(308a) Elongation Kinetics of Fibrillar Polyglutamine Aggregates

Repeat-containing domains are remarkably common in eukaryotes. These domains are frequently natively disordered and play important roles in cellular processes such as transcription, signaling, and formation of multi-protein networks. The most common residues in repeat-containing proteins include glutamine, alanine and (in invertebrates), asparagine. In some cases, expansion of the repeat domain beyond normal length occurs and is associated with a number of disorders; the best known example of this is polyglutamine expansion in Huntington’s disease. Polyglutamine expansion leads to abnormal self-assembly; aggregate formation is causally linked to the neuronal degeneration characteristic of Huntington’s. This link between polyglutamine expansion, aggregation, and disease motivates our detailed examination of the mechanism and kinetics of polyglutamine-mediated aggregation.

Here we present results in which extension of pre-existing polyglutamine fibrils was explored using two techniques: quartz crystal microbalance with dissipation (QCM-D) and online waveguide spectroscopy (OWLS). Polyglutamine fibrils were immobilized on sensor surfaces, and the adsorption of monomeric polyglutamine-containing peptides was monitored over time. We observed a strong length dependence of elongation: peptides containing only 8 glutamine (Q8) associated only weakly and reversibly, while Q20 and Q24 associated to a much larger extent, and association was only partially reversible. We tested the effect of di-amino acid insertions between two Q10 blocks: one insertion (PG) serves as a beta-turn template whereas another insertion (PP) enforces local rigidity. Association rates, and particularly reversibility, were strongly affected by monomer conformation. Specifically, the PG domain increased the association rate and reduced dissociation, whereas PP had the opposite effect. Dissipation measurements provide additional insight into the elongation process via characterization of the viscoelastic properties of the adsorbed layer.

QCM-D measurement of the adsorbed mass includes not only the peptide itself but also any associated water. For comparison, we measured adsorption rates of monomer to immobilized polyglutamine fibrils using OWLS, which measures only peptide mass. The difference between QCM-D and OWLS mass measurements is a direct measure of associated water. We observed that when Q20 added to the fibrils, it brought with it a significant amount of water that remained associated with the fibrils over time. Peptides containing the PG beta-turn template carried less water. Interestingly, after peptide addition was stopped, we observed a further loss of water from the elongating fibrils. We speculate that this is direct observation of fibril maturation, as the beta-sheet propagates through the fibril end and water is expelled. Taken together, our data support an ‘association-conformational conversion’ mechanism of fibril elongation.