Electrostatic Filtering of Polypeptides Through Membrane Protein Pores

Naturally‐occurring membrane proteins have been engineered as nanopore sensors for the single‐molecule detection of various biochemical molecules. Here, we present a natural bacterial porin, CymA containing a dynamic component and densely packed charged residues in the pore, shaping a unique structural conformation and charge feature. Using single‐channel recordings, we investigated the translocation of charged polypeptides through native CymA and truncated CymA lacking the dynamic element. Cationic polypeptides bind to the pore with high affinity, specifically at low salt conditions indicating an electrostatic charge and voltage‐dependent translocation. Anionic peptides did not bind to the pore, confirming the selective binding of polypeptides with the pore due to their specific charge distribution. Further, the distinct peptide translocation kinetics between native and truncated indicated the role of the dynamic segment in molecular transport. We suggest that these natural membrane pores that permit the selective translocation of cationic polypeptides are advantageous for nanopore proteomics applications. Natural bacterial membrane pores of unique structural motifs were used for sensing differently charged polypeptides at single‐molecule resolution. Electrical measurements confirmed the electrostatic effect in charge selective translocation of polypeptides owing to its densely packed charged residues in the pore. This new class of pores will be helpful in several applications in biotechnology for biopolymer sensing and sequencing.