Hydrophobics transport across the outer membrane

疏水性物质穿过外膜的运输

• 批准号: 7252573
• 负责人: BERT VAN DEN BERG
• 金额: $ 29.28万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252573
• 关键词: Address; Binding; Biochemical; Biodegradation; Biological; Biological Assay; Biology; Cells; Class; Environment; Escherichia coli; Family; Family member; Goals; Gram-Negative Bacteria; In Vitro; Knowledge; Lipopolysaccharides; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Modeling; Numbers; Pathway interactions; Play; Research Personnel; Role; Sequence Homology; Site; Site-Directed Mutagenesis; Structure; Substrate Specificity; Testing; Transmembrane Transport; Transport Process; X-Ray Crystallography; Xenobiotics; base; fatty acid transport; fatty acid-transport protein; in vivo; long chain fatty acid; mutant; programs; research study; uptake

Biological membranes form the interface between cells and their environment, and transport of molecules across this barrier is essential for all cells. Whereas a wealth of knowledge exists about the transport of hydrophilic compounds, very little is known about membrane transport of hydrophobic molecules. This is remarkable, since biological membranes pose unique problems for the passage of hydrophobic molecules. The outer membrane (OM) of gram-negative bacteria is a particularly effective barrier against permeation of hydrophobic compounds, due to the presence of lipopolysaccharide (LPS). The only OM proteins that have been shown to transport hydrophobic compounds belong to the FadL transporter family. Members of this family are widespread in gram-negative bacteria and play a role in the uptake of long-chain fatty acids (LCFAs) and xenobiotics. The goal of this project is to understand in structural and mechanistic terms how such molecules are transported across the OM. More specifically, we will address the following questions: 1. Characterization of FadL-mediated LCFA transport. Using the crystal structures of E. coli FadL as a starting point, we will test and further delineate the proposed model of LCFA transport, by using a combination of site-directed mutagenesis, in vivo transport assays and X-ray crystallography. 2. Characterization of substrate specificity of FadL orthologues involved in LCFA and xenobiotics transport by using in vivo and in vitro substrate binding and transport assays. 3. Mechanism of xenobiotics transport by FadL orthologues. By solving crystal structures of xenobiotics transporters we will determine the structural basis for their substrate specificity. These structures will also allow us to determine whether mechanistic differences exist between LCFA and xenobiotics transport.

生物膜形成细胞与其环境之间的界面，分子穿过该屏障的运输对所有细胞都是必不可少的。虽然存在大量的知识关于亲水性化合物的运输，很少有人知道关于疏水性分子的膜运输。这是值得注意的，因为生物膜对疏水分子的通过提出了独特的问题。革兰氏阴性菌的外膜（OM）是一个特别有效的屏障，防止疏水化合物的渗透，由于脂多糖（LPS）的存在。已显示转运疏水化合物的唯一OM蛋白属于FadL转运蛋白家族。这个家族的成员广泛存在于革兰氏阴性菌中，并在长链脂肪酸的摄取中发挥作用 （LCFA）和异生物质。该项目的目标是从结构和机制方面了解这些分子如何通过OM运输。具体而言，我们将讨论以下问题： 1. FadL介导的LCFA转运的表征。利用E.作为起点，我们将测试和进一步描绘LCFA运输的拟议模型，通过使用定点诱变，体内运输试验和X射线晶体学的组合。 2.通过使用体内和体外底物结合和转运测定来表征参与LCFA和外源性物质转运的FadL直向同源物的底物特异性。 3. FadL直向同源物转运外源性物质的机制。通过解决异源物质转运蛋白的晶体结构，我们将确定其底物特异性的结构基础。这些结构也将使我们能够确定LCFA和外源性物质运输之间是否存在机械差异。