Computational design of transmembrane pores

Article

关键词: DE-NOVO DESIGN;    CRYSTAL-STRUCTURE;    ALPHA-HEMOLYSIN;    EVOLUTION;    PACKING;    MODELS;    HOLE;   

DOI  :  10.1038/s41586-020-2646-5

来源: SCIE

【 摘 要 】

An approach for the design of protein pores is demonstrated by the computational design and subsequent experimental expression of both an ion-selective and a large transmembrane pore. Transmembrane channels and pores have key roles in fundamental biological processes(1)and in biotechnological applications such as DNA nanopore sequencing(2-4), resulting in considerable interest in the design of pore-containing proteins. 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 that are capable of conducting ions selectively or are large enough to enable the 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 alpha-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy 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 channels and pores for a wide variety of applications.

【 授权许可】

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