Highly-ordered protein structures have gained interest for future uses for biomaterials. Herein, we constructed a building block protein (BBP) by the circular permutation of the hyperthermostable Aquifex aeolicus cytochrome (cyt) c , and assembled BBP into a triangle-shaped trimer and a tetrahedron. The angle of the intermolecular interactions of BBP was controlled by cleaving the domain-swapping hinge loop of cyt c and connecting the original N- and C-terminal α-helices with an α-helical linker. We obtained BBP oligomers up to ≈40 mers, with a relatively large amount of trimers. According to the X-ray crystallographic analysis of the BBP trimer, the N-terminal region of one BBP molecule interacted intermolecularly with the C-terminal region of another BBP molecule, resulting in a triangle-shaped structure with an edge length of 68 Å. Additionally, four trimers assembled into a unique tetrahedron in the crystal. These results demonstrate that the circular permutation connecting the original N- and C-terminal α-helices with an α-helical linker may be useful for constructing organized protein structures.

A building block protein BBP is designed by circularly permutating thermostable cytochrome c555 (pictured as pink and green pieces on the front cover) and connecting the original N‐ and C‐terminal α‐helices with an α‐helical linker (blue piece). The N‐terminal α‐helix of one BBP molecule interacts intermolecularly with the C‐terminal α‐helix of another BBP molecule in a defined angle, resulting in the formation of a triangle‐shaped trimer and a tetrahedron by four of these trimers. This approach of combining circular permutation and an α‐helical linker insertion is promising for constructing organized protein structures. More information can be found in the https://doi.org/10.1002/asia.201800252

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