Establishing A Reference State For Studying The Aggregation Kinetics Of Polyglutamine Containing Systems

Nine different neurodegenerative diseases including Huntington's disease are associated with the aggregation of proteins containing polyglutamine expansions. Studies of homopolymeric polyglutamine have been important for understanding how and why polyglutamine should be prone to aggregation. There is currently a discrepancy between conclusions from theory and computation versus interpretations of experimental data for homopolymeric polyglutamine. We propose that this discrepancy originates in the effects of intermolecular electrostatic interactions from the flanking lysines in constructs of the form KKQNKK, which are most commonly used for in vitro experiments on polyglutamine. Although the lysines are used to enhance polyglutamine solubility, the working assumption is that they do not alter polyglutamine conformations or aggregation mechanisms. We have performed systematic tests on the effects of flanking lysines on aggregation mechanisms of polyglutamine. Data from a collection of independent experiments based on fluorescence anisotropy, atomic force microscopy, thioflavin T fluorescence, and light scattering provide unequivocal evidence supporting the strong modulating effects of lysines on the mechanisms of polyglutamine aggregation. The extent and nature of modulation depends on polyglutamine length and the number of lysines. Borrowing from the colloidal literature, it appears that the lysines convert the mechanism of polyglutamine aggregation from being diffusion-limited aggregation (DLA) to reaction-limited aggregation (RLA). We were able to unmask the effects of lysines on polyglutamine aggregation with quantitative studies on the effect of titration of mono/divalent phosphate anions on aggregation rates and aggregate morphologies. This work is leading toward a convergent reference state for the aggregation of homopolymeric polyglutamine. This is helping us redefine the role of biologically relevant flanking sequences on polyglutamine aggregation and in controlling aggregation-related neurodegeneration. Our studies suggest that naturally occurring flanking sequences protect functionally relevant, aggregation-prone polyglutamine-rich tracts from aggregation.