1. Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin
- Author
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Fabian Brandl, Lukas Becker, Jasleen Singh Badwal, Wouter P. R. Verdurmen, Andreas Plückthun, University of Zurich, and Plückthun, Andreas
- Subjects
Circular dichroism ,Endosome ,Anthrax toxin ,Protein subunit ,3003 Pharmaceutical Science ,Pharmaceutical Science ,610 Medicine & health ,Article ,cytosolic protein delivery ,03 medical and health sciences ,All institutes and research themes of the Radboud University Medical Center ,Pharmacy and materia medica ,10019 Department of Biochemistry ,030304 developmental biology ,0303 health sciences ,Chemistry ,030302 biochemistry & molecular biology ,circular dichroism ,RS1-441 ,Cytosol ,DARPin ,protein stability ,Biotinylation ,Biophysics ,570 Life sciences ,biology ,anthrax toxin ,Ankyrin repeat ,Nanomedicine Radboud Institute for Molecular Life Sciences [Radboudumc 19] - Abstract
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.
- Published
- 2021
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