Zn(II) metallochaperones in E. coli

描述（由申请人提供）：该项目的长期目标是了解细胞中的Zn（II）运输。本研究的主要目的是鉴定E.大肠杆菌特异性地参与Zn（II）的输入和转运。具体地，我们提出鉴定推定的可溶性Zn（II）-金属伴侣，其被认为将Zn（II）从外膜结合通道/孔蛋白递送至核糖体和/或蛋白质。我们建议利用以下战略来实现我们的目标。DNA微阵列用于鉴定在大肠杆菌中上调的5种转录物（Zn（II）-金属伴侣候选物）。coli细胞用Zn（II）缺乏胁迫。(1)这五种候选蛋白将被过表达、纯化并表征结构和Zn（II）结合。(2)结合Zn（II）的所有候选物将用于下拉实验，以努力鉴定与候选物形成复合物的任何蛋白质。此外，E.将获得缺乏Zn（II）结合候选物的大肠杆菌细胞（敲除系）。(3)在下拉实验中鉴定的所有蛋白质将过表达、纯化并表征Zn（II）结合和结构。将产生针对这些蛋白质的多克隆抗体。(4)野生型和突变型E.大肠杆菌细胞将在65 Zn存在下培养，所得可溶性蛋白将进行天然凝胶电泳。分析凝胶中不同量的65 Zn，并使用上述多克隆抗体分析凝胶的蛋白质印迹。还将通过使用实验室中最近开发的测定法测定敲除系的Zn（II）转运。由于Zn（II）是细菌必需的金属离子，并且金属伴侣将金属离子递送到特定蛋白质，我们相信理解E.大肠杆菌将为设计和制备一类新的抗生素提供新的蛋白质靶点。目前，对新型抗生素的需求对于应对抗生素耐药性的临床危机和对抗CDC生物恐怖主义观察名单上的A类和B类细菌菌株尤为重要。

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.