Construction of a Triangle‐Shaped Trimer and a Tetrahedron Using an α‐Helix‐Inserted Circular Permutant of Cytochrome c₅₅₅

Abstract

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.

Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

“…C-type cytochromes (cyts) are heme proteins that act as electron carriers, functioning in a variety of cellular processes [1,2]. Many c-type cyts can form oligomers by 3D domain swapping (herein, domain swapping) [3][4][5][6][7][8][9][10][11]. In domain swapping, two or more identical protein monomers exchange the same domain or secondary structural elements and fold into dimers or higher oligomers whose units are structurally similar to the original monomer [12][13][14][15].…”

Use of 3D domain swapping in constructing supramolecular metalloproteins

“…In addition, three cyt cb 562 3D-DS dimers formed A building block protein (BBP) was constructed based on hyperthermostable AA cyt c 555 to force intermolecular interactions; circular permutation that cleaved the protein at the 3D-DS hinge loop 74 and a-helical linker insertion at the connection of the original N-and C-terminal a-helices were performed. 101 BBP was expressed as a monomer in E. coli, whereas it formed oligomers as large as B40 mers with a relatively large amount of trimers when refolded at high protein concentrations. BBP molecules formed a 3D-DS trimer, with the N-terminal region of a BBP molecule interacting intermolecularly with the C-terminal region of another BBP molecule, resulting in a triangle-shaped structure with an edge length of 68 Å (PDB ID: 5Z25) (Fig.…”

Oligomerization of cytochrome c, myoglobin, and related heme proteins by 3D domain swapping

“…BBP was expressed as a monomer in E. coli, whereas it formed oligomers up to ~40 mers with a relatively large amount of trimers when refolded under high protein concentration. BBP molecules domain swapped in the trimer, with the N-terminal region of one BBP molecule interacting intermolecularly with the C-terminal region of another BBP molecule, resulting in a triangle-shaped structure with an edge length of 68 Å (PDB ID: 5Z25; Figure 7) [87]. Additionally, four trimers assembled into a unique tetrahedron in the crystal.…”