CONFORMATIONAL EQUILIBRIA OF INTRINSICALLY DISORDERED PROTEINS PROBED BY SINGLE MOLECULE METHODOLOGIES

The structural disorder of the intrinsically-unstructured-proteins is the outcome of a complex ensemble of conformers driven by a rugged energy landscape. Many of these proteins are involved, through their aggregation into amyloid fibrils, in neuro-degenerative pathologies like Parkinson’s, Alzheimer’s and prion diseases. Significant progress has been made recently in characterizing these fibrils at the molecular level. However, the process of aggregation is still poorly understood because traditional bulk methods can only provide ensemble-averaged information for monomers and oligomers alike. We recently demonstrated that by means of single-molecule studies these limitations can be circumvented. (1,2) We applied the AFM-based Single Molecule Force Spectroscopy (AFM-SMFS) methodology to human alpha-synuclein. This methodology proved very effective in characterizing the conformational diversity of wild type (WT) alpha-synuclein and we observed that in several unrelated conditions linked to the pathogenicity of Parkinson’s disease the conformational equilibrium of this protein shifts toward beta-sheet-containing structures (1). The direct relationship of these beta-structures to alpha-synuclein toxicity was confirmed by our single-molecule study of the conformational heterogeneity of its pathologic mutants A30P, A53T and E46K. We found that those mutated sequences have a strongly higher propensity to acquire a monomeric beta-structure with respect to the WT one, and we identified significant differences in their conformational equilibria. These differences were related to the marked differences in the WT and mutant aggregation behaviors, with regard to both fibrillization and oligomerization. (2) Another methodology based on single-molecule-fluorescence-resonance-energy-transfer (SM-FRET) has been recently applied to the same protein. (3) The two methodologies are very complementary: whereas the AFM-based SMFS proposed by us is particularly sensitive to the formation of beta-sheeted secondary structures, and probes time-scales from milliseconds to seconds, the SM-FRET can evidence fast and complex conformational fluctuations in timescales shorter than a few milliseconds. The capability of single-molecule approaches to resolve the properties of individual protein molecules and quantify their sub-populations is most likely going to play a crucial role in studies of the conformational equilibria of intrinsically disordered proteins involved in neurodegenerative diseases