A Protein-Protein Interaction Map of Escherichia coli

大肠杆菌的蛋白质-蛋白质相互作用图

• 批准号: 7467724
• 负责人: Peter Hans Uetz
• 金额: $ 65.46万
• 依托单位: J. CRAIG VENTER INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7467724
• 关键词: Amino Acids; Animal Model; Antibiotics; Bacteria; Bacteriophages; Basic Science; Behavior; Binding; Binding Sites; Biochemical; Biochemistry; Bioinformatics; Biological Process; Biology; C-Peptide; Cataloging; Catalogs; Cells; Chemicals; Classification; Cloning; Communities; Community Actions; Complex; Computer Simulation; Condition; Cytoplasmic Tail; Data; Data Set; Databases; Defect; Epitopes; Escherichia coli; Escherichia coli Proteins; Follow-Up Studies; Gene Expression Regulation; Generations; Genes; Genetic; Goals; Growth; Human; Infection; Integral Membrane Protein; Knowledge; Learning; Length; Literature; Maps; Mass Spectrum Analysis; Mediating; Membrane Proteins; Metabolic; Metabolism; Molecular; Mutagenesis; Mutate; Open Reading Frames; Peptides; Phenotype; Process; Protein C; Protein-Protein Interaction Map; Proteins; Proteome; Quality Control; Reaction; Recording of previous events; Reporting; Research; Research Design; Role; Screening procedure; Signal Transduction; Specific qualifier value; Structure; System; Techniques; Technology; Tertiary Protein Structure; Testing; Time; Yeasts; base; in vivo; interest; microbial; mutant; pathogen; protein protein interaction; synthetic peptide; vector; yeast two hybrid system

DESCRIPTION (provided by applicant): Escherichia coli is both an important pathogen and a primary model organism for microbial biology. Nevertheless, about half of its proteins have not been characterized at any significant level of detail. This project aims at a comprehensive description of protein-protein interactions in E. coli by using exhaustive yeast two-hybrid screening. It will thus add significantly to ongoing attempts to characterize purified protein complexes by mass spectrometry (MS) and other methods. By contrast to the MS studies it will include direct binary interactions. In addition, this project focuses on protein domains of unknown proteins and map their interactions in more detail using both yeast two-hybrid screens and peptide array mapping. Most importantly, such mapping data will be used to identify biologically relevant and essential interactions by mutating specifically epitopes in vivo that are mediating these interactions. An array of cells with such interaction epitope mutants can then be tested for growth defects or more specific phenotypes under various conditions, thus showing which interactions are essential in general or only under certain conditions. The research design will include cloning of all open reading frames (ORFs) into two-hybrid bait and prey vectors and testing all pairwise combinations in an automated, array-based two-hybrid system. In addition to testing all full-length proteins, little-studied protein domains and cytoplasmic loops of transmembrane proteins will be searched for interactions, too. Finally, protein-domain interactions identified in these screens will be analyzed by testing the domain for binding to synthetic peptides from the partner protein. This will allow us to map binding sites to the amino acid level and subsequently study their atomic details using existing crystal or NMR structures. The end product of this project is a comprehensive molecular description of all protein-protein interactions in E. coli. Such maps can be used to integrate other data about metabolic reactions, gene regulation, and signalling networks to achieve a system level understanding of a bacterial cell. Eventually this information will allow us predict the behavior of a cell by computer modelling. With such a level of understanding, we will be both able to target defined processes therapeutically by antibiotics as well as modify the metabolism of the cell in order to use bacteria as chemical factories.

描述（由申请人提供）：大肠杆菌既是重要的病原体，也是微生物生物学的主要模型生物。然而，其大约一半的蛋白质尚未在任何显着的细节上被表征。该项目的目的是通过使用详尽的酵母两杂交筛选来全面描述大肠杆菌中蛋白质蛋白质的相互作用。因此，这将显着增加持续通过质谱（MS）和其他方法来表征纯化蛋白质复合物的尝试。与MS研究相反，它将包括直接的二进制相互作用。此外，该项目着重于未知蛋白质的蛋白质结构域，并使用酵母两杂交筛选和肽阵列映射更详细地绘制其相互作用。最重要的是，此类映射数据将用于通过在体内特异性表位进行介导这些相互作用的特异性表位来识别与生物学相关和基本相互作用。然后，可以在各种条件下测试具有这种相互作用表位突变体的一系列具有相互作用表位突变体的细胞是否存在生长缺陷或更具体的表型，从而表明哪些相互作用在一般或仅在某些条件下是必不可少的。 研究设计将包括将所有开放式阅读框（ORF）克隆到两个杂交诱饵和猎物矢量中，并在一个自动化的，基于阵列的两个杂交系统中测试所有成对组合。除了测试所有全长蛋白外，还将搜索较少的蛋白质结构域和跨膜蛋白的细胞质环，以寻找相互作用。最后，通过测试结构域与伴侣蛋白与合成肽结合的结构来分析这些筛选中确定的蛋白质域相互作用。这将使我们能够将结合位点映射到氨基酸水平，然后使用现有的晶体或NMR结构研究其原子细节。该项目的最终产物是大肠杆菌中所有蛋白质蛋白质相互作用的全面分子描述。此类地图可用于整合有关代谢反应，基因调节和信号网络的其他数据，以实现对细菌细胞的系统水平的理解。最终，这些信息将使我们通过计算机建模来预测单元的行为。有了这样的了解，我们将能够通过抗生素对定义的过程进行靶向定义的过程，并修改细胞的代谢，以便将细菌用作化学工厂。