Computational Design of Transmembrane Pores

Protein pores play key roles in fundamental biological processes(1) and biotechnological applications such as DNA nanopore sequencing(2–4), and hence the design of pore-containing proteins is of considerable scientific and biotechnological interest. Synthetic amphiphilic peptides have been found to form ion channels(5,6), and there have been recent advances in de novo membrane protein design(7,8) and in redesigning naturally occurring channel-containing proteins(9,10). However, the de novo design of stable, well-defined transmembrane protein pores capable of conducting ions selectively or large enough to allow passage of small-molecule fluorophores remains an outstanding challenge(11,12). Here, we report the computational design of protein pores formed by two concentric rings of ɑ-helices that are stable and mono-disperse in both water-soluble and transmembrane forms. Crystal structures of the water-soluble forms of a 12 helical and a 16 helical pore are close to the computational design models. Patch-clamp electrophysiology experiments show that the transmembrane form of the 12-helix pore expressed in insect cells allows passage of ions across the membrane with high selectivity for potassium over sodium, which is blocked by specific chemical modification at the pore entrance. The transmembrane form of the 16-helix pore, but not the 12-helix pore, allows passage of biotinylated Alexa Fluor 488 when incorporated into liposomes using in vitro protein synthesis. A cryo-EM structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer pores for a wide variety of applications.