Photobleaching reveals heterogeneous stoichiometry for equinatoxin II oligomers

Chembiochem. 2014 Sep 22;15(14):2139-45. doi: 10.1002/cbic.201300799. Epub 2014 Aug 19.


Equinatoxin II (EqtII), a sea anemone cytolysin, is known to oligomerize to form pores that spontaneously insert into membranes. Crystallographic and cryo-EM studies of structurally similar cytolysins offer contradictory evidence for pore stoichiometry. Here we used single-molecule photobleaching of fluorescently labeled EqtII to determine the stoichiometry of EqtII oligomers in supported lipid bilayers. A frequency analysis of photobleaching steps revealed a log-normal distribution of stoichiometries with a mean of 3.4±2.3 standard deviations. Comparison of our experimental data with simulations of fixed stoichiometries supports our observation of a heterogeneous distribution of EqtII oligomerization. These data are consistent with a model of EqtII stoichiometry where pores are on average tetrameric, but with large variation in the number of subunits in individual pores.

Keywords: equinatoxin; fluorescence; membrane proteins; photobleaching; pore-forming toxins; single molecule fluorescence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cnidarian Venoms / chemistry*
  • Fluorescence
  • Lipid Bilayers / chemistry
  • Photobleaching
  • Protein Multimerization
  • Sea Anemones / chemistry*


  • Cnidarian Venoms
  • Lipid Bilayers
  • equinatoxin