Zn(II) metallochaperones in E. coli

大肠杆菌中的 Zn(II) 金属伴侣

• 批准号: 7093792
• 负责人: MICHAEL W CROWDER
• 金额: $ 21万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093792
• 关键词: Escherichia coli; binding proteins; gel electrophoresis; gene targeting; immunologic assay /test; intracellular transport; ion transport; metalloproteins; microarray technology; microorganism culture; molecular chaperones; nutrient requirement; western blottings; zinc

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand Zn(ll) trafficking in cells. The main goal of this study is to identify Zn(ll)-metallochaperones in E. coli involved specifically with Zn(ll) import and transport. Specifically, we propose to identify the putative, soluble Zn(ll)-metallochaperones that are thought to deliver Zn(ll) from outer membrane-bound channels/porins to the ribosomes and/or proteins. We propose to utilize the following strategy to achieve our goals. DNA microarrays were used to identify five transcripts (of Zn(ll)-metallochaperone candidates) that were up-regulated in E. coli cells stressed with Zn(ll) deficiency. (1) These five candidate proteins will be over-expressed, purified, and characterized for structure and Zn(ll) binding. (2) All candidates that bind Zn(ll) will be used in pulldown experiments in an effort to identify any proteins that form complexes with the candidates. In addition, E. coli cells lacking the Zn(ll) binding candidates (knockout lines) will be obtained. (3) All proteins identified in the pulldown experiments will be over-expressed, purified, and characterized for Zn(ll) binding and structure. Polyclonal antibodies against these proteins will be generated. (4) Wild-type and mutant E. coli cells will be cultured in the presence of 65Zn, and the resulting soluble proteins will be subjected to native gel electrophoresis. The gels will be analyzed for differential amounts of 65Zn, and western blots of the gels will be analyzed using the aforementioned polyclonal antibodies. The knockout lines will also be assayed for Zn(ll) transport by using a recently developed assay in the lab. Since Zn(ll) is an essential metal ion for bacteria and metallochaperones deliver metal ions to specific proteins, we believe that understanding Zn(ll)-transport in E. coli will present novel protein targets for the design and preparation of a new class of antibiotics. The need for new classes of antibiotics is particularly important currently to combat the clinical crisis of antibiotic resistance and to combat the Category A and B bacterial strains on the CDC's bioterrorism watch list.

描述（由申请人提供）：该项目的长期目标是了解细胞中的锌（LL）运输。这项研究的主要目的是鉴定Zn（LL） - 辅助剂在大肠杆菌中特别涉及的Zn（LL）进口和运输。具体而言，我们建议确定被认为可以从外膜结合的通道/孔蛋白到核糖体和/或蛋白质的推定的可溶性Zn（LL） - 金属伴侣。我们建议利用以下策略来实现我们的目标。 DNA微阵列用于识别五个转录本（Zn（LL） - 辅助伴侣候选），这些转录物在与Zn（LL）缺陷的大肠杆菌细胞中上调。 （1）这五种候选蛋白将过表达，纯化和特​​征于结构和Zn（LL）结合。 （2）所有结合Zn（LL）的候选物将用于下拉实验，以识别与候选者形成复合物的任何蛋白质。另外，将获得缺乏Zn（LL）结合候选物（基因敲除线）的大肠杆菌细胞。 （3）在下拉实验中鉴定出的所有蛋白质都将过表达，纯化和表征Zn（LL）结合和结构。将产生针对这些蛋白质的多克隆抗体。 （4）在65zn存在的情况下将培养野生型和突变大肠杆菌细胞，所得的可溶性蛋白将受到天然凝胶电泳。将分析凝胶的65zn差异，并使用上述多克隆抗体分析凝胶的蛋白质印迹。敲除线还将通过在实验室中使用最近开发的测定法分析Zn（LL）传输。由于Zn（LL）是细菌和金属色蛋白的必需金属离子，将金属离子传递到特定的蛋白质上，因此我们认为，在大肠杆菌中了解Zn（LL） - 转移将为设计和制备新的抗生素靶标提供新颖的蛋白质靶标。目前，对于应对抗生素耐药性的临床危机以及在CDC的生物恐怖观察表上打击A和B类细菌菌株的需求目前尤其重要。