Design of a hyperstable 60-subunit icosahedral nanocage

Abstract

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3–5 for applications ranging from targeted delivery to multivalent immunogen presentation. 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’ with properties custom-tailored to specific applications. Here we describe the computational design of a 25 nm icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly at 2–2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent standard candles for use in light microscopy, and a designed protein pentamer can be placed in the center of each of the twelve pentameric voids to gate macromolecule access to the interior of the nanocage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery6, vaccine design7, and synthetic biology8.