Hydrophobics transport across the outer membrane

生物膜形成了细胞和环境之间的界面，分子的运输通过这一屏障对所有细胞都是必不可少的。虽然关于亲水性化合物的传输有丰富的知识，但对疏水分子的膜传输却知之甚少。这是值得注意的，因为生物膜对疏水分子的通过构成了独特的问题。由于脂多糖的存在，革兰氏阴性细菌的外膜是一种特别有效的屏障，可以阻止疏水化合物的渗透。唯一被证明能运输疏水化合物的OM蛋白属于Fadl转运蛋白家族。该家族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。 (LCFAs)和外源生物。这个项目的目标是从结构和机械的角度了解这些分子是如何通过OM运输的。更具体地说，我们将解决以下问题： 1.Fadl介导的LCFA转运特性。以E.ColiFadl的晶体结构为起点，我们将结合定点突变、体内转运试验和X射线结晶学来测试和进一步描述所提出的LCFA转运模型。 2.通过体内和体外底物结合和转运实验，表征参与LCFA和外源物质转运的Fadl同系物的底物特异性。 3.Fadl同系物的外源物质转运机制。通过解决外源物质转运体的晶体结构，我们将确定其底物专一性的结构基础。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械上的差异。

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.