Free Energy-Guided Design of Aerolysin Nanopore for Single-Molecule Protein Sequencing

Aleksei Aksimentiev, University of Illinois at Urbana-Champaign

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Aleksei Aksimentiev, Christopher Maffeo, Manish Shankla, Han-Yi Chou, David Winogradoff, Shidi Zhao, Kumar Sarthak, Kush Coshic, Adnan Choudhary, Lauren Quednau, Pin-Yi Li, Ashley Knoerdel

Single-molecule protein sequencing would provide unprecedented insights into cellular processes and the diseases that arise from protein misfunction. In collaboration with the Oukhaled Lab in U. Cergy-Pontoise France, we have recently demonstrated that it is possible to statistically discriminate the type of amino acids translocating through the biological nanopore aerolysin from a modulation of the ionic current passing through the pore—a crucial first step toward realizing nanopore protein sequencing. The aerolysin's ability to discriminate between amino acids derives from the long residence of the amino acids within the pore. Building on our vast experience modeling nanopore systems, here we propose a set of molecular dynamics simulations to develop a variant of the aerolysin pore that is capable of distinguishing all twenty amino acids from a single nanopore passage. First, we will determine the free energy landscape underlying amino acid translocation through the nanopore, revealing barriers that slow translocation. Next, we will introduce several mutations around these barriers to increase the residence time of the amino acids, thereby enhancing the ability of the pore to discriminate between them. The most successful aerolysin mutants will be characterized experimentally by our collaborators, paving the way toward the first single-molecule protein sequencing method.