Preorganized cyclic modules facilitate the self-assembly of protein nanostructures.

The rational design of supramolecular assemblies aims to generate complex systems based on the simple information encoded in the chemical structure. Programmable molecules such as nucleic acids and polypeptides are particularly suitable for designing diverse assemblies and shapes not found in nature. Here, we describe a strategy for assembling modular architectures based on structurally and covalently preorganized subunits. Cyclization through spontaneous self-splicing of split intein and coiled-coil dimer-based interactions of polypeptide chains provide structural constraints, facilitating the desired assembly. We demonstrate the implementation of a strategy based on the preorganization of the subunits by designing a two-chain coiled-coil protein origami (CCPO) assembly that adopts a tetrahedral topology only when one or both subunit chains are covalently cyclized. Employing this strategy, we further design a 109 kDa trimeric CCPO assembly comprising 24 CC-forming segments. In this case, intein cyclization was crucial for the assembly of a concave octahedral scaffold, a newly designed protein fold. The study highlights the importance of preorganization of building modules to facilitate the self-assembly of higher-order supramolecular structures.

Coiled-Coil Protein Origami: Design, Isolation, and Characterization.

Coiled-coil protein origami (CCPO) is a rationally designed de novo protein fold, constructed by concatenating coiled-coil forming segments into a polypeptide chain, that folds into polyhedral nano-cages. To date, nanocages in the shape of a tetrahedron, square pyramid, trigonal prism, and trigonal bipyramid have been successfully designed and extensively characterized following the design principles of CCPO. These designed protein scaffolds and their favorable biophysical properties are suitable for functionalization and other various biotechnological applications. To further facilitate the development, we are presenting a detailed guide to the world of CCPO, starting from design (CoCoPOD, an integrated platform for designing CCPO strictures) and cloning (modified Golden-gate assembly) to fermentation and isolation (NiNTA, Strep-trap, IEX, and SEC) concluding with standard characterization techniques (CD, SEC-MALS, and SAXS).