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

描述（由申请人提供）：跨越两层膜的三组分蛋白复合物在革兰氏阴性菌中普遍存在，并涉及多种运输功能，如向宿主传递毒力因子，分泌信号分子和保护细菌细胞免受结构多样的抗菌剂的侵害。这些转运体的一个显著特征是底物转移发生在两层膜上，直接进入外介质，绕过外周质。然而，这种转运的生化机制尚不清楚。表征三组分转运体的主要瓶颈是传统的生化方法不能很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如何在两种膜的情况下发挥作用。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中三组分转运体的生化机制。我们的中心假设是，消除重组蛋白脂质体的拓扑结构和异质性问题将通过各种生化技术显著推进双膜转运体的研究。验证这一假设的方法是将大环内酯和肠毒素转运体MacAB-TolC重组为高密度脂蛋白颗粒，并建立实时检测方法来研究该转运体的作用机制。我们将追求两个具体目标：(i)将MacA、MacB和TolC重组成脂质纳米盘，并表征它们的生化活性；（ii）开发实时结合分析来研究MacAB-TolC复合物的组装。拟议研究的预期结果是发展定量分析，研究组分位于两种不同膜上的多蛋白转运体，并实时表征这些转运体的组装。这一贡献是重要的，因为双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，它们的组装和功能是开发有效抗菌药物的目标。

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.