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

使用晶体学和非晶体结构生物学方法解决肽跨膜转运的机制细节

• 批准号: BB/N006011/1
• 负责人: Anthony Watts
• 金额: $ 130.23万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN006011%2F1
• 关键词: Resolving; mechanistic; details; peptide; transport

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.

细胞被由脂类和蛋白质组成的膜屏障所包裹，这些屏障将有用的物质保留在细胞内，并阻止有害、有毒的化合物进入细胞。一些残留在细胞膜上的蛋白质已经进化成运输机器，将必要的营养物质运送到细胞内，并输出废物。了解这些转运蛋白(转运体)的功能具有重大的生物技术和医学意义，因为这些蛋白中的许多在癌症等疾病中功能异常，这些疾病需要细胞比周围组织吸收更多的营养。蛋白质采用各种不同的状态，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将他们的努力集中在使用成熟的X射线蛋白质结晶学技术来确定转运蛋白的三维结构上。这些结构代表静态快照，但不能提供关于这些蛋白质动态的信息。我们的研究项目旨在通过了解转运的动力学以及膜中存在的脂质如何影响转运体的结构和功能来解决该领域的一个主要概念空白。我们将使用生物光谱学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，这些转运蛋白负责将多肽吸收到细胞中。我们的方法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。利用我们已经获得的晶体结构，以及即将在这里解析的新结构，我们将测量当蛋白质移动多肽穿过膜时这些距离的变化。我们将能够对功能过程中发生的结构变化进行建模，更详细地了解营养和小分子如何有选择地运输到细胞中，以便进一步用于新陈代谢和细胞功能。这项工作不仅对新陈代谢过程，特别是在许多疾病条件下，而且在使用这些蛋白质将药物输送到细胞以及在生物技术方法中使用这些蛋白质，使细胞能够制造选定的化合物用于工业和药学，这是长期目标。

项目成果

Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization.

• DOI: 10.1038/s41467-020-20596-0
• 发表时间: 2021-01-27
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Bada Juarez JF;Judge PJ;Adam S;Axford D;Vinals J;Birch J;Kwan TOC;Hoi KK;Yen HY;Vial A;Milhiet PE;Robinson CV;Schapiro I;Moraes I;Watts A
• 通讯作者: Watts A

A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography.

• DOI: 10.1107/s1600576723006428
• 发表时间: 2023-10-01
• 期刊: JOURNAL OF APPLIED CRYSTALLOGRAPHY
• 影响因子: 6.1
• 作者: Birch, James;Kwan, Tristan O. C.;Judge, Peter J.;Axford, Danny;Aller, Pierre;Butryn, Agata;Reis, Rosana I.;Juarez, Juan F. Bada;Vinals, Javier;Owen, Robin L.;Nango, Eriko;Tanaka, Rie;Tono, Kensuke;Joti, Yasumasa;Tanaka, Tomoyuki;Owada, Shigeki;Sugahara, Michihiro;Iwata, So;Orville, Allen M.;Watts, Anthony;Moraes, Isabel
• 通讯作者: Moraes, Isabel

Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts.

• DOI: 10.1107/s2059798321011220
• 发表时间: 2022-01-01
• 期刊: Acta crystallographica. Section D, Structural biology
• 作者: Axford D;Judge PJ;Bada Juarez JF;Kwan TOC;Birch J;Vinals J;Watts A;Moraes I
• 通讯作者: Moraes I

Detergent-free Lipodisq Nanoparticles Facilitate High-Resolution Mass Spectrometry of Folded Integral Membrane Proteins.

• DOI: 10.1021/acs.nanolett.0c04911
• 发表时间: 2021-04-14
• 期刊: Nano letters
• 影响因子: 10.8
• 作者: Hoi KK;Bada Juarez JF;Judge PJ;Yen HY;Wu D;Vinals J;Taylor GF;Watts A;Robinson CV
• 通讯作者: Robinson CV

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

• DOI: 10.1101/666495
• 发表时间: 2019-06
• 期刊: bioRxiv
• 作者: L. Mendes;M. Batista;P. Judge;A. Watts;C. Redfield;A. Costa-Filho