TY - JOUR TI - Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein AU - Sengupta, Ishita AU - Udgaonkar, Jayant A2 - Martin, Andreas A2 - Kuriyan, John VL - 8 PY - 2019 DA - 2019/06/24 SP - e44698 C1 - eLife 2019;8:e44698 DO - 10.7554/eLife.44698 UR - https://doi.org/10.7554/eLife.44698 AB - 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 OL, which subsequently disaggregated reversibly into small oligomers OS at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL and elongation into β-sheets during the formation of OS. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both OL and OS. KW - prion KW - α to β switch KW - intra-molecular FRET KW - small and large oligomers JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -