Multiparametric fluorescence detection of early stages in the amyloid protein aggregation of pyrene-labeled alpha-synuclein

Journal of Molecular Biology
Shyamala ThirunavukkuarasuThomas M Jovin

Abstract

The aggregation of alpha-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-beta-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of alpha-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of alpha-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 degrees C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (I(I)/I(III)), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molec...Continue Reading

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Alpha-synucleins are small proteins that are believed to restrict the mobility of synpatic vesicles and inhibit neurotransmitter release. Aggregation of these proteins have been linked to several types of neurodegenerative diseases including dementia with Lewy bodies and Parkinson's disease. Here is the latest research on α-synuclein aggregation.

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