Photobleaching Reveals Heterogeneous Stoichiometry for Equinatoxin II Oligomers |
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| Authors: | Dr. Matthew A. B. Baker Dr. Nejc Rojko Dr. Bríd Cronin Prof. Gregor Anderluh Prof. Mark I. Wallace |
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| Affiliation: | 1. Chemistry Research Laboratory, University of Oxford, South Parks Road, OX1 3QZ Oxford (UK);2. Department of Biology, Biotechnical Faculty, University of Ljubljana, Ve?na pot 111, 1000 Ljubljana (Slovenia);3. Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia) |
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| 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. |
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| Keywords: | equinatoxin fluorescence membrane proteins photobleaching pore‐forming toxins single molecule fluorescence |
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