Title: Conformational dynamics and protein folding/misfolding of alpha1-antitrypsin studied by state-of-the-art tandem ion mobility mass spectrometry

标题：通过最先进的串联离子淌度质谱法研究 α1-抗胰蛋白酶的构象动力学和蛋白质折叠/错误折叠

• 批准号: 2414124
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2414124
• 关键词: Title; Conformational; dynamics; protein; folding

We will study the conformational dynamics and oligomerisation of the protein alpha1-antitrypsin (A1AT). We will examine in detail the effect of A1AT sequence variants and also characterise in detail the glycosylation patterns and the effect they have on conformation/oligomerisation. This project is therefore completely aligned with the fundamental mechanisms of disease theme of the DTP. In addition to using more established biochemical and biophysical characterisation, we will study such processes using a novel cyclic ion mobility mass spectrometer (cIMMS). UCL is the 3rd place in the world to have a cIMMS installed, in Dec 2019, and the Thalassinos lab has been the first worldwide to publish a manuscript showcasing how the cIMMS can be used to study native protein structure (Eldrid et al.). This very early access of the Thalassinos lab to this novel technology was facilitated by a previous CASE PhD student, clearly showcasing the importance of these types of studentships for bringing cutting edge technology to academia.The protein A1AT is the most abundant circulating protease inhibitor. It is synthesised in the hepatocyte ER and its key target is the enzyme neutrophil elastase. Mutations in A1AT cause it to polymerise, with both loss and gain of function effects. Polymers accumulated in the liver cause cirrhosis while the lack of circulating A1AT exposes the lungs to uncontrolled elastase activity, predisposing to emphysema. The effect of glycosylation on protein conformation and aggregation propensity has so far not been studied in detail. A1AT has three glycosylation sites containing complex N-glycan structures. A major challenge is the heterogeneity in the glycosylation patterns in addition to multiple possible glycan occupancy at each site. This poses a challenge to established structural and biophysical methods as almost all of them report on ensemble averages. Ion mobility mass spectrometry (IMMS), on the contrary, is an excellent method for studying in detail heterogeneous samples and for isolating particular conformers for further study. The new cIMMS device not only offers great improvements into the resolving power compared to previous IMMS instrumentation but also due to its unique design will allow us to isolate closely related conformers for further higher resolution (tandem ion mobility) conformational analysis, something not possible with any other commercial IMMS instrumentation.We will use the cIMMS to both analyse the effect of different glycosylation patterns on A1AT protein conformation, dynamics and propensity to oligomerise. We will also use it to probe in unprecedented detail the nature of the attached glycans. IMMS can separate glycan isomers from very low amounts of material, something desirable for biological studies, especially those involving patient material.The student will be trained in A1AT sample isolation, preparation and biophysical characterisation in the Lomas lab and in the use of IMMS in the Thalassinos lab. Visits to Waters will provide further training in the use of the cIMMS devise but also in additional cutting-edge technology currently under development at Waters in Wilmslow.We anticipate any findings, and methods developed, to be applicable to other conformational diseases whose incidence increases in ageing populations due to breakdown of protein homeostasis and which have so far been intractable to study by other established techniques.

我们将研究蛋白α1-抗胰蛋白酶（A1AT）的构象动力学和寡聚化。我们将详细研究A1AT序列变体的效果，并详细介绍糖基化模式及其对构象/寡聚化的影响。因此，该项目与DTP疾病主题的基本机制完全一致。除了使用更具成熟的生化和生物物理特征外，我们还将使用新型的环状离子迁移率质谱仪（CIMM）研究此类过程。 UCL是世界上第三名，在2019年12月安装了CIMMS，Thalassinos实验室是全球首次发布手稿，展示了如何使用CIMM来研究天然蛋白质结构（Eldrid等人）。塔拉西诺斯实验室对这项新技术的这种早期访问是由以前的案例博士生促进的，这清楚地表明了这些类型的学生对将尖端技术带入学术界的重要性。蛋白质A1AT是最丰富的循环蛋白酶抑制剂。它是在肝细胞中合成的，其主要靶标是中性粒细胞弹性酶。 A1AT中的突变导致其聚合，并具有功能效应的损失和增益。积累在肝脏中的聚合物会引起肝硬化，而缺乏循环A1AT则暴露于肺部不受控制的弹性酶活性，易于肺气肿。到目前为止，糖基化对蛋白质构象和聚集倾向的影响尚未详细研究。 A1AT具有三个含有复杂N-聚糖结构的糖基化位点。一个主要的挑战是，除了每个位点多种可能的聚糖占用率外，糖基化模式的异质性。这对建立的结构和生物物理方法构成了挑战，因为几乎所有这些方法都报告了整体平均值。相反，离子迁移率质谱法（IMM）是一种详细研究异质样品和分离特定构象异构体进行进一步研究的极好方法。 The new cIMMS device not only offers great improvements into the resolving power compared to previous IMMS instrumentation but also due to its unique design will allow us to isolate closely related conformers for further higher resolution (tandem ion mobility) conformational analysis, something not possible with any other commercial IMMS instrumentation.We will use the cIMMS to both analyse the effect of different glycosylation patterns on A1AT protein conformation, dynamics and propensity to寡聚。我们还将使用它来探测所附的聚糖的性质。 IMM可以将聚糖异构体与非常低的材料分开，这是生物学研究的理想选择，尤其是涉及患者材料的材料。将在Lomas Lab中对A1AT样品隔离，制备和生物物理表征进行培训，并在Thalassinos实验室中使用IMMS。参观沃特斯将提供进一步的培训CIMMS设计，还可以在Wilmslow的Waters目前正在开发的其他最先进的技术中，我们预计，我们预计任何发现和开发的方法适用于其他构型疾病，这些构型疾病的发病率在衰老人群中增加了由于蛋白质稳态而导致的蛋白质稳态导致的衰老人群的增加，并且这些构型疾病的衰老疾病及其在其他建立的技术中都可以使这些技术可行。