Abstract by Hussein Forsberg Chaaban
The self-assembly mechanisms of proteins are implicated in various diseases, serve as obstacles to the development of protein therapeutics and have recently been exploited as biomaterials. Several factors (protein concentration, pH, temperature, additives, ions) can affect the in vitro aggregation process, leading to various aggregate species with different physical and structural properties. Ions are found in a majority of living organisms, and industrially ions are commonly used to stabilize proteins in drug production. Nonetheless, the ion-specific effect on the overall mechanism of action of ions on the stability and aggregation of proteins remain unknown. Thus, it is imperative to understand how different ions affect the different steps of the aggregation mechanism, from the stability of the native protein to the formation of the final aggregate.
In this thesis, we aim to investigate the role of five Hofmeister anions on the aggregation of human insulin (HI) and Hen egg white lysozyme (HEWL) by: 1) Unveiling how the Hofmeister anions affect the balance of colloidal and conformational pathways for the two proteins and 2) probing a library of conditions leading to morphologically and structurally different aggregates.
Firstly, the anion effect on native insulin and lysozyme stability was investigated (Appendix III) by in vitro and in silico approaches. An ion-specific oligomerization of insulin was observed when the different anions were added, suggesting that the anions have altered the aggregation mechanism. In contrast, the anions had a minimal effect on lysozyme, highlighting the importance of the specific ion-protein interaction on the stability of the native protein.
Secondly, we investigated the aggregation process of the two proteins from the aggregation kinetics to the final morphology (Appendix I). Using a microscopy approach covering nm and µm length scales, we presented an amyloid phase diagram for HI and HEWL. Colloidal instability governs the formation of microparticles, while conformational instability governs the formation of amyloid fibrils and spherulites. Interestingly, both proteins displayed a similar phase diagram.
Lastly, the anion effect was studied in relation to the polymorphism of one specific aggregate, namely amyloid fibrils (Appendix II). The anions altered the morphology of lysozyme fibrils, resulting in straight mature fibrils and worm-like fibrils, indicating two alternative aggregation mechanisms. In contrast, only mature straight fibrils were observed for HI. Unlike the secondary structure observed by bulk measurements, the secondary structure between individual HI fibrils showed a significant difference, ranging from an α-helical structure, a β-sheet structure, or a combination of the two.
In conclusion, this work highlights for the first time the existence of an amyloid phase diagram ruled by the Hofmeister anions for the two model proteins, HI and HEWL, by a multi-microscopy approach covering the nm and µm ranges. We reported that proteins with different stabilities undergo aggregation reactions leading to the same type of aggregates and we reported an nm morphological diversity for HEWL fibril and an anion induced structural polymorphism for HI fibril. In addition, this thesis highlights the complexity of the impact of anion-protein interactions on the aggregation mechanism of proteins.