The role of closely-associated lipids in membrane protein structure and function

密切相关的脂质在膜蛋白结构和功能中的作用

• 批准号: BB/R018561/1
• 负责人: Stephen Muench
• 金额: $ 61.19万
• 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: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR018561%2F1
• 关键词: role; closely; associated; lipids; membrane

All cells within living organisms require membranes to function, as they act as a barrier between the cell and the outside environment and also define specific compartments within the cell. To allow small and large molecules to enter and leave the cell, and allow the cell to talk to its neighbours, the membranes also contain proteins that function as receptors, transporters and channels. Membrane proteins are responsible for a broad range of diseases and often act to import and export drugs within the cell. Their importance is highlighted with 60% of therapeutic targets being membrane proteins; however, our understanding of their structure and function lags significantly behind their soluble counterparts. One significant hurdle in studying membrane proteins is that they are rarely stable outside the membrane environment. Therefore, we need to use mimics of the membrane, for example detergents to stabilise the proteins. These membrane mimics are often a crude approximation of the native environment, and membrane proteins do not behave as they would in the native membrane. One reason is that the lipids that make up the membrane are also involved in the function and regulation of membrane proteins. Although the lipids in the membrane have been shown to be important for structure, function and regulation of membrane proteins, only in the last few years have technologies been developed that allow us to better study these interactions. This is highly significant within the therapeutic industries as obtaining high quality medicines is often underpinned by a robust and accurate model systems for assays, with structural information aiding in the modification of drug scaffolds. This project has been developed in collaboration with the industrial partners GSK and UCB who will also support the research financially and by providing guidance and access to their facilities. We propose to use cutting edge technology to analyse the lipids associated with specific membrane proteins (mass spectrometry), conduct structural studies (electron microscopy) and use new membrane protein stabilising scaffolds. This will allow us to ask a number of important questions. The first is how do different methodologies for extracting membrane proteins from their native membrane affect their stability and function? We will next address how the different ways of stabilising membrane proteins may affect the structure and also the efficiency by which a reliable structure can be obtained by electron microscopy. The third question will address which lipids are found tightly associated with membrane proteins and do these lipids change with different expression and extraction methods? These three important questions will be answered using three model systems that represent a GPCR receptor (A2A) that allows the cell to respond to external stimuli, a transporter (AcrB) that exports, amongst other things, antibiotics and is involved in antibiotic resistance and a channel that transports potassium ions and is involved in cell homeostasis (BK channel). By using a broad set of exemplar membrane proteins we can start to understand if the observations we see are unique to a certain family of proteins or translates across a broader area into a range of membrane proteins. To assist with the work the team consists of experts in membrane protein biochemistry, electron microscopy, mass spectrometry, pharmacology and tools for extracting membrane proteins. Moreover, we are collaborating with two major pharmaceutical companies, GlaxoSmithKline and UCB, which highlights the importance of this work not just within the academic field, but also in industry. The industrial support will allow us to translate our research to a very broad audience as it has implications on not just our fundamental understanding of membrane proteins but also in producing more efficient structural biology pipelines and more robust and accurate downstream assays, facilitating the drug design process.

生物体内的所有细胞都需要膜来发挥作用，因为它们充当细胞与外部环境之间的屏障，并且还定义了细胞内的特定隔室。为了允许小分子和大分子进入和离开细胞，并允许细胞与其邻居交谈，膜还包含作为受体，转运蛋白和通道的蛋白质。膜蛋白是导致多种疾病的原因，并且经常在细胞内输入和输出药物。它们的重要性突出了60%的治疗靶点是膜蛋白;然而，我们对它们的结构和功能的理解明显落后于它们的可溶性对应物。研究膜蛋白的一个重要障碍是它们在膜环境外很少稳定。因此，我们需要使用膜的模拟物，例如清洁剂来稳定蛋白质。这些膜模拟物通常是天然环境的粗略近似，并且膜蛋白不像它们在天然膜中那样表现。原因之一是构成膜的脂质也参与膜蛋白的功能和调节。虽然膜中的脂质已被证明对膜蛋白的结构，功能和调节很重要，但只有在过去几年中才开发出技术，使我们能够更好地研究这些相互作用。这在治疗行业中非常重要，因为获得高质量的药物通常由用于测定的稳健且准确的模型系统支撑，其中结构信息有助于药物支架的修饰。该项目是与工业合作伙伴GSK和UCB合作开发的，他们也将在财政上支持研究，并提供指导和使用他们的设施。我们建议使用尖端技术来分析与特定膜蛋白相关的脂质（质谱法），进行结构研究（电子显微镜）并使用新的膜蛋白稳定支架。这将使我们能够提出一些重要的问题。第一个问题是，从天然膜中提取膜蛋白的不同方法如何影响其稳定性和功能？接下来，我们将讨论稳定膜蛋白的不同方法如何影响结构，以及通过电子显微镜获得可靠结构的效率。第三个问题将解决哪些脂质被发现与膜蛋白紧密相关，这些脂质是否会随着不同的表达和提取方法而变化？这三个重要的问题将使用三个模型系统来回答，这三个模型系统代表允许细胞对外部刺激做出反应的GPCR受体（A2 A），输出抗生素并参与抗生素耐药性的转运蛋白（AcrB）以及转运钾离子并参与细胞稳态的通道（BK通道）。通过使用一组广泛的示例性膜蛋白，我们可以开始了解我们看到的观察结果是否是某个蛋白质家族所独有的，或者是否可以在更广泛的领域转化为一系列膜蛋白。 为了协助这项工作，该小组由膜蛋白生物化学、电子显微镜、质谱、药理学和提取膜蛋白的工具方面的专家组成。此外，我们正在与葛兰素史克和UCB两家主要制药公司合作，这不仅突出了这项工作在学术领域的重要性，而且在工业中也是如此。工业支持将使我们能够将我们的研究转化为非常广泛的受众，因为它不仅对我们对膜蛋白的基本理解有影响，而且还可以产生更有效的结构生物学管道和更强大和准确的下游分析，促进药物设计过程。

项目成果

Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis.

• DOI: 10.1038/s42003-021-02834-3
• 发表时间: 2021-11-25
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Higgins AJ;Flynn AJ;Marconnet A;Musgrove LJ;Postis VLG;Lippiat JD;Chung CW;Ceska T;Zoonens M;Sobott F;Muench SP
• 通讯作者: Muench SP