Volume 15, Issue 14 p. 2139-2145
Full Paper

Photobleaching Reveals Heterogeneous Stoichiometry for Equinatoxin II Oligomers

Dr. Matthew A. B. Baker

Dr. Matthew A. B. Baker

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

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Dr. Nejc Rojko

Dr. Nejc Rojko

Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana (Slovenia)

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Dr. Bríd Cronin

Dr. Bríd Cronin

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

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Prof. Gregor Anderluh

Prof. Gregor Anderluh

Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana (Slovenia)

Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia)

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Prof. Mark I. Wallace

Corresponding Author

Prof. Mark I. Wallace

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

Chemistry Research Laboratory, University of Oxford, South Parks Road, OX1 3QZ Oxford (UK)Search for more papers by this author
First published: 19 August 2014
Citations: 35

Graphical Abstract

Poring over the data: Equinatoxin II (EqtII) is known to oligomerize to form pores that spontaneously insert into membranes. Single-molecule photobleaching of fluorescently labeled EqtII was used to determine the stoichiometry of EqtII oligomers in supported lipid bilayers, which suggests that there is a large variation in the number of subunits in individual pores.

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.