Vestibule Engineering of MspA Nanopores Enables Single-Molecule Detection of Protein Tag Reporters and Lysine Acetylation via Geometric Repositioning

Protein fingerprinting with nanopores enables single-molecule proteoform analysis but is often limited by conformational heterogeneity of flexible polypeptides. Here, we reduce this stochasticity through a geometric relocalization strategy by engineering dual hydrophobic anchoring sites (T83F/R118F) in the vestibule of the Mycobacterium smegmatis porin A (M2MspA) nanopore. These substitutions are proposed to create localized aromatic interaction sites that alter the docking geometry of the folded Smt3 domain and improve the reproducibility of sensing-segment presentation near the constriction. We demonstrated that this reconfiguration could convert stochastic, tail-dominated signals into regulated, segment-resolved readouts. Analysis of the single-site mutants suggests that R118F makes the predominant contribution to the observed reporter-positioning phenotype, enabling discrimination of peptide sequences unresolved in M2MspA. Using this spatial control, we achieve single-molecule detection of lysine acetylation in a tau-derived peptide via distinct noise signatures. These results support a nanopore design strategy that emphasizes spatial alignment for high-fidelity proteoform analysis.