来自大肠杆菌的双功能异柠檬酸脱氢酶激酶/磷酸酶与其底物异柠檬酸脱氢酶复合物的纯化、结晶及初步X射线分析
Purification, crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate, isocitrate dehydrogenase, from Escherichia coli.
作者信息
Zheng Jimin, Ji Alan Xian, Jia Zongchao
机构信息
Department of Biochemistry, Queen's University, Kingston, ON, Canada.
出版信息
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Nov 1;65(Pt 11):1153-6. doi: 10.1107/S1744309109038718. Epub 2009 Oct 30.
Abstract
Escherichia coli isocitrate dehydrogenase (ICDH) can be phosphorylated and dephosphorylated by a single bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (AceK), which is encoded by the aceK gene. In order to investigate the regulatory mechanism of (de)phosphorylation of ICDH by AceK, AceK was successfully cocrystallized in complex with its intact protein substrate, ICDH, in the presence of ATP. The complex crystal was obtained by the hanging-drop vapour-diffusion technique using PEG 300 as a precipitant and magnesium sulfate as an additive. SDS-PAGE analysis of dissolved crystals showed that the crystals contained both AceK and ICDH proteins. The complex crystals diffracted to a resolution of 2.9 angstrom in space group P6(3), with unit-cell parameters a = b = 196.80, c = 156.46 angstrom.
摘要
大肠杆菌异柠檬酸脱氢酶（ICDH）可被一种单一的双功能蛋白——异柠檬酸脱氢酶激酶/磷酸酶（AceK，由aceK基因编码）磷酸化和去磷酸化。为了研究AceK对ICDH（去）磷酸化的调控机制，在ATP存在的情况下，AceK与其完整的蛋白质底物ICDH成功共结晶。通过悬滴气相扩散技术，以聚乙二醇300作为沉淀剂、硫酸镁作为添加剂，获得了复合物晶体。对溶解晶体的SDS-PAGE分析表明，晶体中同时含有AceK和ICDH蛋白。该复合物晶体在空间群P6(3)中衍射分辨率达到2.9埃，晶胞参数a = b = 196.80，c = 156.46埃。
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