title: Photobleaching reveals heterogeneous stoichiometry for equinatoxin II oligomers.
abstract: 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.
authors.affiliation: Chemistry Research Laboratory, University of Oxford, South Parks Road, OX1 3QZ Oxford (UK).
authors.forename: Matthew A B
authors.initials: MA
authors.lastname: Baker
authors.forename: Nejc
authors.initials: N
authors.lastname: Rojko
authors.forename: Bríd
authors.initials: B
authors.lastname: Cronin
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Mark I
authors.initials: MI
authors.lastname: Wallace
mesh_terms: D000818:Animals; D003064:Cnidarian Venoms / Q000737:chemistry*; D005453:Fluorescence; D008051:Lipid Bilayers / Q000737:chemistry; D038761:Photobleaching; D055503:Protein Multimerization; D012615:Sea Anemones / Q000737:chemistry*
chemical_list: D003064:Cnidarian Venoms; D008051:Lipid Bilayers; C012307:equinatoxin
keywords: equinatoxin; fluorescence; membrane proteins; photobleaching; pore-forming toxins; single molecule fluorescence