Resolving mechanistic details of peptide transport across membranes using crystallographic and non-crystallographic structural biology approaches

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Cells are enveloped by a membrane barrier composed of lipids and proteins that keep useful materials inside the cell and exclude harmful, toxic compounds from entering. Some of the proteins that residue in the membrane have evolved to function as transport machines, shuttling essential nutrients into the cell and exporting waste products. Understanding how these transport proteins (transporters) function is of major biotechnological and medical significance, as many of these proteins function abnormally in diseases such as cancer, which require cells to take up many more nutrients than surrounding tissue. Proteins adopt a variety of different states which enable them to carry out their specific tasks in cells. However, to date the biomedical science community has largely focused their efforts on determining the three-dimensional structure of transporters using the well-established technique of X-ray protein crystallography. The structures represent static snapshots but fail to provide information on the dynamics of these proteins. Our research project aims at addressing a major conceptual gap in the field, by understanding the dynamics of transport and how lipids present in the membrane impact on the structure and function of transporters. We will use the latest techniques in biological spectroscopy to map out the variety of structural states adopted by an important family of nutrient transporters responsible for the uptake of peptides into the cell. Our methodology will be to label these proteins at selected positions and to measure the distance between the labels in native lipid environments. Using the crystal structures we have already obtained, and new ones to be resolved here, we will measure the changes in these distances as the proteins move peptides across the membrane. We will be able to model the structural changes taking place during function, to understand in much more detail how nutrients and small molecules can be selectively transported into the cell for further use in metabolism and cell function.This work has significant implications for not only metabolic processes, especially in disease conditions, of which there are many, but also in the use of these proteins to deliver drugs into a cell as well as use these proteins in biotechnological ways to allow cells to make selected compounds for use in industry and pharamacology, which are long term aims.