Recombinant anthrax protective antigen: Observation of aggregation phenomena by TEM reveals specific effects of sterols

Negatively stained transmission electron microscope images are presented that depict the aggregation of recombinant anthrax protective antigen (rPA83 monomer and the PA63 prepore oligomer) under varying in vitro biochemical conditions. Heat treatment (50 °C) of rPA83 produced clumped fibrils, but following heating the PA63 prepore formed disordered aggregates. Freeze-thaw treatment of the PA63 prepore generated linear flexuous aggregates of the heptameric oligomers. Aqueous suspensions of cholesterol microcrystals were shown to bind small rPA83 aggregates at the edges of the planar bilayers. With PA63 a more discrete binding of the prepores to the crystalline cholesterol bilayer edges occurs. Sodium deoxycholate (NaDOC) treatment of rPA83 produced quasi helical fibrillar aggregate, similar but not identical to that produced by heat treatment. Remarkably, NaDOC treatment of the PA63 prepores induced transformation into pores, with a characteristic extended ß-barrel. The PA63 pores aggregated as dimers, that aggregated further as angular chains and closed structures in higher NaDOC concentrations. The significance of the sterol interaction is discussed in relation to its likely importance for PA action in vivo.