N-terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina

The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis. A part of this segment was classified as being amphiphilic and membrane seeking, Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia call using T7 RNA polymerase-based expression vector, Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity, The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes, Their specific hemolytic activity was decreased, however, with increasing truncation, The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes, It is very likely that the N-terminus play a role in the formation of functional pores.