Ben-Younis, Aisha; (2024) Probing the conformational dynamics of islet amyloid polypeptide by ion mobility - mass spectrometry. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Early oligomerisation of human islet amyloid polypeptide (hIAPP) is responsible for pancreatic β-cell death and is increasingly considered a primary pathological process linked to type II diabetes (T2D). However, the assembly mechanism remains poorly understood, largely due to the inability of conventional techniques to probe either distributions or detailed structures of early oligomeric species. Rat IAPP (rIAPP) differs from the human sequence at just six amino acid positions but is nonamyloidogenic and non-toxic. Here, high resolution cyclic ion mobility – mass spectrometry (cIM – MS) with electron capture dissociation tandem MS (ECD MS/MS) has been applied to capture and characterise the early oligomers of hIAPP and rIAPP and describe the self-assembly region for their dimers. Mutational analyses of a range of IAPP constructs reveal the extreme sequence specificity of amyloidogenicity and cytotoxicity of IAPP, and has identified residue-specific determinants of IAPP aggregation. cIM – MS has shown that the highly amyloidogenic hIAPP and non-amyloidogenic rIAPP dimers display distinct conformational behaviour, correlating to their solution-phase characteristics. Tandem IM was used to reveal gas-phase behaviour not resolvable by single-stage IM, including conformer-specific unfolding and interconversion. High resolution mobility separation multi-pass experiments were performed for specific conformers, revealing distinct “hidden” conformations present for both hIAPP and rIAPP dimeric species. Native top-down MS by ECD MS/MS has identified the dimerisation interface of the earliest IAPP oligomers. Collectively these data provide important insight into why hIAPP proceeds to form β-sheet fibrils and rIAPP does not. The identification of the binding site between hIAPP units could provide a future therapeutic target to prevent formation of further toxic assemblies. Using amyloidogenic hIAPP as a model system, cIM – MS combined with ECD MS/MS has proved a powerful technique for the indepth study of protein conformation within a single experiment and could be applied to many amyloidogenic systems.

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