Nanoporous Hydrogels for the Observation of Anthrax Exotoxin Translocation Dynamics

The ability to observe lethal anthrax exotoxins translocating through size-constricting nanopores in vitro, combined with detailed sequence and structural data, has aided in elucidated mechanisms of exotoxin cell entry and toxicity. However, due to limited observations of anthrax exotoxins translocating through protective antigen nanopores in vitro and the instability of protective antigen-functionalized suspended lipid bilayers, questions remain regarding the native mechanisms of cell entry. Nanoporous hydrogel membranes offer a robust tool for studying protein translocation with ensemble measurements that complement conventional single-molecule translocation measurements. Here, we utilize nanoporous hydrogel membranes to assess the translocation of full-length anthrax lethal and edema factors through nanopores similar in diameter to protective antigen translocons. We find that, relative to globular serum and other proteins that do not translocate natively through nanopores, anthrax exotoxins demonstrate significantly reduced barriers to pore entry. Computed free-energy barriers to the unfolding of proteins and the dissociation of macromolecular complexes are generally found to coincide with translocation. Finally, a nanopore-blocking strategy is developed that utilizes nonspecific synthetic peptide constructs and effectively prevents LF translocation within the nanoporous hydrogel.