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

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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.

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Abbreviations

T-domain:

Diphtheria toxin T-domain

W:

State membrane-incompetent state populated at neutral pH

W+ :

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

I:

State interfacial state

T:

State(s) transmembrane state(s)

LUV:

Large unilamellar vesicles

POPC:

Palmitoyl-oleoyl-phosphatidylcholine

POPG:

Palmitoyl-oleoyl-phosphatidylglycerol

FCS:

Fluorescence correlation spectroscopy

CD:

Circular dichroism

DSC:

Differential scanning calorimetry

ΔH°:

Enthalpy change

ΔG:

Gibbs free energy

Tm :

Melting temperature

ΔCp:

Change in calorific capacity

Kx :

Partitioning coefficient

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Acknowledgments

We are grateful to Dr. Chiranjib Ghatak for his helpful feedback on the design of thermodynamic schemes. This research was supported by NIH Grant GM-069783 (A.S.L.). M.V.U. was partially supported by Fulbright-CONICYT, Chile.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, 66160, USA

    Mauricio Vargas-Uribe, Mykola V. Rodnin, Alexander Kyrychenko, Yevgen O. Posokhov & Alexey S. Ladokhin

  2. Department of Biochemistry and Biophysics, Stockholm University, 10691, Stockholm, Sweden

    Karin Öjemalm, Aurora Holgado, IngMarie Nilsson & Gunnar von Heijne

  3. VN Karazin Kharkiv National University, Kharkiv, 61022, Ukraine

    Alexander Kyrychenko & Yevgen O. Posokhov

  4. Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12065, USA

    George Makhatadze

Authors
  1. Mauricio Vargas-Uribe
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  2. Mykola V. Rodnin
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  3. Karin Öjemalm
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  4. Aurora Holgado
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  5. Alexander Kyrychenko
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  6. IngMarie Nilsson
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  7. Yevgen O. Posokhov
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  8. George Makhatadze
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  9. Gunnar von Heijne
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  10. Alexey S. Ladokhin
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Corresponding author

Correspondence to Alexey S. Ladokhin.

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Vargas-Uribe, M., Rodnin, M.V., Öjemalm, K. et al. Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain. J Membrane Biol 248, 383–394 (2015). https://doi.org/10.1007/s00232-014-9734-0

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  • DOI: https://doi.org/10.1007/s00232-014-9734-0

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