Chemically tunable permeability of engineered alpha-Hemolysin in synthetic cells

Controlling molecular transport across membranes is a defining feature of living cells, yet replicating this functionality in synthetic systems remains a major challenge. Self-inserting protein nanopores, such as α-hemolysin (αHL), offer a promising route towards programmable membrane permeability due to their robust assembly, compatibility with diverse membrane systems, and intrinsic permeability for diverse biomolecules. Here, we explored the use of chemically functionalized αHL nanopores as tunable transport modules. To quantify translocation of peptide substrates across αHL-containing membranes, we developed a high-throughput luminescence-based breakage-controlled assay using large unilamellar vesicles. With this assay we introduce a one-pot nanopore modification and strategy, compatible with scalable workflows. Electrophysiology and molecular simulations demonstrate that the introduction of cysteine residues at defined pore locations, combined with targeted chemical modification, enables controlled tuning of αHL-nanopore selectivity based on peptide structure and charge. Together, these findings position engineered protein nanopores as versatile and responsive components for controlling membrane transport in synthetic biology.