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

细胞被脂质和蛋白质组成的膜屏障包裹，将有用的物质保持在细胞内，并阻止有害的有毒化合物进入。一些残留在细胞膜上的蛋白质已经进化成了运输机器，将必需的营养物质运送到细胞中并输出废物。了解这些转运蛋白（转运蛋白）的功能具有重大的生物技术和医学意义，因为许多这些蛋白质在癌症等疾病中功能异常，这需要细胞吸收比周围组织更多的营养物质。蛋白质采取各种不同的状态，使它们能够在细胞中执行特定的任务。然而，到目前为止，生物医学科学界主要集中精力使用成熟的X射线蛋白质晶体学技术来确定转运蛋白的三维结构。这些结构代表静态快照，但未能提供有关这些蛋白质的动态信息。我们的研究项目旨在解决该领域的一个主要概念空白，通过了解运输的动力学以及膜中存在的脂质如何影响转运蛋白的结构和功能。我们将使用生物光谱学中的最新技术来绘制出一个重要的营养转运蛋白家族所采用的各种结构状态，该家族负责将肽摄取到细胞中。我们的方法将是在选定的位置标记这些蛋白质，并在天然脂质环境中测量标记之间的距离。使用我们已经获得的晶体结构，以及这里将要解析的新结构，我们将测量蛋白质使肽穿过膜时这些距离的变化。我们将能够模拟功能过程中发生的结构变化，更详细地了解营养物质和小分子如何选择性地转运到细胞中，以进一步用于代谢和细胞功能。这项工作不仅对代谢过程具有重要意义，特别是在疾病条件下，其中有很多，而且还涉及使用这些蛋白质将药物递送到细胞中，以及以生物技术方式使用这些蛋白质以允许细胞制备用于工业和药理学的选定化合物，这是长期目标。

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.