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    The self-assembly of Glucagon


    Joyce, Patrick (2024) The self-assembly of Glucagon. PhD thesis, National University of Ireland Maynooth.

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    Abstract

    Glucagon is a peptide that is known to undergo self-assembly at acidic pH resulting in fibrillar structures. While much is known about the morphology and kinetics in relation to the formation of these fibrillar structures, there is relatively little known about the self-assembly pathways that lead to these end state structures. Small changes in solution conditions are known to have dramatic effects on self-assembly of peptides and this is the case for Glucagon. Glucagon has been shown to assemble via two competing pathways at acidic pH but the complexity of this assembly does not rest solely on the competition between the two assembly pathways. Oligomeric intermediates play a pivotal role in Glucagon’s fibrillogenesis at acidic pH. To date, there has been no detailed description of the changes in the oligomeric species that are present during the lag-phase for fibrillating Glucagon at acidic pH. This thesis aims to address this shortcoming by providing a detailed description of Glucagon’s lag-phase at pH 2.5. Here an analysis of the lag-phase for Glucagon at pH 2.5 is presented using ThT assays, SE-HPLC, SDS-Page, Taylor Dispersion Analysis (TDA), Static Light Scattering (SLS) and Dynamic Light Scattering (DLS). Kinetic measurements from ThT assays demonstrated a reasonably well-defined lag-time for an assembly process that is fundamentally stochastic. Results using multiple techniques (SE-HPLC, SDS-Page, TDA and Light Scattering) showed that the starting point for the assembly process was mostly monomeric but had a small proportion of oligomeric content. The effectiveness of various analytical techniques was examined by monitoring assembly during Glucagon’s lag-phase at the level of oligomer. It was found that while TDA was somewhat effective at monitoring changes in oligomerization, techniques such as SE-HPLC were shown to be ineffective in describing Glucagon’s lag-phase at the level of oligomer. This thesis also demonstrates that SLS and DLS are the best techniques to examine Glucagon’s lag-phase at the level of oligomer compared to the other techniques used. This thesis showed how SLS data can be deconvoluted into a 5-component distribution of the species that are present in the self-assembling system and how this allows for mechanistic insights into Glucagon’s assembly process during the lag-phase. Changes in the distributions of the oligomeric content were shown during the assembly process for Glucagon at pH 2.5. Analysing changes to these distributions a plausible description of the oligomeric distribution of the critical nucleus that precedes fibril formation was deduced. This thesis shows how a shift in the proportion of oligomers between two low order populations coincides with the nucleation step with the emerging population providing a size distribution for the critical nucleus. The kinetics for the self-assembling system was analysed and the most transient species occurring during the aggregation reaction were identified. The kinetic description allowed for the identification of the most likely rate limiting step for the aggregation reaction. Glucagon’s self-assembly at pH 3.6 was studied using acetate buffers. Kinetic experiments from ThT assays demonstrated that variation of both ionic strength and the buffering cation profoundly affected Glucagon’s self-assembly. TEM data showed that stable amorphous aggregates can form under low ionic strength conditions that are not precursors to any fibril formation. The ThT fluorescent probe was shown to accelerate the assembly of these amorphous aggregates. At higher ionic strengths Glucagon was shown to fibrillate at pH 3.6 Specific interactions with fibrillating Glucagon at pH 3.6 were observed for acetate buffers using sodium, potassium, and magnesium cations, leading to varying degrees of peptide stabilization.
    Item Type: Thesis (PhD)
    Keywords: self-assembly; Glucagon;
    Academic Unit: Faculty of Science and Engineering > Chemistry
    Item ID: 19954
    Depositing User: IR eTheses
    Date Deposited: 06 Jun 2025 14:53
    URI: https://mural.maynoothuniversity.ie/id/eprint/19954
    Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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