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

我们将研究蛋白质α1-抗胰蛋白酶(A1AT)的构象动力学和寡聚作用。我们将详细研究A1AT序列变体的影响，并详细描述糖基化模式及其对构象/寡聚的影响。因此，该项目完全符合DTP的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外，我们还将使用新型循环离子迁移率质谱仪(CIMMS)来研究这些过程。伦敦大学学院是世界上第三个于2019年12月安装CIMMS的地方，Thalassinos实验室也是世界上第一个发表手稿展示CIMMS如何用于研究天然蛋白质结构的地方(Eldrid等人)。Thalassinos实验室很早就接触到了这项新技术，这是由之前的一名博士生促成的，清楚地表明了这些类型的学生对将尖端技术带给学术界的重要性。蛋白质A1AT是最丰富的循环蛋白酶抑制物。它是在肝细胞内质网中合成的，其关键靶点是中性粒细胞弹性蛋白酶。A1AT的突变导致它聚合，功能效应既有损失，也有增加。聚集在肝脏中的聚合物会导致肝硬变，而循环中A1AT的缺乏会使肺暴露于不受控制的弹力酶活性，从而容易患上肺气肿。糖基化对蛋白质构象和聚集倾向的影响到目前为止还没有详细的研究。A1AT有三个糖基化位点，含有复杂的N-糖链结构。一个主要的挑战是糖基化模式的异质性，以及每个位点上多个可能的多糖占有率。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了集合平均值。相反，离子迁移质谱(IMMS)是详细研究非均相样品和分离特定构象以便进一步研究的一种很好的方法。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进行进一步的更高分辨率(串联离子迁移率)构象分析，这是任何其他商业IMMS仪器都不可能做到的。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。我们还将利用它来以前所未有的细节探索附着的多糖的性质。IMMS可以从极少量的物质中分离出葡聚糖异构体，这对于生物学研究是可取的，特别是那些涉及患者材料的研究。学生将在洛马斯实验室接受A1AT样品分离、制备和生物物理表征方面的培训，并在Thalassinos实验室接受IMMS使用方面的培训。参观Waters将提供有关使用CIMMS设备的进一步培训，以及Wilmlow Waters目前正在开发的其他尖端技术。我们预计任何发现和开发的方法都将适用于其他构象疾病，这些疾病的发病率因蛋白质稳态的破坏而在老龄化人口中增加，到目前为止，这些疾病很难通过其他现有技术进行研究。

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.