Reconstitution of Two-membrane Transporters into High-density Lipoprotein Particl

双膜转运蛋白重构为高密度脂蛋白颗粒

• 批准号: 8220711
• 负责人: HELEN I ZGURSKAYA
• 金额: $ 18.49万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220711
• 关键词: Affect; Antibiotic Resistance; Antibiotics; Bacterial Infections; Binding; Biochemical; Biological Assay; Bypass; Carrier Proteins; Cells; Chimeric Proteins; Complex; Cytoplasm; Development; Drug Delivery Systems; Drug Efflux; Emerging Technologies; Enterotoxins; Goals; Gram-Negative Bacteria; Heterogeneity; High Density Lipoproteins; Human; Immobilization; Individual; Infection; Ion Channel; Link; Lipids; Literature; Maca; Macrolides; Membrane; Membrane Fusion; Membrane Proteins; Membrane Transport Proteins; Methods; Modeling; Molecular; Monitor; Multi-Drug Resistance; Outcome; Periplasmic Proteins; Permeability; Pharmaceutical Preparations; Predisposition; Protein Family; Protein Inhibition; Proteins; Public Health; Reporting; Resistance; Signaling Molecule; Staging; Surface; Surface Plasmon Resonance; System; Techniques; Technology; Testing; Time; Transmembrane Transport; Virulence; Virulence Factors; Work; antimicrobial drug; bacterial resistance; cofactor; inhibitor/antagonist; nanodisk; news; novel; particle; pathogen; periplasm; protein complex; proteoliposomes; public health relevance; reconstitution; tool

DESCRIPTION (provided by applicant): Three-component protein complexes spanning two membranes are universally spread among Gram-negative bacteria and have been implicated in such diverse range of transport functions as delivery of virulence factors into the hosts, secretion of signaling molecules and protection of bacterial cells against structurally diverse antimicrobial agents. A remarkable feature of these transporters is that the substrate transfer occurs across two membranes directly into external medium bypassing the periplasm. However the biochemical mechanism of such transport remains unclear. The major bottleneck in characterization of three-component transporters is that traditional biochemical methods cannot be readily applied to two-membrane systems. Our long-term goal is to understand how three-component transporters function in the context of two membranes. The objective of this application is to develop a novel experimental approach to study the biochemical mechanism of three-component transporters from Gram-negative pathogens. Our central hypothesis is that elimination of topological and heterogeneity problems of reconstituted proteoliposomes will significantly advance studies of two-membrane transporters by various biochemical techniques. The approach used to test this hypothesis is to reconstitute the macrolide and enterotoxin transporter MacAB-TolC into high-density lipoprotein particles and to develop real-time assays to study the mechanism of this transporter. We will pursue two specific aims: (i) Reconstitute MacA, MacB and TolC into lipid nanodiscs and characterize their biochemical activities; (ii) Develop real-time binding assays to study assembly of MacAB-TolC complex. The expected outcome of the proposed studies is development of quantitative assays to study multi-protein transporters with components located in two different membranes and characterization of assembly of such transporters in real-time. This contribution is significant because two-membrane transporters are responsible for the intrinsic antibiotic resistance and virulence of Gram-negative pathogens and their assembly and functions are targeted in development of effective antimicrobial agents. PUBLIC HEALTH RELEVANCE: Gram-negative pathogens cause devastating infections in humans and are intrinsically resistant to a broad range of clinically important antibiotics. This proposal is focused on the development of experimental tools to study the molecular mechanisms of proteins that contribute in the major way to both bacterial virulence and antibiotic resistance.

描述（由申请人提供）：跨越两个膜的三组分蛋白质复合物普遍分布在革兰氏阴性细菌中，并且涉及多种运输功能，例如将毒力因子递送到宿主中、分泌信号分子以及保护细菌细胞免受结构不同的抗菌剂的侵害。这些转运蛋白的一个显着特征是底物转移穿过两个膜直接进入外部介质，绕过周质。然而这种运输的生化机制仍不清楚。三组分转运蛋白表征的主要瓶颈是传统的生化方法不能轻易应用于两膜系统。我们的长期目标是了解三组分转运蛋白如何在两种膜的背景下发挥作用。本申请的目的是开发一种新的实验方法来研究革兰氏阴性病原体三组分转运蛋白的生化机制。我们的中心假设是，消除重构蛋白脂质体的拓扑和异质性问题将显着推进通过各种生化技术对二膜转运蛋白的研究。用于检验这一假设的方法是将大环内酯和肠毒素转运蛋白 MacAB-TolC 重组为高密度脂蛋白颗粒，并开发实时检测方法来研究该转运蛋白的机制。我们将追求两个具体目标：（i）将 MacA、MacB 和 TolC 重构为脂质纳米盘并表征其生化活性； (ii) 开发实时结合测定法来研究 MacAB-TolC 复合物的组装。拟议研究的预期结果是开发定量分析方法来研究具有位于两种不同膜中的成分的多蛋白转运蛋白，并实时表征此类转运蛋白的组装。这一贡献意义重大，因为两膜转运蛋白负责革兰氏阴性病原体的内在抗生素耐药性和毒力，并且它们的组装和功能是有效抗菌药物开发的目标。 公共卫生相关性：革兰氏阴性病原体会引起人类毁灭性感染，并且对多种临床上重要的抗生素具有内在耐药性。该提案的重点是开发实验工具来研究蛋白质的分子机制，这些蛋白质对细菌毒力和抗生素耐药性有主要贡献。