Biochemistry of Fatty Acid Transport in Escherichia Coli

长链脂肪酸是能量生产和大分子生物合成的重要底物，是重要的调节分子。将脂肪酸转运到细胞中代表了大多数细菌和动物系统共同的重要代谢过程。在革兰氏阴性细菌大肠杆菌中发现了最好理解的脂肪酸转运过程。在大肠在大肠杆菌中，长链脂肪酸被一个与利用和脂肪酸依赖性基因表达有关的过程吸收。该过程需要脂肪酸转运蛋白FadL和脂肪酰辅酶A合成酶（FACS）。FadL的功能是使脂肪酸穿过外膜，而FACS激活这些化合物以进一步代谢，使该过程单向。这项工作的技术和科学意义是多重的。首先，它将定义转运蛋白FadL如何定位在膜内。这项研究将涉及修改蛋白质的检测和引入氨基酸取代，以评估功能。第二，这项工作将使用荧光技术来定义使用人工膜的FadL的活性。更具体地说，这将涉及监测脂肪酸穿过含有FadL的膜的运动，从而可以确定指导活性的基本性质。第三，这项工作将研究流式细胞仪如何进入膜，在那里它激活脂肪酸，因为它们被运输。第四，我们希望确定FACS的结构，以进一步了解这种蛋白质在脂肪酸转运中的功能。这项工作的目的是提供基本的见解的分子机制支持跨膜运动的脂肪酸，以及它们如何随后作为效应分子调节脂肪酸响应基因表达。这个实验系统可以作为理解其他系统（包括哺乳动物）中这一过程的范例。此外，该研究计划将为研究生、本科生和高中生提供分子遗传学、分子生物学和生物化学方面的培训环境。

Long-chain fatty acids are important substrates for energy production and macromolecular biosynthesis and are important regulatory molecules. The transport of fatty acids into the cell represents an important metabolic process common to most bacterial and animal systems. The most well understood fatty acid transport process is found in the gram-negative bacterium bacteria, Escherichia coli. In E. coli, long-chain fatty acids are taken up by a process that is linked to utilization and fatty acid-dependent gene expression. This process requires the fatty acid transport protein FadL and the enzyme fatty acyl CoA synthetase (FACS). FadL functions to move fatty acids across the outer membrane while FACS activates these compounds for further metabolism rendering this process unidirectional. The technical and scientific significance of this work is several-fold. First, it will define how the transport protein FadL is positioned within the membrane. This research will involve modifying the protein for detection and the introduction of amino acid substitutions to assess function. Second, this work will use fluorescent techniques to define the activity of FadL using artificial membranes. More specifically, this will involve monitoring the movement of fatty acids across membranes containing FadL so that the fundamental properties guiding activity can be determined. Third, this work will investigate how FACS moves into the membrane, where it activates fatty acids as they are transported. And fourth, we expect to define the structure of FACS to further understand how this protein functions in fatty acid transport. This work is designed to provide fundamental insights into the molecular mechanisms underpinning the movement of fatty acids across the membrane and how they subsequently function as effecter molecules regulating fatty acid responsive gene expression. This experimental system serves as a paradigm for understanding this process in other systems, including mammals. In addition, this research program will provide a training environment in molecular genetics, molecular biology and biochemistry for graduate, undergraduate and high school students.