(750g) Kinetics of Aggregation of Polyglutamine and Polyalanine Peptides

Nucleotide repeat elements are commonplace in the human genome. Trinucleotide repeats are one type of these elements, which appear in both coding and non-coding regions; when in coding regions, the trinucleotide repeats produce proteins with homo-aminoacid repeat tracts. Several proteins with such repeat tracts have been linked to neurodegenerative disorders. Best known are the expanded CAG disorders such as Huntington's disease, which result in proteins with abnormally long polyglutamine (polyQ) domains. A broad polymorphism of polyQ length is observed; the length is inversely correlated with the age of onset and severity of symptoms. In these polyglutamine diseases, nuclear and cytoplasmic aggregates are observed and it is believed that aggregation is related to toxicity. Less common, although still observed, are proteins containing expanded polyalanine domains. Because the common feature of these disease-related proteins is the abnormally long homo-aminoacid repeat, there is considerable interest in understanding the folding and aggregation properties of this domain. In this light, we have undertaken a systematic investigation of the folding and aggregation properties of synthetic polyglutamine and polyalanine peptides, and the development of statistically valid methods for interpreting aggregation data. We will present results from three related studies. First, we developed a statistical model discrimination method to analyze protein aggregation data in light of competing mechanisms. We show that, with only monomer loss kinetic data, multiple models provide equivalent fits, making mechanistic determination impossible. We also define the type and quality of experimental data needed to make more definitive conclusions about the mechanism of aggregation. Specifically, direct measurement of fibril size is demonstrated to provide robust discrimination. We also demonstrate a method for extracting aggregate morphology and characteristic dimensions from static light scattering data. Second, we synthesized peptides containing the sequence Q10XXQ10, where XX is either PP, AA or dPG. Using a combination of FRET, circular dichroism, light scattering, electron microscopy and sedimentation assays, we demonstrate the strong conformational influence of these different interrupting residues, and the resulting influence on aggregation kinetics. We also show that ?seeding? a solution of Q20 peptide with a β-sheet/turn template does not grossly accelerate aggregation; this result along with other data challenges the notion that aggregation is initiated by a monomer nucleation event. Third, we synthesized polyalanine peptides of diverse length, and characterized their conformational and aggregational properties. A comparison of polyA and polyQ peptides reveals insights into the role of the side chain in regulating aggregation.