Barker Ryan A, Tisnado Jerrell, Lambert Lisa A, Gärdes Astrid, Carrano Mary W, Carrano Paul N, Gillian Christopher, Carrano Carl J
Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA, 92182-1030, USA.
Biometals. 2015 Feb;28(1):197-206. doi: 10.1007/s10534-014-9815-7. Epub 2014 Dec 21.
Full length recombinant iron regulatory protein, Fur, has been isolated and characterized from the algal-associated marine bacterium Marinobacter algicola DG893. Under nondenaturing conditions the Fur protein behaves on size exclusion chromatography as a dimer while it is monomeric under SDS PAGE conditions. ICP-MS and fluorescence quenching experiments show that Mb-Fur binds a single metal ion (Zn, Mn, or Co) per monomer. Electrophoretic mobility shift assays were used to probe the interaction of Mb-Fur with the purported Fur box in the promoter region upstream of the vibrioferrin biosynthetic operon. Interaction of Mb-Fur with a 100 bp DNA fragment containing the Fur box in the presence of 10 µM Mn, Co or Zn(II) resulted in decreased migration of DNA on a 7.5% polyacrylamide gel. In the absence of the Fur protein or the metal, no interaction is seen. The presence of EDTA in the binding, loading or running buffers also abolished all activity demonstrating the importance of the metal in formation of the promoter-repressor complex. Based on a high degree of similarity between Mb-Fur and its homolog from Pseudomonas aeruginosa (PA) whose X-ray structure is known we developed a structural model for the former which suggested that only one of the several metal binding sites found in other Fur's would be functional. This is consistent with the single metal binding stoichiometry we observed. Since the purported metal binding site was one that has been described as "structural" rather than "functional" in PA and yet the monometallic Mb-Fur retains DNA Fur box binding ability it reopens the question of which site is which, or if different species have adapted the sites for different purposes.
已从与藻类相关的海洋细菌嗜藻海杆菌DG893中分离并鉴定出全长重组铁调节蛋白Fur。在非变性条件下，Fur蛋白在尺寸排阻色谱上表现为二聚体，而在SDS-PAGE条件下为单体。电感耦合等离子体质谱（ICP-MS）和荧光猝灭实验表明，Mb-Fur每个单体结合一个金属离子（锌、锰或钴）。电泳迁移率变动分析用于探究Mb-Fur与铁载体生物合成操纵子上游启动子区域中假定的Fur盒的相互作用。在存在10 μM锰、钴或锌（II）的情况下，Mb-Fur与包含Fur盒的100 bp DNA片段相互作用，导致DNA在7.5%聚丙烯酰胺凝胶上的迁移率降低。在没有Fur蛋白或金属的情况下，未观察到相互作用。结合缓冲液、上样缓冲液或运行缓冲液中存在乙二胺四乙酸（EDTA）也会消除所有活性，这表明金属在启动子-阻遏物复合物形成中的重要性。基于Mb-Fur与其来自铜绿假单胞菌（PA）的同源物之间的高度相似性（已知PA的X射线结构），我们为前者建立了一个结构模型，该模型表明在其他Fur中发现的几个金属结合位点中只有一个具有功能。这与我们观察到的单金属结合化学计量一致。由于在PA中，假定的金属结合位点被描述为“结构”而非“功能”位点，但单金属Mb-Fur仍保留DNA Fur盒结合能力，这重新引发了关于哪个位点是哪种类型，或者不同物种是否将这些位点用于不同目的的问题。
