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(申请人提供)：膜蛋白复合体仍然是生物化学和分子生物学的最后一个主要挑战。尽管在膜蛋白和膜复合体的结晶方面已经取得了很大的进展，但仍有许多未知功能的膜蛋白有待研究。对于膜中的复合体来说，情况更是如此。在这里，我们建议通过对大肠杆菌膜蛋白复合体的综合分析来解决这个问题，大肠杆菌膜蛋白复合体不仅是一个主要的微生物模型系统，而且是唯一一个通过实验确定了所有膜蛋白拓扑结构的物种。在这个项目的过程中，我们将纯化大部分或全部的大肠杆菌膜蛋白复合体，并用质谱仪分析它们的组成。我们已经成功地在酵母中进行了这样的分析，从而预测获得数百个蛋白质复合体。我们将独立地使用多个酵母双杂交系统来筛选大肠杆菌膜蛋白的所有细胞质结构域之间的相互作用。此外，我们将使用细菌双杂交系统绘制膜蛋白的相互作用图，以确保体内分析条件。这些研究将使我们能够绘制膜蛋白复合体的拓扑图，并将这些蛋白质与细胞质中的可溶性蛋白质联系起来。最后，我们将重点研究在上述筛选中发现的未鉴定的膜蛋白，特别是转运蛋白，并使用突变和专门的分析方法详细分析它们在体内的相互作用和功能，以使用高通量协议确定它们的活性(如转运蛋白)。生物信息学分析将对所有三个分项目提供帮助。据我们所知，这种规模的综合研究以前从未尝试过。该项目的结果将对微生物和真核生物的膜蛋白生物学产生广泛的影响，包括生物技术和医学应用。例如，膜蛋白是抗菌药物的常见靶点，该项目将确定新的靶点，但也将阐明许多这些(迄今尚未确定的)跨膜蛋白的分子功能。