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, 4, 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-nanometre 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 between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of 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 centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery6, vaccine design7 and synthetic biology8.
Figures
![Design methodology and biochemical characterization.]()
Figure 1: Design methodology and biochemical characterization. a, b, Icosahedral three-fold axis in red and aligned trimeric building block in green. c, Optimization of r and ω yields closely opposed interfaces between subunits. d, Sequence design yields low-energy interfaces; in the I3-01 case, composed of five designed residues (thick representations) and two native residues (thin representations). e, I3-01 appears larger by SEC than the similarly sized I3-01(L33R) and wild-type trimer (1wa3). f, DLS measurement of hydrodynamic radius (note logarithmic scale in f and h) of 1wa3 (3.5 nm) and I3-01 (14 nm). I3-01 remains assembled in 6.7 M GuHCl and in 2 M GITC. g, Extremely sharp disassociation to trimeric building blocks at 2.25 M GITC. Data points represent independent measurements. h, I3-01 icosahedron disassembles into the trimeric building blocks at 3 M GITC, and reassembles following dilution to 1 M.
![Cryo-EM.]()
Figure 2: Cryo-EM. a, Field-of-view cryo-EM micrograph showing homogeneous icosahedral particles in various orientations. b, Back-projections of I3-01 from the design model. c, Cryo-EM class averages closely match the design projections along all three symmetry axes. d, e, The calculated initial, unrefined density (blue, 3.22σ) closely matches the design model (green).
![Tuning nanocage structure and function with genetic fusions.]()
Figure 3: Tuning nanocage structure and function with genetic fusions. a, The left panel shows a cryo-EM micrograph of I3-01(ctGFP); the top right panel shows a computational model with sfGFP in green; the bottom right panel shows the class average along the five-fold axis. b, Fluorescence microscopy fields of view. c, Fluorescence intensity histograms. AFU, arbitrary fluorescence units; ± standard deviation. d, Correlation between the mean fluorescence intensity and sfGFP copy number for nanoparticles with different numbers of fused sfGFP molecules. Error bars are s.e.m. (n = 3). e, f, Computational model and class averages along the five-fold axis of negatively stained I3-01 (e) and I3-01(HB) (f); the helical bundle is shown in red. Weak density in the centre of the pentameric faces in I3-01 may reflect randomly packaged material. There is clear density in the centre of the pentameric faces in the I3-01(HB) class averages consistent with the model.
![I3-01 tolerance to temperature.]()
Extended Data Fig. 1: I3-01 tolerance to temperature. DLS measurements as I3-01 is subjected to heating to 90 °C (solid line), then cooling to 25 °C (dotted line) in TBS (a), 6.7 M GuHCl (b) and 2 M GITC (c). Under all three conditions, any indications of aggregation or increase in size due to temperature appear to be completely reversible.
![Reproducibility of I3-01 transition in 2 M to 2.25 M GITC.]()
Extended Data Fig. 2: Reproducibility of I3-01 transition in 2 M to 2.25 M GITC. Four examples each of independent measurements at 2 M (blue) and 2.25 M (red) GITC using DLS show the reproducibility of the cage disassociation. Histograms are plotted offset by 1% intensity from each other for clarity.
![SEC of T33-21 and I3-01 fused with sfGFP.]()
Extended Data Fig. 3: SEC of T33-21 and I3-01 fused with sfGFP. Size exclusion chromatography traces for T33-21 (12mer in red and 24mer in blue) and I3-01 (60mer in green and 120mer in purple) sfGFP fusions, display increased particle sizes with increasing copies of GFP, but retain monodispersed populations. The N-terminal fusion of sfGFP (dashed line) is expected to extend mostly outward from the icosahedron, thus greatly increasing the hydrodynamic radius while the C-terminal fusion is predicted to occupy the internal void space. A230, ultraviolet absorbance at 230 nm; mAU, milli-absorbance units.
![Tolerance of I3-01–sfGFP fusions to GuHCl.]()
Extended Data Fig. 4: Tolerance of I3-01–sfGFP fusions to GuHCl. N-terminal (red) and C-terminal (blue) sfGFP fusions were equilibrated to 0–6.4 M GuHCl. Ultraviolet absorbance at 490 nm (A490) monitors the unfolding of sfGFP (top, solid line and crosses). DLS experiments (top, dotted line and dots) reveal as sfGFP unfolds, the hydrodynamic radius increases slightly, and then stabilizes. The bottom panels show that in 1 M GuHCl (solid line) and in 6 M GuHCl (dotted line), the icosahedral assemblies remain relatively monodisperse.
![I3-01 C-terminal fusions with other fluorescent proteins.]()
Extended Data Fig. 5: I3-01 C-terminal fusions with other fluorescent proteins. Fluorescent proteins mTurquoise2 (in blue) or sYFP2 (in green) were fused to the C terminus of I3-01. The field of view using widefield fluorescence microscopy shows distinct signals of each type when the two types are mixed together.
![I3-01 retains native enzyme activity.]()
Extended Data Fig. 6: I3-01 retains native enzyme activity. Coupled KDPG aldolase assay showing native-like enzymatic activity in I3-01. The K129A knockout shows no enzyme activity, similar to buffer alone. UV339, absorbance at 339 nm; error bars are standard deviation.
