The aim of this thesis is to investigate and better understand the mechanisms of protein self-assembly. Specifically, I study three protein systems which form morphologically and structurally distinct brillar protein aggregates. The first of these studies is concerned with the self-assembly of amyloid brils formed from bovine insulin. Amyloid brils are associated with human diseases such as Alzheimers Disease and type-2 diabetes, and are also garnering interest in biomaterial applications. Fragmentation-dominated models for the self-assembly of amyloid brils have had important successes in explaining the kinetics of amyloid bril formation but predict bril length distributions that do not match experimental observations. Here I resolve this inconsistency using a combination of experimental kinetic measurements and computer simulations. I provide evidence for a structural transition demarcated by a critical bril mass concentration, or CFC, above which fragmentation of the brils is suppressed. Our simulations predict the formation of distinct bril length distributions above and below the CFC, which I confirm by electron microscopy. These results point to a new picture of amyloid bril growth in which structural transitions that occur during self-assembly have strong effects on the final population of aggregate species with small, and potentially cytotoxic, oligomers dominating for long periods of time at protein concentrations below the CFC. I further show that the CFC can be modulated by environmental conditions, pointing to possible in vivo strategies for controlling cytotoxicity. I probe the structural nature of the transition by performing small angle neutron scattering. Secondly, I study the formation of amyloid-like brils from the protein ovalbumin. I undertake kinetic experiments of self-assembly and find two key features emerge: the lack of a lag time and the existence of a slow growth regime in the long-time limit. I observe, using TEM, that these brils are worm-like in nature and form closed-loops. I find the growth kinetics are intimately connected to this particular morphology. I present a simple kinetic model which captures the features of the kinetics found in experiments by incorporating end-to-end association of brils. I comment on the ramifications this type of amyloid bril assembly may have on oligomeric toxicity. Thirdly, the DNA-mimic protein ocr is highly charged (-56e at pH 8) and forms non-amyloid brillar assemblies at very high ammonium sulphate concentrations (3.2M). The fact that ocr forms translucent brillar gels at such high salt concentrations is extremely unique. Typically under such high salt conditions, non-specific amorphous aggregates are formed. In order to better understand the mechanism of why ocr forms specific bril aggregates, I used variants of the wile-type protein in which extensive regions of surface have been removed or modified. The structural characteristics of gels formed from the variants were probed using microrheological techniques. I find that non-specific electrostatic charge screening plays an important role in ocr aggregation. However, I also locate a potentially important α-helical region which may play a part in establishing specific interactions so that ocr may form ordered brillar assemblies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:615346 |
| Date | January 2013 |
| Creators | Ryan, Morris |
| Contributors | Macphee, Cait; Allen, Rosalind; Poon, Wilson |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/8878 |
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