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
Article

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.

Keywords

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

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

Supplementary material

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

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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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