title: Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin.
abstract: Many molluscs transport oxygen using a very large cylindrical multimeric copper-containing protein named hemocyanin. The molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330-450kDa subunits, resulting in a molecular mass >3.3MDa. Therefore, molluscan hemocyanin is one of the largest proteins. The reason why these organisms use such a large supermolecule for oxygen transport remains unclear. Atomic-resolution X-ray crystallographic analysis is necessary to unveil the detailed molecular structure of this mysterious large molecule. However, its propensity to dissociate in solution has hampered the crystallization of its intact form. In the present study, we successfully obtained the first crystals of an intact decameric molluscan hemocyanin. The diffraction dataset at 3.0-Å resolution was collected by merging the datasets of two isomorphic crystals. Electron microscopy analysis of the dissolved crystals revealed cylindrical particles. Furthermore, self-rotation function analysis clearly showed the presence of a fivefold symmetry with several twofold symmetries perpendicular to the fivefold axis. The absorption spectrum of the crystals showed an absorption peak around 345nm. These results indicated that the crystals contain intact hemocyanin decamers in the oxygen-bound form.
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Matsuno
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Zuoqi
authors.initials: Z
authors.lastname: Gai
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kato
authors.affiliation: Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan.
authors.forename: Sanae
authors.initials: S
authors.lastname: Kato
authors.affiliation: Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Katoh
authors.affiliation: Hokkaido Industrial Technology Center, Hakodate 041-0801, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: Hokkaido Industrial Technology Center, Hakodate 041-0801, Japan.
authors.forename: Takeya
authors.initials: T
authors.lastname: Yoshioka
authors.affiliation: Graduate School of Fisheries Science, Hokkaido University, Hakodate 041-8611, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kishimura
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan. Electronic address: tanaka@sci.hokudai.ac.jp.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
mesh_terms: D000818:Animals; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D006433:Hemocyanins / Q000737:chemistry*; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D015394:Molecular Structure; D008974:Mollusca / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D014965:X-Rays
chemical_list: D006433:Hemocyanins; D010100:Oxygen
keywords: Crystallization; Decamer; Hemocyanin; Self-rotation function; Supermolecule