Hideaki Ogata

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.

Overexpression of antibody light chains in small plasma cell clones can lead to misfolding and aggregation. On the other hand, the formation of amyloid fibrils from antibody light chains is related to amyloidosis. Although aggregation of antibody light chain is an important issue, atomic-level structural examinations of antibody light chain aggregates are sparse. In this study, we present an antibody light chain that maintains an equilibrium between its monomeric and tetrameric states. According to data from X-ray crystallography, thermodynamic and kinetic measurements, as well as theoretical studies, this antibody light chain engages in 3D domain swapping within its variable region. Here, a pair of domain-swapped dimers creates a tetramer through hydrophobic interactions, facilitating the revelation of the domain-swapped structure. The negative cotton effect linked to the β-sheet structure, observed around 215 nm in the circular dichroism (CD) spectrum of the tetrameric variable region, is more pronounced than that of the monomer. This suggests that the monomer contains less β-sheet structures and exhibits greater flexibility than the tetramer in solution. These findings not only clarify the domain-swapped structure of the antibody light chain but also contribute to controlling antibody quality and advancing the development of future molecular recognition agents and drugs.

We report the first neutron structure of [NiFe]-hydrogenase in its oxidized state. This study leads to new insights into the oxidized active site and visualization of the protons characteristic of the oxidized enzyme.