The conversion of the cellular prion protein (PrPC) to its infectious counterpart (PrPSc) is the initial step of prion diseases. These neurodegenerative disorders are characterized by different incubation times, sympthoms and disease phenotypes. Structural heterogenity of PrP aggregates is responsible for this biological diversity. Understanding the structural rearrangements of PrP at the monomeric and oligomeric level is essential to gain insights into its aggregation processes. However traditional “in-bulk” techniques can only provide ensemble-averaged information for monomer and oligomer structures. We applied single-molecule force spectroscopy to characterize the heterogeneous structural ensemble of the murine PrP at the monomeric and at the oligomeric level. By stretching chimeric protein construct carrying one MoPrP molecule we found that the protein folds with a two state mechanism. Less frequently the protein can adopt more extended conformations that encompass also the N-terminal domain. These structures might be involved in subsequent aggregation processes. We also developed an assay to characterize the oligomerization processes using multiple PrP constructs. By analyzing the extension of these constructs under tension we characterized the structure between different PrP moieties, under different conditions. We found that reciprocal PrP orientation affects the length and mechanical resistance of these structures but their events frequency. Comparing the structures observed from monomers, dimers, trimers and tetramers we found that their frequency of events and their average length increased by increasing the number of PrP moieties. Remarkably, decreasing pH to more acidic values resulted in a higher frequency of events that involved structures between PrP moieties only in multimeric constructs. Instead, increasing the ionic strength significantly diminished their frequency, indicating how solution conditions can strongly alter the conformational transitions. These results provide a new scenario on PrP misfolding and aggregation processes, characterizing their early aggregation steps under different reaction conditions.
