Position-dependent hydrophobic "molecular brakes" in M2-MspA nanopores enable label-free differentiation of oppositely-charged analytes under symmetric conditions.

Nanopore-based single-molecule sensing has emerged as a powerful tool for analyzing proteins, peptides, and nucleic acids. However, label-free control of analyte transport has predominantly relied on electrophoretic and electro-osmotic forces, whereas the deliberate engineering of hydrophobic interactions to modulate translocation kinetics remains underexplored. Here, we introduce aromatic residues at strategically distinct positions (T83F and S116F) within the Mycobacterium smegmatis Porin A (M2-MspA) constriction region to spatially segregate hydrophobic trapping from electrostatic steering, thereby establishing a position-dependent 'molecular brake' mechanism. These mutations were strategically introduced to modulate hydrophobic interactions and electrostatic forces within the pore. S116F elongates dwell times by ∼2.5-fold and deepens current blockades by 8-11% for negatively charged analytes, consistent with cooperative hydrophobic-electrostatic trapping near the R118 vestibule. Conversely, for positively-charged lysozyme, T83F prolongs dwell times by ∼3.2-fold while sharpening blockade distributions (ΔI/I0 = 63.97 ± 1.00%), whereas S116F introduces electrostatic repulsion that partially offsets hydrophobic attraction, thereby reducing residence time and revealing charge-dependent modulation of analyte-pore interactions. Notably, the combination of T83F and S116F enables the differentiation of complex mixtures, such as single-stranded DNA (ssDNA) and lysozyme, under symmetric salt conditions and neutral pH. Our work highlights the importance of precise spatial positioning of aromatic residues and shaping of charge distributions in nanopore engineering, offering a foundation for designing multifunctional nanopores. These position-dependent hydrophobic 'molecular brakes' enable label-free differentiation of oppositely-charged analytes under symmetric, neutral-pH conditions, advancing M2-MspA toward peptide profiling and protein fingerprinting.