ATP-COUPLED ARSENICAL PUMP OF ESCHERICHIA COLI

大肠杆菌的 ATP 耦合砷泵

• 批准号: 2061029
• 负责人: BARRY P. ROSEN
• 金额: $ 28.56万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2061029
• 关键词: Escherichia coli; adenosine triphosphate; anions; arsenic; chemical binding; chimeric proteins; drug resistance; ion transport; membrane transport proteins; molecular cloning; protein engineering; protein reconstitution; protein structure function; stoichiometry

The overall goals of this research are first, elucidation of the molecular mechanisms of an ion pump whose genes are natural components of a bacterial resistance plasmid, and second, the role of this transport system in bacterial resistance to antibiotics and toxic compounds. A bacterial anion-translocating TPase has been identified as the product of the arsenical resistance (ars) operon of resistance plasmid R773. When expressed in Escherichia coli the ATP-driven pump catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. This anion-translocating ATPase serves as an excellent model system for ion-translocating ATPases. The ars operon has been cloned and sequenced, and most of the protein components have been identified-and purified. Two of the structural genes, the arsA and arsB genes, encode the two subunits of the pump. This two-component inner membrane complex binds and transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony. Specific aims of the project include determination of the structural elements of the ArsA and ArsB proteins which contribute toward their function as ATPase and ion transport system. Domains of interest in the ArsA protein are a) nucleotide binding sites, b) anion binding sites, and c) sites of self interaction during dimerization. The ArsB protein will be characterized by a) purification and b) development of an in vitro assay for 73AsO2- transport. The ArsA and ArsB proteins form a complex. The methodology for reconstitution of a functional pump from isolated ArsA and ArsB proteins will be optimized. The stoichiometry of the subunits in the pump will be determined, and the contact regions of the two proteins will be identified. The operon is metalloregulated by a diverse set of inducers, including various salts of arsenic, antimony and bismuth. Characterization of the transcriptional regulators, the ArsR and ArsD proteins, will be carried out with the following aims: a) purification of the two proteins, b) determination of their role in regulation, including binding sites on the proteins for DNA and ionic substrates, and c) determination of the repressor binding sites on the DNA.

这项研究的总体目标是首先阐明 离子泵的分子机制，其基因是天然成分 细菌抗性质粒，其次，该转运的作用 对抗生素和有毒化合物的细菌耐药性系统。 一个 细菌阴离子 - 转化TPase已被确定为 电阻质粒R773的砷耐药性（ARS）操纵子。 什么时候 在大肠杆菌中表达的ATP驱动泵催化了 氧化砷，抗肌和砷酸盐，因此提供 对有毒化合物的抗性。 这个阴离子转换ATPase 是用于离子ATP酶的极好的模型系统。 ARS操纵子已被克隆和测序，大多数蛋白质 已经确定并纯化组件。 两个结构 基因，ARSA和ARSB基因编码泵的两个亚基。 这种两分量的内膜复合物结合并运输砷 和抗肌者，砷或砷氧化态的氧 锑。 该项目的具体目的包括确定 ARSA和ARSB蛋白的结构元素有助于 它们作为ATPase和ION运输系统的功能。 感兴趣的领域 在ARSA蛋白中是a）核苷酸结合位点，b）阴离子结合 位点，c）二聚化期间自我相互作用的位点。 ARSB 蛋白质的特征是a）纯化和b）开发 73ASO2运输的体外测定。 ARSA和ARSB蛋白形成A 复杂的。 重建功能泵的方法 分离的ARSA和ARSB蛋白将被优化。 化学计量的 将确定泵中的亚基，并 将确定两种蛋白质。 操纵子由多种诱导剂组合，包括 各种砷，锑和鞭毛的盐。 表征 转录调节剂，ARSR和ARSD蛋白将被携带 以以下目的出现：a）两种蛋白质的纯化，b） 确定其在调节中的作用，包括在 DNA和离子底物的蛋白质，c）确定 DNA上的阻遏物结合位点。