Designing Protein Assemblies

The oligomeric fusion strategy for designing self-assembling protein materials or 'nanohedra'. (Adapted from Lai, et al. 2012)
The oligomeric fusion strategy for designing self-assembling protein materials or ‘nanohedra’. (Adapted from Lai, et al. 2012)

An outstanding goal in bioengineering is to design macromolecules that assemble into complex higher order structures. Considerable progress has been demonstrated in the last 20 years in designing DNA (and RNA) sequences to form interesting structures and materials. Progress in using proteins as the building blocks has been much more challenging, owing in part to the much greater complexity of the rules that govern the native structures of proteins. Nonetheless, Nature has achieved spectacular assemblies using protein molecules as her building blocks; virus shells, microtubules, and bacterial S-layers provide a few striking examples.

Three overlapped crystal structures of the 12-subunit designed cage (Lai, et al., 2012). The hypothetical center sphere is shown to illustrate the open nature of the cage.
Three overlapped crystal structures of the 12-subunit designed cage (Lai, et al., 2012). The hypothetical center sphere is shown to illustrate the open nature of the cage.

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.I222_cyan_30degrees_small

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structural, computational, and synthetic biology