The Sticholysin I mutants St I E2C and St I R52C show similar binding to liposomal vesicles but differ in their permeabilizing activity

the mechanism of pore formation by actinoporins is a multistep process, involving binding of water soluble monomer to membrane and subsequent oligomerization of monomers on the membrane surface, forming a functional pore. however, molecular details of membrane insertion mechanism and oligomerization are not clear. a phosphocholine-binding site and a surface cluster of aromatic rings, together with a basic region, are important to the initial interaction with membrane and the n-terminal region is relevant in the pore formation. aiming to deepen into the structure-function relationship in sticholysins, we designed and produced two cys mutants of recombinant sticholysin i (rst i) in relevant functional regions for membrane interaction: st i e2c (in the n-terminal region) and st i r52c (in the membrane binding site). conformational studies suggested that the replacement of glu-2 and arg-52 by a cys residue in rst i not noticeably changes protein conformation as assessed by fluorescence and cd spectroscopy, the first change not affecting toxin？s permeabilizing ability. the relative decrease in the pore forming capacity of st i r52c is not related with a smaller binding capacity of this mutant to membrane. in summary, st i e2c and st i r52c retain the main conformational properties of the wild type and show similar binding to liposomal vesicles while differing in their permeabilizing activity. st i e2c and st i r52c constitute good tools to study those steps of the permeabilizing mechanism of sticholysins that take place after binding to membrane, using thiol-specific probes such as fluorescent and spin labels.