Conformational transition of DnaA protein by ATP: structural analysis of DnaA protein, the initiator of Escherichia coli chromosome replication.
Conformational transition of DnaA protein by ATP: structural analysis of DnaA protein, the initiator of Escherichia coli chromosome replication.
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ATP 引起的 DnaA 蛋白构象转变：大肠杆菌染色体复制启动子 DnaA 蛋白的结构分析。
DOI：
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发表时间：
1997
期刊：
影响因子：
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通讯作者：
K. Sekimizu
中科院分区：
文献类型：
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作者：
T. Kubota;T. Katayama;Y. Ito;T. Mizushima;K. Sekimizu
DnaA 蛋白与染色体起点 (oriC) 结合以启动 DNA 复制。我们开发了一种有效的 DnaA 蛋白质纯化系统，这将有助于蛋白质的理化分析。与现有方法相比，DnaA 蛋白的产量增加了至少 6 倍，仅从 100 g 细胞中即可获得超过 22 mg 的蛋白。通过该程序纯化的 DnaA 蛋白显示出对 ATP 的不可区分的亲和力以及 oriC 质粒的体外复制活性。通过内在荧光和圆二色性监测被 ATP 阻断的 DnaA 蛋白变性过程。圆二色性分析表明，DnaA蛋白富含α螺旋，ATP结合导致蛋白质构象发生显着转变，α螺旋含量减少。这是第一个证据表明 ATP 结合深刻影响 DnaA 蛋白的构象。
DnaA protein binds to the chromosomal origin (oriC) to initiate DNA replication. We developed an efficient system for purification of DnaA protein which will facilitate physicochemical analysis of the protein. The yield of DnaA protein was increased at least 6-fold compared to an available method being used, and over 22 mg of the protein were obtained from only 100 g of cells. DnaA protein purified by this procedure showed an indistinguishable affinity for ATP, and activity for in vitro replication of oriC plasmid. The process of denaturation of DnaA protein, which was blocked by ATP, was monitored by intrinsic fluorescence and circular dichroism. Analysis of circular dichroism revealed that DnaA protein is rich in alpha-helices, and that ATP-binding leads to a significant transition of protein conformation in that the content of alpha-helices is decreased. This is the first evidence indicating that ATP-binding profoundly affects conformation of DnaA protein.