The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3–5, for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery6, vaccine design7, and synthetic biology8.

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.

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids, and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1,2 or proteins3–5, but strategies for engineering hybrid biological materials are only beginning to emerge6,7. Here, we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as Enveloped Protein Nanocages (EPNs). Robust EPN biogenesis required protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induced EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical and electron cryomicroscopic (cryo-EM) analyses revealed that one design, EPN-01, comprised small (~100 nm) vesicles containing multiple protein nanocages that closely matched the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporated the vesicular stomatitis viral glycoprotein (VSV-G) could fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These studies show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a novel class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.