Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin

Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.