BPS2026 - Reverse translocation and constriction engineering improve MspA nanopore protein fingerprinting

Nanopore sensing has recently advanced as a powerful approach for protein recognition and fingerprinting at the single-molecule level. Among biological nanopores, Mycobacterium smegmatis porin A (MspA), which features a spacious vestibule and a narrow constriction, is particularly promising for full-length protein detection. However, protein translocation through MspA in the forward direction (vestibule-to-stem) often produces inconsistent signals, likely due to electrophoretical trapping and conformational fluctuations within the vestibule. Here, we show for the first time that reverse translocation of full-length proteins (stem-to-vestibule) through MspA yields higher capture rates and more uniform current blockades than forward transport. To further tune pore performance and probe the role of constriction charges, we systematically mutated residues N90 and N91 to different charged amino acids. All mutants expressed efficiently, and single-channel recordings revealed that constriction charge strongly modulates conductance, ion selectivity, protein translocation dynamics, and gating behavior. Together, these findings reveal that pore orientation and constriction-site engineering as key determinants for optimizing MspA nanopores, providing critical insights to guide their development for protein fingerprinting.