Barrel expansion of outer membrane protein G nanopore through β-hairpin duplication

Outer membrane β-barrel proteins (OMPs) are channels found in the outer membranes of Gram-negative bacteria characterized by a stable and diverse barrel architecture, which has made them attractive for nanopore sensing applications. Here, we systematically investigated the feasibility of expanding outer membrane protein G (OmpG) from its native 14-stranded β-barrel to an enhanced conductance variant by independently duplicating each of its seven hairpin units and inserting them downstream of their endogenous positions. Most combinations did not increase pore diameter, but duplication of the terminal seventh hairpin exhibited a rare population of pores with enhanced conductance, suggesting barrel enlargement. Further engineering efforts to optimize the terminal β-turn sequence have resulted in up to 50% of pores with increased conductance. Importantly, the enlarged pores retained the sensing functionality of the original scaffold, highlighting the potential of this approach for developing β-barrel OMP sensors with tunable dimensions.