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 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.

Mutations of evolutionarily conserved aromatic residues suggest that misfolding of the mouse prion protein may commence in multiple ways

“…For all the moPrP variants, the kinetics of misfolding appeared monophasic and fit well to a single exponential equation. It should be noted that the misfolding of moPrP at pH 4 is accompanied by oligomerization (Sabareesan & Udgaonkar, 2016; Sengupta & Udgaonkar, 2019). Earlier studies had shown that the kinetics of CD‐monitored misfolding and the kinetics of oligomerization monitored by size exclusion chromatography (SEC) were identical for wt moPrP, suggesting that both processes occur concurrently (Sabareesan & Udgaonkar, 2016).…”

“…It has been proposed that during the misfolding of the prion protein, the separation of the α2‐α3 and the β1‐α1‐β2 subdomains must occur before the conversion of α2 and α3 to the β‐conformation later during the misfolding process (Goluguri et al., 2019; Hadži et al., 2015; Sengupta & Udgaonkar, 2019). Locking of the α2‐α3 subdomain to the β1‐α1‐β2 subdomain by binding to anti‐prion drugs (Kamatari et al., 2013; Kuwata et al., 2007), prevents misfolding and oligomerization, presumably by stabilizing the N state with respect to an aggregation‐competent PUF.…”

“…If PUF1 is indeed populated on the pathways of unfolding from the N state to PUF2*, and from the N state to PUF2**, it is likely that it is the structure lost during the unfolding of the N state to PUF1, which makes all three PUFs competent to misfold. If PUF1 is not populated on the pathways of unfolding from the N state to PUF2*, and from the N state to PUF2**, then when misfolding commences from PUF1, additional structural loss and conformational conversion would occur only during the oligomerization process (Sengupta & Udgaonkar, 2019).…”

Destabilization of polar interactions in the prion protein triggers misfolding and oligomerization

“…The salt bridge between K193‐E195 breaks in the monomer PrP upon the addition of salt 26 . Subsequently, the segments spanning α2 and α3 first contract and then elongate 64 . The β1–α1–β2 subdomain separates from α2 and α3, which is essential during PrP oligomerization 51,60,65,66 .…”

Isobaric crosslinking mass spectrometry technology for studying conformational and structural changes in proteins and complexes

“…The indirect strategies are not able to directly observe the structural changes but can be used to deduce/infer structural changes based on changes in the output of the approach. These methods include limited proteolysis, immunochemical assays, fluorescence resonance energy transfer (FRET), chemical footprinting, and chemical crosslinking, some of which can be performed on proteins/complexes under physiological conditions [14][15][16][17] . The main challenge for the indirect strategies is interpreting the data to infer the correct conformational/structural change out of numerous possibilities.…”