Electrogenic Reactions during Lactose/proton Symport Catalyzed by LacY

智力上的优点。在生物能量学的广泛领域中，主要挑战之一是了解糖和氨基酸等营养物质是如何穿过生物膜并在细胞内积累的。人们现在知道，大多数活细胞的功能就像电池一样。因此，像呼吸作用或三磷酸腺苷水解这样的化学反应与将氢离子或钠泵出细胞相联系。以这种方式，细胞或细胞内细胞器，如线粒体，产生氢离子或钠的电化学梯度，其中细胞内部是电负性的，与外部相比，氢离子或钠的含量较低。在这种情况下，氢离子或钠离子就会受到驱动力，沿着它们的电化学梯度扩散回细胞内。嵌入在膜中的运输蛋白，如本提案中描述的那样，利用这种能量下坡运动释放的自由能来驱动特定营养物质的转运和积累，在这种情况下，糖乳糖，众所周知，每个乳糖分子的跨膜运输伴随着一个氢离子。本研究的目的是通过研究乳糖渗透酶(Lacy)的电学性质来了解这一基本而普遍的生物过程的分子机制。乳糖渗透酶是主要促进者超家族成员的模型，是一大类相关的膜运输蛋白。该项目包括应用一项重要的新技术，使用新开发的固体支撑膜电极来观察这种运输器的电性能。以Lacy为范式的主动转运和生物能量学的研究使膜转运领域发生了革命性的变化。从(I)最初发现膜小泡可用作研究转运的模型系统，到(Ii)发展用于量化微观系统中的电势和氢离子梯度的探针，到(Iii)定点和细胞扫描突变，到(Iv)在功能状态下提纯Lacy至均一，(V)获得X射线晶体结构，(Vi)设计用于各种生化和光谱研究的共转体，本实验室在该领域的发展已超过45年。这项研究历史上的大多数突破都被广泛选择，纳入世界各地本科生和研究生教学的各种教科书、参考书和多种语言的教材。PI将继续向高中和大学观众演讲，向年轻人传递科学知识，激发他们对基础科学的兴趣。在国内和国际的研讨会、大学和多学科会议上的应邀讲座将成为向社会及时传达这一新信息的多种渠道。

Intellectual merit. One of the major challenges in the broad field of bioenergetics is to understand how nutrients such as sugars and amino acids cross biological membranes and accumulate inside of cells. It is now understood that most living cells function like batteries. Thus, chemical reactions like respiration or ATP hydrolysis are coupled to the pumping of hydrogen ions or sodium out of cells. In this manner, cells or intracellular organelles like mitochondria create an electrochemical gradient of either hydrogen ions or sodium in which the inside of the cell is electrically negative and low in hydrogen ions or sodium relative to the outside. Given this scenario, there is then a driving force on hydrogen ions or sodium to diffuse back into the cell down their electrochemical gradients. Transport proteins embedded in the membrane like the one described in this proposal utilize the free energy released by this energetically downhill movement to drive translocation and accumulation of a specific nutrient in this case, the sugar lactose and it is known that transport of each lactose molecule across the membrane is accompanied by one hydrogen ion. It is the aim of this research to understand the molecular mechanism of this basic and universal biological process by studying the electrical properties of the lactose permease (LacY), a model for the members of the Major Facilitator Superfamily, a huge group of related membrane transport proteins. The project includes the application of an important new technique to observe the electrical properties of this transporter using newly developed solid-supported membrane electrodes.Broader impacts. Studies on active transport and bioenergetics with LacY as the paradigm have revolutionized the field of membrane transport. From (i) the initial discovery that membrane vesicles are useful as a model system to study transport to (ii) the development of probes for quantifying electrical potentials and hydrogen ion gradients in microscopic systems to (iii) site-directed and Cys-scanning mutagenesis to (iv) purifying LacY to homogeneity in a functional state to (v) obtaining X-ray crystal structures to (vi) engineering the symporter for all manner of biochemical and spectroscopic studies, this laboratory has pioneered developments in the field for over 45 years. Most of the breakthroughs in the history of the research have been widely selected for inclusion in various textbooks, reference books and teaching materials in many languages for both undergraduate and graduate teaching worldwide. The PI will continue to speak to high school and university audiences in order to convey scientific knowledge to young people and stimulate their interest in basic science. Invited lectures in symposia, at universities and multi-disciplinary conferences nationally and internationally will serve as multiple channels to convey this novel information to society in a timely manner.