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 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.
Highlights
The structural characterization of kinetic intermediates in protein aggregation is a challenging task
fluorescence resonance energy transfer (FRET) pairs to monitor site-specific misfolding in the monomeric unit of the oligomer
All native Trp residues are solvent-exposed in the WT mouse prion protein (moPrP) monomer
Summary
The structural characterization of kinetic intermediates in protein aggregation is a challenging task. Most experimental probes, used to study misfolding and aggregation kinetics, track either the acquisition of b-structure, or global changes in size. Equilibrium and kinetic measurements using multi-site FRET, to probe conformational changes in different parts of a protein, while it folds, unfolds, forms functional oligomers or interacts with its binding partner, have been a rich source of site-specific information, usually invisible to global probes (Lakshmikanth et al, 2001; Lillo et al, 1997; Lin et al, 2013). The mostly a-helical and monomeric prion protein (PrP) undergoes drastic secondary and tertiary structural re-arrangements upon aggregation into a variety of misfolded b-sheet-rich multimers (Pan et al, 1993), most of which are not infectious. The pathogenic misfolded aggregates formed in vivo are highly heterogeneous, with the most infectious oligomers composed of 14–28 monomers (Silveira et al, 2005)
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