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

我们将研究α - 1抗胰蛋白酶（A1AT）蛋白的构象动力学和寡聚化。我们将详细研究A1AT序列变异的影响，并详细描述糖基化模式及其对构象/寡聚化的影响。因此，该项目完全符合DTP疾病主题的基本机制。除了使用更成熟的生化和生物物理表征，我们将使用一种新型的循环离子迁移质谱（cIMMS）来研究这些过程。UCL是世界上第三个安装cIMMS的地方，于2019年12月安装，Thalassinos实验室是全球第一个发表手稿的地方，展示了如何使用cIMMS来研究天然蛋白质结构（Eldrid等人）。Thalassinos实验室很早就接触到了这项新技术，这是由CASE之前的一名博士生促成的，这清楚地展示了这类学生为学术界带来尖端技术的重要性。A1AT蛋白是最丰富的循环蛋白酶抑制剂。它在肝细胞内质网中合成，其关键靶点是中性粒细胞弹性酶。A1AT的突变导致其聚合，同时丧失和获得功能效应。聚合物在肝脏中积累导致肝硬化，而循环A1AT的缺乏使肺部暴露于不受控制的弹性酶活性，易患肺气肿。糖基化对蛋白质构象和聚集倾向的影响迄今尚未得到详细研究。A1AT有三个糖基化位点，含有复杂的n -聚糖结构。一个主要的挑战是糖基化模式的异质性以及每个位点上可能存在的多个聚糖占用。这对现有的结构和生物物理方法提出了挑战，因为几乎所有这些方法都报告集合平均值。相反，离子迁移率质谱（IMMS）是详细研究非均质样品和分离特定构象以作进一步研究的一种极好的方法。与以前的IMMS仪器相比，新的cIMMS设备不仅在分辨率上有了很大的提高，而且由于其独特的设计，我们可以分离出密切相关的构象，以获得更高的分辨率（串联离子迁移率）构象分析，这是任何其他商用IMMS仪器都无法做到的。我们将使用cIMMS来分析不同糖基化模式对A1AT蛋白构象、动力学和寡聚倾向的影响。我们还将用它以前所未有的细节来探测所附聚糖的性质。IMMS可以从非常少量的物质中分离出聚糖异构体，这对于生物学研究来说是非常理想的，特别是那些涉及患者材料的研究。该学生将在Lomas实验室接受A1AT样品分离、制备和生物物理表征的培训，并在Thalassinos实验室接受IMMS的使用培训。参观沃特斯将提供使用cIMMS设计的进一步培训，以及目前正在Wilmslow沃特斯开发的其他尖端技术。我们期望任何发现和开发的方法都适用于其他构象疾病，这些疾病由于蛋白质稳态的破坏而在老龄化人口中发病率增加，并且迄今为止难以通过其他既定技术进行研究。

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.