Abstract
The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation.
Highlights
The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s
We employed either ATTO 488 (A488 as the donor) or ATTO 647N (A647N as the acceptor) to label the N47A-SH3 monomers on a designed N-terminal cysteine residue located after a flexible six-residue (Gly-Ser-Gly-Ser-Gly-Cys) tail
The incubation of equimolar mixtures of the donor- and acceptor-labelled monomers (N47A-SH3-DA) under amyloidogenic conditions (0.1 M glycine and 0.1 M NaCl, pH 3.2, 37 °C, hereafter referred to as ‘aggregation buffer’) for several hours resulted in the effective co-aggregation and formation of mature fibrils
Summary
The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, at low protein concentrations. The study of the aggregation process is currently focused on determining the roles of these intermediates as well as their complete characterization[3,7] because they are potential targets that could define molecular treatment strategies These early stages of aggregation represent a challenge for conventional biophysical techniques because these techniques only provide average information about the ensemble of the population and cannot provide insight into the heterogeneity of the samples. Our single-molecule experiments allowed us to quantify the thermodynamic constant for this first equilibrium
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