Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein.

Abstract

During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers O_L, which subsequently disaggregated reversibly into small oligomers O_S at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of O_L and elongation into β-sheets during the formation of O_S. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both O_L and O_S.