In order to design a novel protein to self-assemble into a complex but well-defined architecture, the protein molecule must contain multiple self-associating interfaces. It turns out, somewhat surprisingly, that two distinct self-associating interfaces is sufficient to create a wide range of outcomes, from cages to extended three-dimensional materials. The designed assembly problem therefore boils down to desiging a protein molecule having two geometrically precise interaction interfaces. A design idea we put forward in 2001 was to create protein molecules of this type by fusing together two simpler oligomeric proteins, e.g. a dimer and a trimer. The resulting fusion protein inherits the self-associating interfaces, and thereore the symmetry properties, of the two daughter components. The key problem is how to make such a fusion in a way that the two oligomeric components are held in a specific geometry required to make some desired architecture. One solution is to genetically fuse an oligomer that ends in an alpha helix to an oligomer that begins in an alpha helix, using helix-preferring amino acids for any extra linking residues. If the two oligomeric components are connected by a continuous helix running between them, then their relative orientation can be anticipated in advance. In initial proof of concept experiments, two students, Jennifer Padilla and Chris Colovos, identified a dimer and trimer that could be fused in a way that was predicted to give the right geometry for self-assembly of a 12-subunit tetrahedral cage, about 160 Å in diameter and a half-megadalton in mass. Those experiments gave rise to assemblies that were roughly consistent with the design, but which were too polymorphic to crystallize and confirm the results.

In 2012, a new student (Yen-Ting Lai) re-inspected the originally designed protein sequence and identified a few amino acids that could be causing steric classes and preventing correct assembly. A new construct was obtained, differening from the original design by just two amino acids, which assembled into homogeneous 12-subunit complexes. The protein was crystallized and shown to match the designed tetrahedral cage structure overall, but with substantial deviations arising from alternative packing modes of the dimeric component, combined with helix flexibility. This success was a decisive demonstration of the ability to design highly symmetric self-assembling materials of the type we originally dubbed 'nanohedra'. At essentially the same time, former student Neil King, as a postdoc in David Baker's laboratory, was able to design two novel protein assemblies of comparable size and complexity by using computational interface design. A natural oligomer (a trimer) provided one self-associating interface, and a second geometrically specific (dimeric) interface was designed computationally using the Rosetta-Design program; several computationally designed constructs had to be screened to find successful designs. These design strategies and others being developed in various laboratories are opening the way to a broad range of engineered protein materials with biomedical and nanotechnology applications.

References:

• King NP, Sheffler W, Sawaya MR, Vollmar BS, Sumida JP, André I, Gonen T, Yeates TO, Baker D. (2012). Computational design of self-assembling protein nanomaterials with atomic level accuracy. Science. Jun 2012. 336(6085):1171-4. [Abstract]
  We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.
• Lai YT, Cascio D, Yeates TO. (2012). Structure of a 16-nm cage designed by using protein oligomers. Science. Jun 2012. 336(6085):1129. [Abstract]
  Designing protein molecules that will assemble into various kinds of ordered materials represents an important challenge in nanotechnology. We report the crystal structure of a 12-subunit protein cage that self-assembles by design to form a tetrahedral structure roughly 16 nanometers in diameter. The strategy of fusing together oligomeric protein domains can be generalized to produce other kinds of cages or extended materials.
• Yeates TO. (2011). Nanobiotechnology: protein arrays made to order. Nat Nanotechnol. Sep 2011. 6(9):541-2. [Abstract]
• Padilla JE, Colovos C, Yeates TO. (2001). Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U.S.A.. Feb 2001. 98(5):2217-21. [Abstract]
  A general strategy is described for designing proteins that self assemble into large symmetrical nanomaterials, including molecular cages, filaments, layers, and porous materials. In this strategy, one molecule of protein A, which naturally forms a self-assembling oligomer, A(n), is fused rigidly to one molecule of protein B, which forms another self-assembling oligomer, B(m). The result is a fusion protein, A-B, which self assembles with other identical copies of itself into a designed nanohedral particle or material, (A-B)(p). The strategy is demonstrated through the design, production, and characterization of two fusion proteins: a 49-kDa protein designed to assemble into a cage approximately 15 nm across, and a 44-kDa protein designed to assemble into long filaments approximately 4 nm wide. The strategy opens a way to create a wide variety of potentially useful protein-based materials, some of which share similar features with natural biological assemblies.