Biochemistry of Fatty Acid Transport in Escherichia Coli

长链脂肪酸是能量产生和大分子生物合成的重要底物，是重要的调节分子。脂肪酸进入细胞是大多数细菌和动物系统共有的重要新陈代谢过程。最广为人知的脂肪酸运输过程是在革兰氏阴性细菌--大肠杆菌中发现的。在大肠杆菌中，长链脂肪酸被与利用和脂肪酸依赖的基因表达相联系的过程所摄取。这一过程需要脂肪酸转运蛋白Fadl和脂肪酰辅酶A合成酶(FACS)。FADL的功能是将脂肪酸移过外膜，而FACS则激活这些化合物进行进一步的新陈代谢，使这一过程具有单向性。这项工作的技术和科学意义是多方面的。首先，它将定义转运蛋白Fadl在膜内的定位。这项研究将包括修改用于检测的蛋白质，并引入氨基酸替代来评估功能。其次，这项工作将使用荧光技术通过人造膜来确定Fadl的活性。更具体地说，这将涉及监测脂肪酸在含有FADL的膜上的移动，以便确定指导活性的基本性质。第三，这项工作将研究FAC是如何进入细胞膜的，在那里它激活了脂肪酸的运输。第四，我们希望定义FACS的结构，以进一步了解这种蛋白质在脂肪酸运输中的功能。这项工作旨在为支撑脂肪酸跨膜移动的分子机制以及它们如何随后作为调节脂肪酸响应基因表达的效应分子发挥作用提供基本的见解。这一实验系统为理解包括哺乳动物在内的其他系统的这一过程提供了一个范例。此外，该研究项目还将为研究生、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.