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

Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in 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 in deciphering the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques utilized so far to solve this problem point to a direct correlation between the αSyn unique conformational properties and its propensity to aggregate but 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 αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu, 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 significant 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 αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to directly test the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn.