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: 8112360
• 负责人: HELEN I ZGURSKAYA
• 金额: $ 22.25万
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8112360
• 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; 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络合物普遍传播在革兰氏阴性细菌之间，并且与将毒力因子输送到宿主的分泌，信号分子的分泌以及对结构上不同的细胞对结构上不同的抗生素抗菌剂的保护等多样化的运输功能。这些转运蛋白的一个显着特征是，底物转移发生在两个膜上直接绕过绕着周围的膜。但是，这种运输的生化机制尚不清楚。三组分转运蛋白表征的主要瓶颈是传统的生化方法不能容易地应用于两膜系统。我们的长期目标是了解三组分转运蛋白在两个膜的背景下如何发挥作用。该应用的目的是开发一种新型的实验方法，以研究革兰氏阴性病原体的三组分转运蛋白的生化机制。我们的中心假设是消除重建蛋白脂质体的拓扑和异质性问题将通过各种生物化学技术显着提高两种膜转运蛋白的研究。检验该假设的方法是重建大花环和肠毒素转运蛋白MACAB-TOLC中的高密度脂蛋白颗粒，并开发实时测定以研究该转运蛋白的机制。我们将追求两个具体的目标：（i）将MACA，MACB和TOLC重新构成脂质纳米散发，并将其特征在于其生化活动； （ii）开发实时结合测定，以研究MacAb-TOLC复合物的组装。拟议的研究的预期结果是开发定量测定，以研究多蛋白转运蛋白，其成分位于两个不同的膜中，并实时表征这种转运蛋白的组装。这种贡献很重要，因为两个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及其组装和功能的目标是开发有效的抗菌剂。 公共卫生相关性：革兰氏阴性病原体会引起人类毁灭性的感染，并且对广泛的临床重要抗生素具有本质上的抗性。该建议的重点是开发实验工具，以研究蛋白质的分子机制，这些蛋白质以主要方式促进细菌毒力和抗生素耐药性。