Integrated microscopy approach to protein assembly on and in membranes

膜上和膜内蛋白质组装的集成显微镜方法

基本信息

批准号：
BB/N015487/1
负责人：
Bart Hoogenboom
金额：
$ 58.42万
依托单位：
University College London
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%2FN015487%2F1
关键词：
Integrated microscopy approach protein assembly

项目摘要

Proteins are key building blocks and the working horses of the living cell. They generate energy, make muscles contract, and translate light in our eyes into an image as perceived by our brain, to name but a few of many examples. In many cases, proteins need to form larger assemblies to carry out their biological function. Incorrect assembly causes biological malfunction and disease. Hence there is a general interest to understand how proteins form such larger assemblies. However, though we have an extensive toolkit to determine the structures of proteins and their assemblies, it is much harder to trace the processes by which these assemblies are being built.In this project, we will therefore develop new methodology to visualise protein assembly at length and time scales that will enable us to determine how such processes take place. Because the therefore required resolution can not be achieved by one technique alone, we will use a combination of different microscopy techniques: Single-molecule fluorescence microscopy, which can track individual labelled proteins as they move about; atomic force microscopy, which can visualise protein assemblies as they are being formed; and electron microscopy, which can provide static snapshots of the structure of proteins and protein assemblies.To test this correlative and integrated microscopy approach, we will apply it to a protein (named perforin) that is used by the immune system to attack virally infected and cancerous cells in the human body, essentially by perforating the membranes that protect cells from their environment. Perforin drills holes in these membranes by forming assemblies of several tens of proteins that span the membrane.We aim to understand the mechanisms of membrane pore formation by these proteins, out of a fundamental interest in how this nanometre-scale machinery works, to better understand diseases or enhanced vulnerability to cancer caused by malfunctioning perforin, and because mechanistic understanding can facilitate the design of drugs that prevent such pores from being formed when the immune response needs to be suppressed transplant, for example during organ transplantation.

蛋白质是活细胞的关键组成部分和工作马。它们产生能量，使肌肉收缩，并将我们眼中的光转化为我们大脑感知的图像，仅举几个例子。在许多情况下，蛋白质需要形成更大的组装体才能发挥其生物学功能。不正确的组装会导致生物功能障碍和疾病。因此，人们普遍有兴趣了解蛋白质如何形成如此大的组装体。然而，尽管我们有一个广泛的工具包来确定蛋白质及其组装的结构，但追踪这些组装的构建过程要困难得多。因此，在这个项目中，我们将开发新的方法来详细可视化蛋白质组装以及时间尺度，使我们能够确定这些过程如何发生。由于单独使用一种技术无法实现所需的分辨率，因此我们将结合使用不同的显微镜技术：单分子荧光显微镜，可以跟踪单个标记蛋白质的移动；原子力显微镜，可以在蛋白质组装体形成时将其可视化；和电子显微镜，它可以提供蛋白质和蛋白质组装体结构的静态快照。为了测试这种相关和集成的显微镜方法，我们将其应用于免疫系统用来攻击病毒感染和感染的蛋白质（称为穿孔素）。人体中的癌细胞，主要是通过刺穿保护细胞免受环境影响的膜来实现的。穿孔素通过形成数十个跨膜蛋白质的组合体在这些膜上钻孔。我们的目标是了解这些蛋白质形成膜孔的机制，出于对这种纳米级机械如何工作的根本兴趣，以便更好地理解穿孔素功能失常引起的疾病或增加患癌症的可能性，并且因为对机制的理解可以促进药物的设计，当需要抑制免疫反应的移植时，例如在器官移植期间，防止这种孔的形成。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Lipid specificity of the immune effector perforin

免疫效应器穿孔素的脂质特异性

DOI：
10.1101/2020.04.22.054890
发表时间：
2020
期刊：
影响因子：
0
作者：
Hodel A
通讯作者：
Hodel A

Polypeptide Materials - Methods and Protocols

多肽材料 - 方法和方案

DOI：
10.1007/978-1-0716-0928-6_15
发表时间：
2021
期刊：
影响因子：
0
作者：
Hammond K
通讯作者：
Hammond K

Antimicrobial peptide capsids of de novo design.

DOI：
10.1038/s41467-017-02475-3
发表时间：
2017-12-22
期刊：
Nature communications
影响因子：
16.6
作者：
De Santis E;Alkassem H;Lamarre B;Faruqui N;Bella A;Noble JE;Micale N;Ray S;Burns JR;Yon AR;Hoogenboom BW;Ryadnov MG
通讯作者：
Ryadnov MG