Hydrophobics transport across the outer membrane

疏水性物质穿过外膜的运输

• 批准号: 7084409
• 负责人: BERT VAN DEN BERG
• 金额: $ 30.15万
• 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/7084409
• 关键词: X ray crystallography; bacterial proteins; biological transport; cell biology; cell membrane; hydropathy; long chain fatty acid; membrane proteins; site directed mutagenesis

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于所有细胞都至关重要。尽管关于亲水性化合物的运输有丰富的知识，但对于疏水性分子的膜运输却知之甚少。这是值得注意的，因为生物膜给疏水分子的通过带来了独特的问题。由于脂多糖 (LPS) 的存在，革兰氏阴性细菌的外膜 (OM) 是防止疏水性化合物渗透的特别有效的屏障。唯一已被证明能够转运疏水性化合物的 OM 蛋白属于 FadL 转运蛋白家族。该家族的成员广泛存在于革兰氏阴性细菌中，并在长链脂肪酸的摄取中发挥作用 （LCFA）和异生素。该项目的目标是从结构和机制方面理解这些分子如何在 OM 中运输。更具体地说，我们将解决以下问题： 1. FadL 介导的 LCFA 运输的表征。以大肠杆菌 FadL 的晶体结构为起点，我们将结合使用定点诱变、体内转运测定和 X 射线晶体学，测试并进一步描述所提出的 LCFA 转运模型。 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 和异生物质转运的 FadL 直系同源物的底物特异性。 3. FadL 直系同源物转运外源物质的机制。通过解决外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。这些结构还将使我们能够确定 LCFA 和外源物质运输之间是否存在机制差异。