs free energy


Melting temperature


Change in calorific capacity


Partitioning coefficient

Supplementary material

232_2014_9734_MOESM1_ESM.docx (64 kb)
Supplementary material 1 (DOCX 64 kb)


  1. Bennett MJ, Eisenberg D (1994) Refined structure of monomeric diphtheria toxin at 2.3 Å resolution. Protein Sci 3:1464–1475PubMedCentralCrossRefPubMedGoogle Scholar
  2. Clamme JP, Azoulay J, Mely Y (2003) Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy. Biophys J 84:1960–1968PubMedCentralCrossRefPubMedGoogle Scholar
  3. Eftink MR (1994) The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys J 66:482–501PubMedCentralCrossRefPubMedGoogle Scholar
  4. Haugland RP (1996) Handbook of fluorescent probes and research chemicals. Molecular Probes, Inc., EugeneGoogle Scholar
  5. Hayashibara M, London E (2005) Topography of diphtheria toxin A chain inserted into lipid vesicles. Biochemistry 44:2183–2196CrossRefPubMedGoogle Scholar
  6. Hessa T, Kim H, Bihlmaler K, Lundin C, Boekel J, Andersson H, Nilsson I, White SH, von Heijne G (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433:377–381CrossRefPubMedGoogle Scholar
  7. Hessa T, Meindl-Beinker NM, Bernsel A, Kim H, Sato Y, Lerch-Bader M, Nilsson I, White SH, von Heijne G (2007) Molecular code for transmembrane-helix recognition by the Sec61 translocon. Nature 450:1026–1030CrossRefPubMedGoogle Scholar
  8. Hope MJ, Bally MB, Mayer LD, Janoff AS, Cullis PR (1986) Generation of multilamellar and unilamellar phospholipid vesicles. Chem Phys Lipids 40:89–107CrossRefGoogle Scholar
  9. Kurnikov IV, Kyrychenko A, Flores-Canales JC, Rodnin MV, Simakov N, Vargas-Uribe M, Posokhov YO, Kurnikova M, Ladokhin AS (2013) pH-triggered conformational switching of the diphtheria toxin T-domain: the roles of N-terminal histidines. J Mol Biol 425:2752–2764PubMedCentralCrossRefPubMedGoogle Scholar
  10. Kyrychenko A, Posokhov YO, Rodnin MV, Ladokhin AS (2009) Kinetic intermediate reveals staggered pH-dependent transitions along the membrane insertion pathway of the diphtheria toxin T-domain. Biochemistry 48:7584–7594PubMedCentralCrossRefPubMedGoogle Scholar
  11. Ladokhin AS (2009) Fluorescence spectroscopy in thermodynamic and kinetic analysis of pH-dependent membrane protein insertion. Methods Enzymol 466:19–42CrossRefPubMedGoogle Scholar
  12. Ladokhin AS (2013) pH-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin T-domain. Toxins (Basel) 5:1362–1380CrossRefGoogle Scholar
  13. Ladokhin AS, Jayasinghe S, White SH (2000) How to measure and analyze tryptophan fluorescence in membranes properly, and why bother? Anal Biochem 285:235–245CrossRefPubMedGoogle Scholar
  14. Ladokhin AS, Legmann R, Collier RJ, White SH (2004) Reversible refolding of the diphtheria toxin T-domain on lipid membranes. Biochemistry 43:7451–7458CrossRefPubMedGoogle Scholar
  15. Lopez MM, Makhatadze GI (2002) Differential scanning calorimetry. Methods Mol Biol 173:113–119PubMedGoogle Scholar
  16. Lundin C, Kim H, Nilsson I, White SH, von Heijne G (2008) Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N(in)-C(out) and N(out)-C(in) transmembrane helices. Proc Natl Acad Sci USA 105:15702–15707PubMedCentralCrossRefPubMedGoogle Scholar
  17. Makhatadze GI, Medvedkin VN, Privalov PL (1990) Partial molar volumes of polypeptides and their constituent groups in aqueous solution over a broad temperature range. Biopolymers 30:1001–1010CrossRefPubMedGoogle Scholar
  18. Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161–168CrossRefPubMedGoogle Scholar
  19. Murphy JR (2011) Mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process. Toxins (Basel) 3:294–308CrossRefGoogle Scholar
  20. Oh KJ, Senzel L, Collier RJ, Finkelstein A (1999) Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain. Proc Natl Acad Sci USA 96:8467–8470PubMedCentralCrossRefPubMedGoogle Scholar
  21. Palchevskyy SS, Posokhov YO, Olivier B, Popot JL, Pucci B, Ladokhin AS (2006) Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin. Biochemistry 45:2629–2635CrossRefPubMedGoogle Scholar
  22. Posokhov YO, Rodnin MV, Das SK, Pucci B, Ladokhin AS (2008a) FCS study of the thermodynamics of membrane protein insertion into the lipid bilayer chaperoned by fluorinated surfactants. Biophys J 95:L54–L56PubMedCentralCrossRefPubMedGoogle Scholar
