4.6 Article

Conformational equilibria in monomeric alpha-synuclein at the single-molecule level

Journal

PLOS BIOLOGY
Volume 6, Issue 1, Pages 99-108

Publisher

PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pbio.0060006

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Human alpha-Synuclein (alpha Syn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of alpha Syn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between alpha Syn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy-based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant alpha Syn. The conformational heterogeneity of monomeric alpha Syn was characterized at the single-molecule level. Three main classes of conformations, including disordered and beta-like'' structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the beta-like'' structures significantly increased in different conditions promoting the aggregation of alpha Syn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric alpha Syn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric alpha Syn.

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