Wainwright Vickers, Sarah Rose; (2024) Conformational Dynamics and Protein Folding/Misfolding of Alpha 1 Antitrypsin Studied by State of the Art Mass Spectrometry Techniques. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Alpha 1 antitrypsin is a protein, synthesised in the liver, primarily responsible for the regulation of proteases in the lungs. Its mechanism of action involves a large conformational change which results in the permanent inhibition of elastase. This mechanism is reliant on the thermodynamic instability balanced with the kinetic stability of the native conformation of alpha 1 antitrypsin, which during inhibition of its target protease forms a thermodynamically hyperstable conformation. Due to the metastability of the native conformation of alpha 1 antitrypsin, it is vulnerable to misfolding and aggregation when destabilised, typified by the Z alpha 1 antitrypsin variant that accumulates within hepatocytes, causing liver disease. The polymerisation of alpha 1 antitrypsin has been investigated since it was first linked to the alpha 1 antitrypsin deficiency and associated liver diseases. Several models for the progression of structural changes in the formation of polymers have been presented, but there is a need for further understanding of the conformational dynamics involved in alpha 1 antitrypsin’s misfolding. Ion mobility mass spectrometry is a technique well-suited to the study of misfolding of proteins due to its capabilities to resolve complex mixtures that are conformationally heterogenous. This thesis reports the development of methods using ion mobility mass spectrometry techniques coupled to collisional activation and electron capture dissociation for the study of protein misfolding. These methods were applied to understand the misfolding pathway of alpha 1 antitrypsin, where a novel misfolding intermediate was identified and ex-vivo samples from patients suffering from alpha 1 antitrypsin associated liver disease. Overall, the data presented aids our understanding of the misfolding of alpha 1 antitrypsin and presents methods that could be expanded to the plethora of protein misfolding diseases.

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