  23. Posokhov YO, Rodnin MV, Lu L, Ladokhin AS (2008b) Membrane insertion pathway of annexin B12: thermodynamic and kinetic characterization by fluorescence correlation spectroscopy and fluorescence quenching. Biochemistry 47:5078–5087CrossRefPubMedGoogle Scholar
  24. Rodnin MV, Posokhov YO, Contino-Pepin C, Brettmann J, Kyrychenko A, Palchevskyy SS, Pucci B, Ladokhin AS (2008) Interactions of fluorinated surfactants with diphtheria toxin T-domain: testing new media for studies of membrane proteins. Biophys J 94:4348–4357PubMedCentralCrossRefPubMedGoogle Scholar
  25. Rodnin MV, Kyrychenko A, Kienker P, Sharma O, Posokhov YO, Collier RJ, Finkelstein A, Ladokhin AS (2010) Conformational switching of the diphtheria toxin T domain. J Mol Biol 402:1–7PubMedCentralCrossRefPubMedGoogle Scholar
  26. Rodnin MV, Kyrychenko A, Kienker P, Sharma O, Vargas-Uribe M, Collier RJ, Finkelstein A, Ladokhin AS (2011) Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain. Biophys J 101:L41–L43PubMedCentralCrossRefPubMedGoogle Scholar
  27. Rosconi MP, London E (2002) Topography of helices 5–7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin. J Biol Chem 277:16517–16527CrossRefPubMedGoogle Scholar
  28. Rosconi MP, Zhao G, London E (2004) Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-streptavidin: At low pH, helices 8 and 9 form a transmembrane hairpin but helices 5–7 form stable nonclassical inserted segments on the cis side of the bilayer. Biochemistry 43:9127–9139CrossRefPubMedGoogle Scholar
  29. Senzel L, Huynh PD, Jakes KS, Collier RJ, Finkelstein A (1998) The diphtheria toxin channel-forming T domain translocates its own NH2-terminal region across planar bilayers. J Gen Physiol 112:317–324PubMedCentralCrossRefPubMedGoogle Scholar
  30. Senzel L, Gordon M, Blaustein RO, Oh KJ, Collier RJ, Finkelstein A (2000) Topography of diphtheria toxin’s T domain in the open channel state. J Gen Physiol 115:421–434PubMedCentralCrossRefPubMedGoogle Scholar
  31. Vargas-Uribe M, Rodnin MV, Kienker P, Finkelstein A, Ladokhin AS (2013a) Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state. Biochemistry 52:3457–3463CrossRefPubMedGoogle Scholar
  32. Vargas-Uribe M, Rodnin MV, Ladokhin AS (2013b) Comparison of membrane insertion pathways of the apoptotic regulator Bcl-xL and the diphtheria toxin translocation domain. Biochemistry 52:7901–7909CrossRefPubMedGoogle Scholar
  33. Walter P, Blobel G (1982) Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol 96:84–93CrossRefGoogle Scholar
  34. Walters J, Milam SL, Clark AC (2009) Practical approaches to protein folding and assembly: spectroscopic strategies in thermodynamics and kinetics. Methods Enzymol 455:1–39PubMedCentralCrossRefPubMedGoogle Scholar
  35. Wang J, London E (2009) The membrane topography of the diphtheria toxin T domain linked to the a chain reveals a transient transmembrane hairpin and potential translocation mechanisms. Biochemistry 48:10446–10456PubMedCentralCrossRefPubMedGoogle Scholar
  36. Wang Y, Malenbaum SE, Kachel K, Zhan HJ, Collier RJ, London E (1997) Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width. J Biol Chem 272:25091–25098CrossRefPubMedGoogle Scholar
  37. White SH, Wimley WC, Ladokhin AS, Hristova K (1998) Protein folding in membranes: determining the energetics of peptide-bilayer interactions. Methods Enzymol 295:62–87CrossRefPubMedGoogle Scholar
  38. Zhan H, Oh KJ, Shin Y-K, Hubbell WL, Collier RJ (1995) Interaction of the isolated transmembrane domain of diphtheria toxin with membranes. Biochemistry 34:4856–4863CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mauricio Vargas-Uribe
    • 1
  • Mykola V. Rodnin
    • 1
  • Karin Öjemalm
    • 2
  • Aurora Holgado
    • 2
  • Alexander Kyrychenko
    • 1
    • 3
  • IngMarie Nilsson
    • 2
  • Yevgen O. Posokhov
    • 1
    • 3
  • George Makhatadze
    • 4
  • Gunnar von Heijne
    • 2
  • Alexey S. Ladokhin
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Kansas Medical CenterKansas CityUSA
  2. 2.Department of Biochemistry and BiophysicsStockholm UniversityStockholmSweden
  3. 3.VN Karazin Kharkiv National UniversityKharkivUkraine
  4. 4.Department of BiologyRensselaer Polytechnic InstituteTroyUSA

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