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

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.