?-SYNUCLEIN FIBRIL

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ?-Synuclein is a 14.5 kDa protein that aggregates into amyloid fibrils within dopaminergic neurons comprising the brains of Parkinson disease patients, putatively resulting in neurodegeneration. Amyloid fibrils are an intermolecular cross ?-sheet quaternary structure adopted by numerous proteins associated with human aggregation-associated degenerative diseases. Amyloid fibrils prepared in vitro by incubating recombinant proteins are typically long, straight, unbranched fibers 8-12 nm in diameter. These can grow to several microns in length. ?-Synuclein aggregates into amyloid fibrils when shaken in PBS in the presence of 1% seed (sonicated pre-formed fibrils to which monomers can add to elongate into fibrils). We have found that when these fibrils are diluted into high concentrations (5M) of guanidine hydrochloride (GdnHCl) they denature to monomer . The amount of monomer and aggregate in solution can be determined by ultracentrifugation of the sample at 60,000 rpm, collecting the supernatant and pellet, dissolving the pellet in 8M GdnHCl, and injecting both the supernatant and pellet samples on a reverse phase HPLC column. The area of the peak of ?-synuclein can be compared to a standard curve to determine the amount of material in each fraction and, assuming only monomer remains in the supernatant, the amount of protein aggregated. Interestingly, when ?-synuclein fibrils are diluted into 3M GdnHCl they denature to mostly monomer but upon further incubation, re-aggregate to roughly 50% aggregated fibers. Additionally, ?-synuclein aggregated from monomer will also aggregate to the extent of about 50% in 3M GdnHCl, either in the presence or absence of seed. Because the fibrils formed in PBS do not seem to be stable in 3M GdnHCl, as they at first disaggregate to monomer in the presence of 3M GdnHCl, the aggregates that later form in 3M GdnHCl ought to have different properties and likely a different structure than the fibrils formed in PBS. We are trying to compare the two types of aggregates to investigate those differences.