A Fluorescent-Based Approach to Unravel Protein-Protein Interactions in Actinoporins

Abstract

Actinoporins are a group of toxins produced by sea anemones. They specifically bind to sphingomyelin in the cell membranes of their targets. Upon binding to the bilayer, they oligomerize and form cation-selective pores. Despite years of research, many details about actinoporin-membrane interaction remain elusive. Here, we used a fluorescence-based approach in order to gain molecular knowledge of different details of the process. We took advantage of the fact that, except for one known exception, actinoporins are naturally cysteinless. Thus, we engineered two single-cysteine mutants of the proteins sticholysin I and II (StnI and StnII), produced by the Caribbean Sea anemone Stichodactyla helianthus. Once purified, these mutants were labelled with the appropriate small fluorophores. Using different combinations of mutant and wild-type proteins, as well as different degrees of labelling, we studied several intriguing aspects of the sticholysin-membrane interaction. Although oligomerization is certainly known to occur, resolving the stoichiometry of the complex has been a major challenge within the field, particularly in model membranes, since those are closer to in vivo conditions. It has also been demonstrated that StnI and StnII can act synergistically, but the details of their collaboration remain obscure. In our novel approach, using FRET in combination with mathematical modelling, we are able to provide new data on the mechanism by which these proteins associate to form pores.