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Updated: Jun 19, 2026
Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
Published on: March 5, 2017
Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II
Alison Drechsler1, Gregor Anderluh, Raymond S Norton
1School of Chemistry, Bio21 Institute, University of Melbourne, VIC 3010, Australia.
Abstract:
Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or "rafts" in PC membranes, (31)P and (2)H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T(2) relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.
Insights
Equinatoxin II (EqtII) causes membrane disruption, particularly in lipid bilayers containing sphingomyelin (SM) and cholesterol (Chol). Solid-state NMR reveals EqtII alters lipid order and bilayer structure, impacting membrane integrity.
Area of Science:
- Biochemistry
- Biophysics
- Membrane Biology
Background:
- Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina.
- EqtII lyses red blood cells and model membranes, with activity dependent on sphingomyelin (SM).
- Cholesterol (Chol) and SM interactions form domains in phosphatidylcholine (PC) membranes.
Purpose of the Study:
- Investigate EqtII's effects on lipid order and bilayer morphology.
- Determine the role of SM and Chol in EqtII-induced membrane disruption.
- Utilize solid-state NMR to probe lipid-protein interactions.
Main Methods:
- (31)P and (2)H solid-state NMR spectroscopy.
- Analysis of multilamellar vesicles with varying ratios of DMPC, SM, and Chol.
- Monitoring changes in lipid phase transitions, vesicle morphology, and relaxation times.
Main Results:
- EqtII alters lipid acyl chain phase transition temperatures and phospholipid headgroup T(2) relaxation times.
- The toxin promotes the formation of small vesicle structures at higher temperatures.
- Cholesterol stabilizes DMPC bilayers but exacerbates disruption in SM-containing bilayers.
Conclusions:
- EqtII-induced membrane disruption is enhanced by the presence of both SM and Chol.
- Domain boundaries between liquid-ordered and liquid-disordered phases may facilitate membrane disruption by EqtII.
- EqtII's mechanism involves altering lipid phase behavior and bilayer structure.