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Methods. 2011 Mar;53(3):295-305. doi: 10.1016/j.ymeth.2010.12.008. Epub 2010 Dec 14.

Assays for α-synuclein aggregation.

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1
Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, Gustav Wieds Vej 10C, DK - 8000 Aarhus C, Denmark.

Abstract

This review describes different ways to achieve and monitor reproducible aggregation of α-synuclein, a key protein in the development of Parkinson's disease. For most globular proteins, aggregation is promoted by partially denaturing conditions which compromise the native state without destabilizing the intermolecular contacts required for accumulation of regular amyloid structure. As a natively disordered protein, α-synuclein can fibrillate under physiological conditions and this process is actually stimulated by conditions that promote structure formation, such as low pH, ions, polyamines, anionic surfactants, fluorinated alcohols and agitation. Reproducibility is a critical issue since α-synuclein shows erratic fibrillation behavior on its own. Agitation in combination with glass beads significantly reduces the variability of aggregation time curves, but the most reproducible aggregation is achieved by sub-micellar concentrations of SDS, which promote the rapid formation of small clusters of α-synuclein around shared micelles. Although the fibrils produced this way have a different appearance and secondary structure, they are rich in cross-β structure and are amenable to high-throughput screening assays. Although such assays at best provide a very simplistic recapitulation of physiological conditions, they allow the investigator to focus on well-defined molecular events and may provide the opportunity to identify, e.g. small molecule inhibitors of aggregation that affect these steps. Subsequent experiments in more complex cellular and whole-organism environments can then validate whether there is any relation between these molecular interactions and the broader biological context.

PMID:
21163351
DOI:
10.1016/j.ymeth.2010.12.008
[Indexed for MEDLINE]

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