Abstract
The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.
Highlights
Introduction aSynuclein is a small (140 amino acids) protein, highly expressed in the central nervous system where it constitutes about 0.5–1.0% of the entire cytosolic protein content
The amino acidic sequence of aS includes a conserved Nterminal domain containing a series of amphipathic repeats that modulates membrane binding; a central region, between residues 61–95, that comprises the highly aggregation-prone NAC sequence [10,11]; a C-terminal region enriched in acidic residues and prolines. aS lacks of ordered secondary structure under physiological conditions, as deduced by far-UV circular dichroism (CD) and Fourier transform IR (FT-IR) measurements [12]
Structural characterization of dimers in solution The secondary structure of NN, CC, NC and DC in PBS buffer was evaluated by far-UV CD, FT-IR and Nuclear magnetic resonance (NMR) spectroscopy
Summary
Synuclein (aS) is a small (140 amino acids) protein, highly expressed in the central nervous system where it constitutes about 0.5–1.0% of the entire cytosolic protein content In fibrillar form, it is the major component of intracellular deposits of proteins and lipids called Lewy bodies and Lewy neurites, which are hallmarks of Parkinson’s disease (PD) and other related neurological disorders [1,2]. AS lacks of ordered secondary structure under physiological conditions, as deduced by far-UV circular dichroism (CD) and Fourier transform IR (FT-IR) measurements [12] For this reason, it has been widely accepted that aS is an intrinsically disordered protein [13,14]. The clarification of aS physiological conformation remains fundamental to the understanding of its protein function
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