Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments

Protein structures evolved through a complex interplay of cooperative interactions and it is still very challenging to design new protein folds de novo. Here, we present a strategy to design self-assembling polypeptide nanostructured polyhedra, based on modularization using orthogonal dimerizing segments. We designed end experimentally demonstrated formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled-coil-forming segments separated by flexible peptide hinges. Path of the polypeptide chain is guided by the defined order of segments that traverse each of the 6 edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. Coincidence of the polypeptide termini in the same vertex is demonstrated by reconstitution of the split fluorescent protein by the polypeptide with the correct tetrahedral topology, while polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides the basis for construction of new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.