Direct observation of multiple misfolding pathways in a single prion protein molecule

Abstract

Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a highresolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into offpathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.protein folding | optical tweezers

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates

“…Landscape reconstructions based on high-bandwidth equilibrium measurements of the extension at constant force or trap separation typically provide higher spatial resolution (20,22). However, equilibrium trajectories present interpretation difficulties for PrP, because in addition to native folding they also contain transitions into shortlived, nonnative states accessible only from the unfolded state (28,30), which would distort the reconstructed landscape. In contrast, unfolding FECs always go first from N to U, with subsequent refolding into the misfolded states suppressed by virtue of the nonequilibrium conditions caused by the fast force-ramp rate.…”

“…As an independent test of whether this overall picture is correct, we analyzed equilibrium measurements of the extension at constant force, in the range approximately 8-10 pN. Previous work has shown that the native folding pathway did not include any intermediate state (28,30). Representative records at different forces (Fig.…”

“…Representative records at different forces (Fig. 4A) were filtered to remove the short-lived off-pathway states (28) and the lifetimes of the two states were determined by threshold analysis (19). The lifetimes were single-exponentially distributed at each force (Fig.…”

“…2A, red) reveal a nonlinear rise in force with extension arising from the elasticity of the handles, interrupted by a sudden increase in extension and concomitant drop in force as PrP unfolded in a single step. The contour length change from folded (N) to unfolded (U) was determined by fitting the FECs to two wormlike chains (WLCs) in series, one for the DNA handles and one for the protein (28). The value obtained from fitting 3,250 unfolding FECs, 34.1 AE 0.4 nm (all errors: standard error on the mean), agrees well with the 34.3 nm expected from the NMR structure of the native state (29), confirming that the PrP was indeed natively folded before each pull.…”

“…Truncated, His-tagged, wild-type hamster PrP(90-231) was engineered with cysteine residues at each terminus, expressed in Escherichia coli, purified by affinity FPLC, and refolded on-column as described (28). Protein identity was checked by Western blotting, and folding into the native structure verified by CD spectroscopy.…”