Electrogenic Reactions during Lactose/proton Symport Catalyzed by LacY

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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.