Zn(II) metallochaperones in E. coli

描述(由申请者提供)：本项目的长期目标是了解锌(11)在细胞内的贩运。本研究的主要目的是在大肠杆菌中鉴定与锌(11)的输入和转运有关的锌(11)-金属配位体。具体地说，我们建议鉴定可能的、可溶的锌(11)金属配位体，它们被认为是将锌(11)从外膜结合的通道/孔蛋白输送到核糖体和/或蛋白质中。我们建议利用以下战略来实现我们的目标。DNA微阵列被用来鉴定在缺锌胁迫下的大肠杆菌细胞中上调表达的五个转录本(候选锌(11)-金属配位体)。(1)这五个候选蛋白将被过度表达、纯化，并对其结构和锌离子结合进行鉴定。(2)所有与锌(11)结合的候选蛋白都将用于下拉实验，以努力识别与候选蛋白形成络合物的任何蛋白质。此外，还将获得缺乏锌(11)结合候选(基因敲除株)的大肠杆菌细胞。(3)在下拉实验中确定的所有蛋白质都将被过度表达、纯化，并对锌(11)结合和结构进行鉴定。将产生针对这些蛋白质的多克隆抗体。(4)野生型和突变型的大肠杆菌细胞将在65Zn的存在下进行培养，所产生的可溶性蛋白将进行天然凝胶电泳。将分析凝胶中不同数量的65Zn，并将使用上述多克隆抗体分析凝胶的蛋白质印迹。基因敲除品系还将利用实验室最近开发的一种分析方法来检测锌(11)的运输。由于锌(11)是细菌必需的金属离子，而金属配位蛋白将金属离子输送到特定的蛋白质中，我们相信了解锌(11)在大肠杆菌中的转运将为设计和制备新型抗生素提供新的蛋白质靶点。目前，对于对抗抗生素耐药性的临床危机以及疾控中心生物恐怖主义观察名单上的A类和B类细菌菌株来说，对新类别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.