Amyloid Fibril Formation - Dye Detection and Effects of Lipids
Amyloid fibrils are long fibrillar homopolymers of self-assembled proteins. They can be formed by essentially any polypeptide, but are of particular interest because of their occurrence in several incurable and debilitating human diseases, e.g. Alzheimer’s disease (AD). A central pathological event in AD is build-up of amyloid fibrils by the amyloid-β (Aβ) peptide. This thesis describes my work in the field of biochemistry and biophysics to understand the molecular mechanisms and events that lead to amyloid fibril formation in general, and Aβ assembly in particular.
One of the most widely used fluorescent dyes in amyloid research is thioflavin-T (ThT); despite this, its fibril-binding and fluorescent properties are not completely understood at a molecular level. In this thesis, I show that the binding of ThT to fibrils, and consequently its fluorescence, can vary depending on fibril morphology, and that ThT exhibits significant self-quenching at high binding density. These findings are important contributions to the understanding of how ThT functions as an amyloid dye, and can therefore help in the experimental design of biophysical assays involving ThT fluorescence. In addition, I also detail the use of YOYO-1 for amyloid fibril detection.
Biogenesis of Aβ occurs in and around lipid-rich environments, and lipids have been proposed to have a central role in AD pathology. In this thesis I have investigated fibril-lipid interactions. I show that DOPC lipid vesicles augment Aβ fibril formation, and that they do so through interaction with growing fibrils, thereby reinforcing secondary mechanisms such as fibril fragmentation and secondary nucleation. This demonstrates how, on a molecular level, lipids may contribute to the pathogenesis of AD.
Finally, I have studied amyloid fibril formation by the hormone insulin, a blood-glucose regulating protein used in treatment of diabetes. I demonstrate that mildly acidic solutions and presence of alcohols as co-solvents has a retarding effect on fibril formation. This has strong implications on the purification and long-term stability of pharmaceutical insulin.
atomic force microscopy