Equinatoxin II is a toxin from a protein family of actinoporins . It was found in beadlet anemone (Actinia equina), which uses it to prey and to defend itself from predators. Eqt II forms cation selective pores in target lipid membranes. The pore-forming process includes binding of Eqt II to sphingomyelin (SM) in lipid membranes, insertion of N-terminal α- helix into the membrane, oligomerization and eventual formation of pore. The purpose of our research was to examine the differences between toxin's binding on lipid bilayer of large unilamelar vesicles (LUV) and lipid monolayers of lipid droplets (LK). We also examined the role of N-terminal α-helix in the initial binding to lipid membrane. We multiplied plasmids, containing information for Eqt IIV8C,K69C mutant, in Escherichia coli DH5α strain. The Eqt IIV8C,K69C was then isolated by ion-exchange and gel chromatography. The oxidised and reduced forms of the toxin mutant were prepared. N-terminal α-helix of the oxidized form of Eqt IIV8C,K69C is unable to move because of the cysteine bond between the helix and the core of the molecule. N-terminal α-helix of reduced form of Eqt IIV8C,K69C can move freely. The concentrations of lipids in prepared LUV and LK were determined by the use of special kits for determining concentration of lipids. In order to study the binding of Eqt II on lipid membranes, we used stopped flow fluorescence spectroscopy. When tryptophan residues (that are present in the binding site of the Eqt II) are inserted into the nonpolar lipid membrane, the intensity of fluorescence increases. To describe the changes in fluorescence intensity we used the pseudo-first order rate equation. We fitted monoexponential curves to the experimental curves. When reduced form of Eqt IIV8C,K69C was bound to either LUV or LK, we measured higher values of maximal fluorescence intensity than in the case of oxidized form of Eqt IIV8C,K69C. The latter has the N-terminal α-helix connected to the core of the molecule, which disables its movement. We suppose that this rigid structure poses a hindrance that disables deeper insertion of tryptophan residues into the lipid membrane, which results in a bit lower maximal fluorescence intensity. Higher values of maximal fluorescence intensities were also measured when either oxidized of reduced form of Eqt IIV8C,K69C was bound on LUV as when it was bound to LK. We suppose that SM in the membranes of lipid bilayer of LUV might be a bit more accessible than SM in the lipid monolayer of LK. However, the greater binding ability of Eqt II to LUV might be the result of a higher cholesterol to SM ratio in LUV than it is in LK. Cholesterol in cell membranes is known to increase the binding ability of actinoporins to SM. We did not discover significant differences between binding of Eqt II to LUV and LK.
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