Abstract
Studying the interaction of pore-forming toxins, including perfringolysin O (PFO), with lipid is crucial to understanding how they insert into membranes, assemble, and associate with membrane domains. In almost all past studies, symmetric lipid bilayers, i.e., bilayers having the same lipid composition in each monolayer (leaflet), have been used to study this process. However, practical methods to make asymmetric lipid vesicles have now been developed. These involve a cyclodextrin-catalyzed lipid exchange process in which the outer leaflet lipids are switched between two lipid vesicle populations with different lipid compositions. By use of alpha class cyclodextrins, it is practical to include a wide range of sterol concentrations in asymmetric vesicles. In this article, protocols for preparing asymmetric lipid vesicles are described, and to illustrate how they may be applied to studies of pore-forming toxin behavior, we summarize what has been learned about PFO conformation and its lipid interaction in symmetric and in asymmetric artificial lipid vesicles.