Exploring magnetically aligned bilayers as a novel tool for membrane protein crystallisation

探索磁性排列双层作为膜蛋白结晶的新工具

• 批准号: BB/R021759/1
• 负责人: Ioannis Vakonakis
• 金额: $ 19.2万
• 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: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR021759%2F1
• 关键词: Exploring; magnetically; aligned; bilayers; novel

Membrane-embedded proteins (or membrane proteins) are amongst the most influential for the survival, correct behaviour and function of cells. They form the means by which cells interact with their environments, by which they import nutrients and expel potentially poisonous molecules, and communicate with each other. In humans, membrane proteins comprise almost a third of all known proteins, and over half of the currently available drugs act on membrane proteins. Thus, studying the roles of membrane proteins and how they function is a pursuit shared by both academic research scientists and the pharmaceutical industry alike.A powerful means for studying the role and function of proteins is visualising their three-dimensional shape with enough detail to distinguish individual chemical groups and atoms. Such analysis allows us to understand the purpose of each protein component as if we were observing a machine, and to envision ways of assisting or disrupting its mechanism that can then be translated into drugs and therapies for diseases. X-ray crystallography is the premier method by which we visualise proteins at this level of detail; however, this method requires the formation of highly ordered crystals where protein molecules pack against each other in a predictable and regular manner. Due to the fact that membrane proteins need to be extracted from their natural membrane environment in order to be crystallised, they are often damaged and therefore they do not easily form crystals. It is for this reason that, despite their enormous importance in living organisms, membrane proteins make up a very small proportion, less than 2%, of proteins for which the detailed shape is known. Thus, developing novel tools that induce membrane proteins to form crystals could tremendously expand our detailed understanding of cellular mechanisms.Traditionally, membrane proteins were isolated and handled in the presence of soap-like detergent molecules; however, such detergents make the proteins less likely to function correctly or to form crystals. For this reason, researchers have been developing advanced methods that provide a more membrane-like environment for the proteins during crystallisation, e.g. by the addition of lipids that are similar to those in the cell membrane. When membrane proteins do crystallise through these methods the crystals often consist of spontaneously formed stacks of lipid bilayers, an arrangement that vaguely resembles the situation the protein would encounter in the membrane of a living cell. In this proposal we aim to develop a new method that assists the regular packing of membrane proteins in such stacked lipid bilayers, and thereby increases the probability that they form crystals. To do so we will utilise strong superconducting magnets, which are known to impose order in membranes by forcing their orientation to follow the direction of the magnetic field. We hypothesise that in this way membrane-embedded proteins will also be forced toward a particular direction, and this spatial restriction may induce them to pack more readily into crystals. Should this magnetic alignment crystallisation ('MAX') approach prove successful, we aim to further develop the use of magnets in the crystallisation of membrane proteins into a tool widely available in the academic community and industry.

膜包埋蛋白（或膜蛋白）是对细胞的存活、正确行为和功能最有影响的蛋白之一。它们形成了细胞与环境相互作用的方式，通过这种方式，它们输入营养物质并排出潜在的有毒分子，并相互交流。在人类中，膜蛋白几乎占所有已知蛋白质的三分之一，目前可用的药物中有一半以上作用于膜蛋白。因此，研究膜蛋白的作用及其功能是学术研究科学家和制药行业共同追求的目标。研究蛋白质作用和功能的一种强大手段是可视化其三维形状，并具有足够的细节以区分单个化学基团和原子。这种分析使我们能够像观察机器一样了解每个蛋白质组分的目的，并设想如何协助或破坏其机制，然后将其转化为药物和治疗疾病的方法。X射线晶体学是我们在这种细节水平上可视化蛋白质的首要方法;然而，这种方法需要形成高度有序的晶体，其中蛋白质分子以可预测和规则的方式相互堆积。由于膜蛋白需要从其天然膜环境中提取以结晶的事实，它们经常被损坏，因此它们不容易形成晶体。正是由于这个原因，尽管膜蛋白在生物体中具有巨大的重要性，但它们在已知详细形状的蛋白质中所占的比例很小，不到2%。因此，开发新的工具，诱导膜蛋白形成晶体可以极大地扩展我们的细胞机制的详细理解。传统上，膜蛋白分离和处理的肥皂样洗涤剂分子的存在下，然而，这样的洗涤剂使蛋白质不太可能正确的功能或形成晶体。出于这个原因，研究人员一直在开发先进的方法，在结晶过程中为蛋白质提供更像膜的环境，例如通过添加与细胞膜中类似的脂质。当膜蛋白通过这些方法结晶时，晶体通常由自发形成的脂质双层堆叠组成，这种排列与蛋白质在活细胞膜中遇到的情况大致相似。在这项提案中，我们的目标是开发一种新的方法，帮助膜蛋白在这种堆叠的脂质双层中的规则包装，从而增加它们形成晶体的可能性。为了做到这一点，我们将利用强超导磁体，众所周知，这种磁体通过迫使膜的方向遵循磁场的方向来在膜中施加秩序。我们假设，以这种方式，膜包埋的蛋白质也将被迫向一个特定的方向，这种空间限制可能会导致他们更容易包装成晶体。如果这种磁性排列结晶（“MAX”）方法被证明是成功的，我们的目标是进一步开发磁铁在膜蛋白结晶中的应用，使其成为学术界和工业界广泛使用的工具。

项目成果

Structures of the Plasmodium falciparum heat-shock protein 70-x ATPase domain in complex with chemical fragments identify conserved and unique binding sites.

• DOI: 10.1107/s2053230x21007378
• 发表时间: 2021-08-01
• 期刊: Acta crystallographica. Section F, Structural biology communications
• 作者: Mohamad N;O'Donoghue A;Kantsadi AL;Vakonakis I
• 通讯作者: Vakonakis I