A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation
A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation
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DOI：
10.1016/j.jmb.2003.08.047
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发表时间：
2003-10-31
影响因子：
5.6
通讯作者：
Perkins, SJ
中科院分区：
文献类型：
--
作者：
Lee, YC;Flora, R;Perkins, SJ
在细菌RuvABC系统的背景下，RuvA蛋白结合并参与同源重组期间形成的称为霍利迪连接的四向DNA结构的后续加工。来自大肠杆菌的RuvA（EcoRuvA）的四种晶体结构表明它是四聚体，而来自麻风分枝杆菌的RuvA（MleRuvA）和EcoRuvA的中子散射和另外两种晶体结构表明它是八聚体。为了澄清这种差异，通过分析超离心进行沉降平衡实验，结果表明，MleRuvA在0.1 M NaCl中以0.2-0.5 mg/ml的四聚体-八聚体平衡存在，解离常数为4 μ M，并且在较高浓度下为八聚体。在0.3 M NaCl中的相同实验表明，MleRuvA是高达3.5 mg/ml的四聚体，表明盐桥参与八聚体形成。与EcoRuvA的沉降平衡实验表明，它是四聚体在低浓度的两种盐缓冲液，但蛋白质是不溶的高蛋白质浓度在0.1 M NaCl。可以得出结论，游离RuvA存在于细菌细胞中发现的典型浓度范围和缓冲液中的四聚体和八聚体形式之间的平衡中。(C)2003 Elsevier Ltd.保留所有权利。
In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells. (C) 2003 Elsevier Ltd. All rights reserved.