De novo design of semisynthetic conduction pores.

Protein pores used for molecular sensing are generally assembled from large, multi-subunit natural proteins. Their ion conductance, and thus performance in sensing applications, depends on lumen geometry and chemistry. Here rather than designing a new pore or relying only on mutations to a natural scaffold, we show that installing additional de novo subunits to native protein pore complexes enables large-scale architectural changes. We design de novo proteins that integrate seamlessly with CsgG, a pore widely used in sensing applications, to form 18-subunit, 315-kilodalton semisynthetic conduction pores. This required designing within a confined, nonuniform pore under ninefold symmetry and maintaining an open conducting lumen with a stable, low-noise baseline current. The complexes exhibit distinct current-voltage responses relative to the native pore, including clear rectification, while cryo-electron microscopy confirms the designed lumen architecture, establishing a strategy for modifying existing nano-assemblies with designed protein components under stringent symmetry and structural constraints.