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Title: Characterising the early aggregation events of human lysozyme using single-molecule microscopy
Author: Bernardo Gancedo, Ana
ISNI:       0000 0004 7961 8590
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Human lysozyme (HuL) is a widely characterised protein whose mutational variants misfold, forming amyloid fibrils that are associated with a rare systemic amyloidosis. Given that a number of proteins, including lysozyme, can misfold and give rise to disorders such as Alzheimer's disease, Parkinson's disease, and type II diabetes, it is vital to understand the mechanistic features by which this process occurs, in order to attempt to cure or regulate these diseases. Work on lysozyme and other globular proteins has revealed that they require a process of global or partial unfolding to initiate protein aggregation, which is different from the natively unfolded proteins known as intrinsically disordered proteins (IDPs). Although the processes of folding and fibril formation for HuL have been well studied, the details of the early events leading to aggregation have proven difficult to study. Recent advances in site-specific labelling of HuL have made it possible to introduce Alexa fluorophores into the I59T variant of HuL without perturbing the process of in vitro fibril formation as compared to the unlabelled protein. Using this fluorescently labelled protein, we have used single-molecule fluorescence microscopy techniques including two-colour coincidence detection (TCCD) and fluorescence resonance energy transfer (FRET) to determine the oligomer population distributions present during the aggregation of HuL. Our data showed that HuL populates disordered small low-FRET oligomers (< 10 monomers) which in turn can undergo a conformational change to form more stable and structured high-FRET oligomers over the course of the aggregation. The second type of oligomers can then grow to form amyloid fibrils. We fitted our data from the early stages of the aggregation reaction to an early-time kinetic model, in which the monomer concentration can be assumed constant during the selected period of time. Using this model, we calculated the different rate constants associated with the microscopic steps along the aggregation pathway that we were able to monitor using SM techniques. In addition, the interactions between HuL oligomers and the extracellular chaperone clusterin were investigated. Previous studies have shown that clusterin inhibits I59T HuL aggregation by interacting with species formed early in the aggregation pathway. Here, we show that clusterin enhances the population of small high-FRET oligomers, trapping them and preventing their growth into larger aggregates over the time-course of our analysis. These results allow us to directly compare the mechanism of protein aggregation in a globular system versus that of IDPs, providing potential insight into targeting these pathways for therapeutic interventions.
Supervisor: Dobson, Christopher M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Aggregation ; lysozyme ; single-molecule ; amyloid ; Alexa ; fluorophore ; laser ; clusterin ; oligomer ; human