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Zn（ll）的贩运。本研究的主要目的是鉴定大肠杆菌中锌(ll)-金属伴侣蛋白特异性参与锌（ll）的进口和运输。具体来说，我们建议鉴定推定的可溶性Zn(ll)-金属伴侣，它们被认为可以将Zn（ll）从外膜结合通道/孔蛋白传递到核糖体和/或蛋白质。我们建议利用以下策略来实现我们的目标。DNA微阵列技术鉴定了5个Zn(ll)-金属伴侣候选物转录本，这些转录本在Zn（ll）缺乏的大肠杆菌细胞中表达上调。(1)这5个候选蛋白将被过表达、纯化，并对其结构和Zn（ll）结合进行表征。(2)所有结合Zn（ll）的候选物将用于下拉实验，以识别与候选物形成复合物的任何蛋白质。此外，将获得缺乏Zn（ll）结合候选者（敲除系）的大肠杆菌细胞。(3)所有在下拉实验中鉴定的蛋白都将被过表达、纯化，并对Zn（ll）的结合和结构进行表征。将产生针对这些蛋白的多克隆抗体。(4)将野生型和突变型大肠杆菌细胞在65Zn存在下培养，得到的可溶性蛋白进行天然凝胶电泳。将分析凝胶中65Zn的差异量，并使用上述多克隆抗体分析凝胶的western blots。基因敲除系还将使用最近在实验室开发的检测方法检测Zn（ll）的转运。由于锌（ll）是细菌必需的金属离子，金属伴侣蛋白将金属离子传递给特定的蛋白质，我们相信了解锌（ll）在大肠杆菌中的转运将为设计和制备一类新的抗生素提供新的蛋白质靶点。为了应对抗生素耐药性的临床危机，以及对抗疾病预防控制中心生物恐怖主义观察名单上的A类和B类细菌菌株，目前对新型抗生素的需求尤为重要。

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.