Structure-Guided Conversion of a TonB-Dependent Transporter into a Functional β-Barrel Nanopore

Abstract

Protein nanopores are valuable platforms for single-molecule sensing; however, expanding the range of structurally defined nanopores remains challenging. We describe a minimal, structure-guided redesign strategy that converts BtuB, a TonB-dependent vitamin B12 transporter, into a functional β-barrel nanopore. Using protein structure prediction and molecular dynamics, we eliminated the internal plug domain, selected extracellular loops, and preserved the 22-stranded β-barrel scaffold. BtuBΔ1–136_Δloops formed single channels in planar lipid bilayers, exhibiting an approximately linear current–voltage relationship and a single-channel conductance of ∼1.2 nS. Polymer exclusion analysis revealed an apparent PEG-accessible diameter of approximately 4 nm, indicating a highly accessible pore entrance. Noise analysis showed that extracellular loop architecture influences the stability of ion transport. These findings highlight the distinction between geometric pore accessibility and functional ion-conducting pathways in redesigned β-barrel nanopores, suggesting that TonB-dependent transporters may serve as useful scaffolds for β-barrel nanopore engineering.