Integrative functional mapping of the Escherichia coli membrane interactome

大肠杆菌膜相互作用组的综合功能图谱

• 批准号: 9129760
• 负责人: MILTON H. SAIER
• 金额: $ 27.96万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9129760
• 关键词: Amino Acid Motifs; Binding; Biochemistry; Bioinformatics; Biological Assay; Biological Models; Biology; C-terminal; Chimeric Proteins; Collaborations; Complex; Cytoplasm; Cytoplasmic Protein; Cytoplasmic Tail; Data; Dissection; Ensure; Epitopes; Escherichia coli; Eukaryota; Fermentation; Gene Fusion; Genome; Growth; Health; Integral Membrane Protein; Knock-out; Link; Maps; Mass Spectrum Analysis; Medical; Membrane; Membrane Proteins; Microbe; Modeling; Molecular Biology; Molecular Genetics; Mutate; Mutation; Nutrient; Operon; Phenotype; Physiology; Proteins; Protocols documentation; Regulon; Reporter Genes; Reproducibility; Reverse Transcriptase Polymerase Chain Reaction; Site; Structure; System; Tertiary Protein Structure; Two-Hybrid System Techniques; Work; Yeasts; antimicrobial; base; environmental change; feeding; flexibility; improved; in vivo; interest; microbial; mutant; overexpression; pathogen; protein E; protein complex; research study; transcriptome; uptake; vector; yeast two hybrid system

DESCRIPTION (provided by applicant): Membrane protein complexes remain the last major challenge in biochemistry and molecular biology. Although major advances have been made crystallizing selected membrane proteins and complexes, many membrane proteins of unknown function remain to be studied. This is even truer for complexes in the membrane. Here we propose to tackle this problem by an integrated analysis of membrane protein complexes in E. coli, not only a major microbial model system but also the only species in which the topology of all membrane proteins has been determined experimentally. In the course of this project, we will purify most or all membrane protein complexes of E. coli and analyze their composition by mass spectrometry. We have successfully carried out such an analysis in yeast and thus predict to obtain several hundred protein complexes. Independently, we will screen all cytoplasmic domains of E. coli membrane proteins for interactions using multiple yeast two-hybrid systems. In addition, we will map interactions of membrane proteins using a bacterial two-hybrid system to ensure in vivo assay conditions. These studies will allow us to map the topologies of membrane protein complexes and link those proteins to soluble proteins in the cytoplasm. Finally, we will focus on uncharacterized membrane proteins found in the aforementioned screens, especially transporters, and analyze their in vivo interactions and functions in detail using mutations and specialized assays to determine their activities (such as transport) using high throughput protocols. Bioinformatic analysis will assist all of the three subprojects. To our knowledge no such integrated study of this scale has been attempted before. The results of this project will have a broad impact on membrane protein biology in both microbes and eukaryotes, including biotechnological and medical applications. For instance, membrane proteins are common targets of antimicrobials and this project will identify new targets but also elucidate the moleculr function of many of these (hitherto uncharacterized) transmembrane proteins.

描述（由申请人提供）：膜蛋白复合物仍然是生物化学和分子生物学的最后一个主要挑战。尽管已经使所选膜蛋白和复合物结晶的重大进展已经使许多功能的膜蛋白尚有待研究。对于膜中的复合物来说，这甚至更真实。 在这里，我们建议通过对大肠杆菌中的膜蛋白复合物进行综合分析来解决这个问题，这不仅是主要的微生物模型系统，而且是唯一通过实验确定所有膜蛋白拓扑的物种。 在该项目的过程中，我们将纯化大肠杆菌的大多数或所有膜蛋白复合物，并通过质谱分析其组成。 我们已经成功地在酵母中进行了这样的分析，因此预测将获得数百个蛋白质复合物。独立地，我们将使用多种酵母两种杂交系统的相互作用来筛选大肠杆菌蛋白的所有细胞质结构域。此外，我们将使用细菌两杂交系统来绘制膜蛋白的相互作用，以确保体内测定条件。这些研究将使我们能够绘制膜蛋白复合物的拓扑结构，并将这些蛋白质与细胞质中的可溶性蛋白联系起来。最后，我们将重点关注上述筛选（尤其是转运蛋白）中发现的未表征的膜蛋白，并使用突变和专业测定法对其在体内相互作用和功能进行详细分析，以确定其活动（例如运输）使用高吞吐量方案。生物信息学分析将有助于所有三个副标题。据我们所知，从未尝试过这种对此规模的综合研究。该项目的结果将对微生物和真核生物（包括生物技术和医学应用）的膜蛋白生物学产生广泛的影响。例如，膜蛋白是抗菌剂的常见靶标，该项目将确定新靶标，但也阐明了许多（迄今未表征）跨膜蛋白的分子功能。