title: Purification, crystallization and preliminary crystallographic studies of Lys48-linked polyubiquitin chains.
abstract: Post-translational modification of proteins by covalent attachment of ubiquitin regulates diverse cellular events. A Lys48-linked polyubiquitin chain is formed via an isopeptide bond between Lys48 and the C-terminal Gly76 of different ubiquitin molecules. The chain is attached to a lysine residue of a substrate protein, which leads to proteolytic degradation of the protein by the 26S proteasome. In order to reveal the chain-length-dependent higher order structures of polyubiquitin chains, Lys48-linked polyubiquitin chains were synthesized enzymatically on a large scale and the chains were separated according to chain length by cation-exchange column chromatography. Subsequently, crystallization screening was performed using the hanging-drop vapour-diffusion method, from which crystals of tetraubiquitin, hexaubiquitin and octaubiquitin chains were obtained. The crystals of the tetraubiquitin and hexaubiquitin chains diffracted to 1.6 and 1.8 A resolution, respectively. The tetraubiquitin crystals belonged to space group C222(1), with unit-cell parameters a = 58.795, b = 76.966, c = 135.145 A. The hexaubiquitin crystals belonged to space group P2(1), with unit-cell parameters a = 51.248, b = 102.668, c = 51.161 A. Structural analysis by molecular replacement is in progress.
authors.affiliation: Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8015, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Morimoto
authors.forename: Shin
authors.initials: S
authors.lastname: Isogai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tenno
authors.forename: Hidehito
authors.initials: H
authors.lastname: Tochio
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
authors.forename: Mariko
authors.initials: M
authors.lastname: Ariyoshi
mesh_terms: D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D008239:Lysine / Q000235:genetics; D051379:Mice; D025821:Polyubiquitin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D025821:Polyubiquitin; D008239:Lysine