Purification, crystallization and preliminary crystallographic studies of Lys48-linked polyubiquitin chains
Purification, crystallization and preliminary crystallographic studies of Lys48-linked polyubiquitin chains
复制标题
Lys48 连接的多聚泛素链的纯化、结晶和初步晶体学研究
DOI：
10.1107/s1744309110018804
复制
发表时间：
2010
期刊：
影响因子：
--
通讯作者：
M.
中科院分区：
文献类型：
--
作者：
Morimoto;D.;Isogai;S.;Tenno;T.;Tochio;H.;Shirakawa;M.;Ariyoshi;M.
蛋白质的翻译后修饰通过泛素的共价连接调节不同的细胞事件。Lys48与不同泛素分子的C-末端Gly76之间通过异肽键形成Lys48连接的多泛素链。该链连接到底物蛋白质的赖氨酸残基上，从而导致26S蛋白酶体对蛋白质的降解。为了揭示多泛素链与链长相关的高阶结构，在酶促条件下大规模合成了Lys48连接的多泛素链，并用阳离子交换柱层析法按链长进行分离。随后，采用悬滴气相扩散法进行结晶筛选，得到了四泛素链、六泛素链和八泛素链晶体。四泛素链和六泛素链的晶体的衍射率分别达到1.6%和1.8% ä。四泛素晶体属于C2221空间群，晶胞参数a=58.795，b=76.966，c=135.145 ä。晶体属P21空间群，晶胞参数为a=102.668，b=51.248，c=51.161 ä。通过分子置换进行结构分析正在进行中。
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 Å resolution, respectively. The tetraubiquitin crystals belonged to space group C2221, with unit-cell parameters a = 58.795, b = 76.966, c = 135.145 Å. The hexaubiquitin crystals belonged to space group P21, with unit-cell parameters a = 51.248, b = 102.668, c = 51.161 Å. Structural analysis by molecular replacement is in progress.