Formation of a nanoporous crystal structure of domain-swapped cytochrome c555 trimer by I8K/T14D mutation

Various building block proteins have been designed to construct unique nanoporous materials. We have previously developed a cytochrome (cyt) [Formula: see text]-based trimer (cp-[Formula: see text] and a relevant closed cyclic trimer CL-([Formula: see text], where (cp-[Formula: see text] and CL-([Formula: see text] assembled into a tetrahedron and a nanoporous structure, respectively. In this study, we aimed to reinforce the tetrahedron by introducing I8K and T14D mutations to cp-[Formula: see text], which was designed to form salt bridges at the trimer interfaces. However, the X-ray crystallographic structure of the cp-[Formula: see text] I8K/T14D trimer ((cp-[Formula: see text] I8K/T14D)[Formula: see text] revealed a nanoporous structure similar to that of CL-([Formula: see text]. The cp-[Formula: see text] I8K/T14D variant exhibited a Soret band at approximately 407 nm, identical to that of CL-([Formula: see text], indicating a similar His/Met heme coordination structure. The X-ray structure of (cp-[Formula: see text] I8K/T14D) 3 showed that the introduced amino acids did not form a direct hydrogen bond with neighboring amino acids, where the packing of the trimers was stabilized by Gln–Gln hydrogen bonds analogous to those in the nanoporous structure of CL-([Formula: see text]. The introduced amino acids may induce the cp-[Formula: see text] I8K/T14D molecules to assemble into a nanoporous structure by long-range electrostatic interactions and/or may stabilize the nanoporous structure by facilitating the construction of a hydrogen bond network with water molecules at distances of 2.2–3.5 Å at the interface of (cp-[Formula: see text] I8K/T14D) 3 .