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

Photobleaching reveals heterogeneous stoichiometry for equinatoxin II oligomers.

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Chemistry Research Laboratory, University of Oxford, South Parks Road, OX1 3QZ Oxford (UK).


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.


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

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