权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Interrogating the folding and function of membrane proteins by mass spectrometry

通过质谱分析膜蛋白的折叠和功能

基本信息

批准号：
BB/K000659/1
负责人：
Alison Ashcroft
金额：
$ 63.53万
依托单位：
University of Leeds
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2013
资助国家：
英国
起止时间：
2013 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FK000659%2F1
关键词：
Interrogating folding function membrane proteins

项目摘要

A membrane protein is a protein that is attached to, or associated with, the membrane of a cell. The membrane is the cell's impermeable surface through which many ions, nutrients, hormones, drugs and proteins enter and exit the cell, and membrane proteins are responsible for transportation across the membrane and cell signalling, the latter of which governs the actions of the cell. Membrane proteins are important medically. Examples of maladies in which membrane proteins are implicated include: cancer, which can involve over-production of membrane proteins and errors in cell signalling pathways; heart disease, where the regulation of ions across the cell membrane may be failing; diabetes, which has been linked with membrane protein mutations; and depression, where there is an inbalance of the levels of specific molecules in cells. Due to their association with such diseases, and their convenient location at the cell surface where they are accessible to small molecule drugs, membrane proteins account for >50 % of all known drug targets. They are also important targets for anti-bacterial agents. Despite membrane proteins representing ~30% of all proteins, details of their characteristics and mode of action are scarce as they are notoriously difficult species to handle and analyse because of their hydrophobicity and insolubility. Compared with water-soluble proteins, few membrane proteins have been characterised structurally: only ~309 out of a total of >65,000 protein structures archived in the Protein Data Bank are of membrane proteins. Our understanding of many diseases, and our subsequent ability to treat these diseases and improve our quality of life, is reliant on a better knowledge of the structure of these proteins, the ways in which they interact with ions and small molecules, and their mode of function. Our aim is to understand the way in which membrane proteins function by developing analytical methods with which we can monitor structural changes taking place during a folding or functional event. We will use electrospray ionisation-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry, a combined technique which produces information about mass, stoichiometry, shape and stability of individual protein species present within complex mixtures in a single, rapid experiment. It is ideal for monitoring the way a protein changes its 3D shape and for monitoring reactions between proteins and other species, where the starting materials, reaction intermediates and products can be analysed simultaneously. We have pioneered this technique to provide insights into water-soluble protein functioning events in real-time and now wish to exploit its use for membrane proteins. Prior to ESI-MS analysis we will use chemical labelling techniques to probe the 3D structures of membrane proteins at different times during a functioning event. Such methods modify the protein which results in a change of mass which we can detect. We will then use ESI-MS to analyse the individual species within the reaction and identify exactly which regions of the protein are exposed and available to react (the labelled areas), and those regions which are shielded within its 3D structure or by interactions with other species. By labelling and analysing at different times during the reaction we will produce a series of "snapshots" of the event in real-time. Together the snapshots will provide a complete picture revealing insights into the functional properties of membrane proteins. To carry out the chemical labelling and ESI-MS analysis we will purchase laser equipment to couple to our existing mass spectrometers and then develop robust protocols that we can apply to a wide range of membrane proteins. For this development work, we have chosen two different proteins, each an example from one of the two major classes of membrane proteins, the all alpha-helical and beta-barrel, which we have considerable experience of handling.

膜蛋白是一种附着在细胞膜上或与细胞膜相关的蛋白质。膜是细胞的不可渗透表面，许多离子、营养物质、激素、药物和蛋白质通过它进入和离开细胞，膜蛋白负责跨膜运输和细胞信号传递，后者控制细胞的行动。膜蛋白在医学上很重要。与膜蛋白有关的疾病的例子包括：癌症，可能涉及膜蛋白的过度生产和细胞信号通路的错误；心脏病，跨细胞膜的离子调节可能失效；糖尿病，与膜蛋白突变有关；以及抑郁症，细胞中特定分子水平失衡。由于它们与这类疾病有关，以及它们位于细胞表面的方便位置，小分子药物可以接触到它们，膜蛋白占所有已知药物靶点的50%。它们也是抗菌剂的重要靶点。尽管膜蛋白约占所有蛋白质的30%，但由于它们的疏水性和不溶性，它们是出了名的难以处理和分析的物种，因此对它们的特征和作用方式的详细信息很少。与水溶性蛋白质相比，很少有膜蛋白质具有结构特征：在蛋白质数据库中记录的65,000个蛋白质结构中，只有309个是膜蛋白质。我们对许多疾病的了解，以及随后我们治疗这些疾病和提高生活质量的能力，都依赖于对这些蛋白质的结构、它们与离子和小分子相互作用的方式以及它们的功能模式的更好了解。我们的目标是通过开发分析方法来了解膜蛋白的功能，利用这些方法我们可以监测折叠或功能事件期间发生的结构变化。我们将使用电喷雾电离-质谱仪(ESI-MS)与离子迁移率光谱相结合，这是一项综合技术，可以在单一、快速的实验中产生关于复杂混合物中存在的单个蛋白质物种的质量、化学计量比、形状和稳定性的信息。它非常适合于监测蛋白质改变其3D形状的方式，以及监测蛋白质与其他物种之间的反应，在这种情况下，可以同时分析起始材料、反应中间体和产物。我们开创了这项技术，以提供对水溶性蛋白质功能事件的实时洞察，现在希望利用它来研究膜蛋白质。在ESI-MS分析之前，我们将使用化学标记技术来探索膜蛋白在功能事件中不同时间的三维结构。这样的方法可以修饰蛋白质，从而导致我们可以检测到的质量变化。然后我们将使用ESI-MS来分析反应中的单个物种，并准确地确定蛋白质的哪些区域暴露并可用于反应(标记区域)，以及哪些区域在其3D结构中被屏蔽或通过与其他物种的相互作用而被屏蔽。通过在反应过程中的不同时间进行标记和分析，我们将实时产生一系列事件的“快照”。这些快照将提供一幅完整的图景，揭示膜蛋白的功能特性。为了进行化学标记和电喷雾质谱(ESI-MS)分析，我们将购买激光设备，与我们现有的质谱仪连接，然后开发可应用于各种膜蛋白的可靠方案。在这项开发工作中，我们选择了两种不同的蛋白质，每种蛋白质都来自两大类膜蛋白中的一种，全α-螺旋和β-桶，我们在处理这两种蛋白质方面有相当的经验。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.

DOI：
10.1021/acs.analchem.7b01310
发表时间：
2017-09-05
期刊：
Analytical chemistry
影响因子：
7.4
作者：
Calabrese AN;Jackson SM;Jones LN;Beckstein O;Heinkel F;Gsponer J;Sharples D;Sans M;Kokkinidou M;Pearson AR;Radford SE;Ashcroft AE;Henderson PJF
通讯作者：
Henderson PJF

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

使用标签转移光交联剂快速绘制蛋白质相互作用图

DOI：
10.1002/ange.201809149
发表时间：
2018
期刊：
Angewandte Chemie
影响因子：
0
作者：
Horne J
通讯作者：
Horne J

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers.

DOI：
10.1002/anie.201809149
发表时间：
2018-12-17
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
0
作者：
Horne JE;Walko M;Calabrese AN;Levenstein MA;Brockwell DJ;Kapur N;Wilson AJ;Radford SE
通讯作者：
Radford SE

Using hydroxyl radical footprinting to explore the free energy landscape of protein folding.