緒方 英明

Protein‐based supramolecules require precise arrangement of building blocks. A regular‐triangle trimer (cp‐c555)3 has been constructed using an α‐helix‐inserted‐circular permutant (cp‐c555) of Aquifex aeolicus cytochrome (cyt) c555, where the trimers may dissociate to monomers. In this study, we stabilized the regular‐triangle structure by constructing a cyclic regular‐triangle of three α‐helix‐linked cyt c555 molecules using sortase‐mediated ligation (SML). Comparing SML using sortase A for six cp‐c555 variant trimers, the variant with GGG at the N‐terminus and LPETG at the C‐terminus reacted most efficiently. OP‐(c555)3 was designed, in which two cyt c555 molecules were fused using an α‐helix. The cyt c555 C‐terminal region was attached to the N‐terminus of the dimer and the cyt c555 N‐terminal region was attached to the C‐terminus of the dimer using the same α‐helix. OP‐(c555)3 was expressed in Escherichia coli cells and the termini were connected by SML, forming a cyclic regular‐triangle, CL‐(c555)3. CL‐(c555)3 showed higher thermostability than (cp‐c555)3 and OP‐(c555)3. CL‐(c555)3 structural stability was confirmed using high speed‐atomic force microscopy. The X‐ray crystal structure of CL‐(c555)3 showed a cyclic structure and a nanoporous supramolecular assembly. These results demonstrate that a nanoporous supramolecular assembly can be constructed by designing a cyclic molecule with a central hole using SML.