A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein

Abstract

SUMMARY Transient folding intermediates and/or partially unfolded equilibrium states are thought to play a key role in the formation of protein aggregates. However, there is only indirect evidence linking accumulation of folding intermediates to aggregation, and the underlying mechanism remains to be elucidated. Here we show that a partially unfolded state of the prion protein accumulates both as a stable equilibrium state at acidic pH (A-state) and as a late folding intermediate. With a time resolution of approximately 60 μs, we systematically studied the kinetics of folding and unfolding, starting from various initial conditions including the U-, N-, and A-states. Quantitative modeling showed that the observed kinetic data are completely consistent with a sequential four-state mechanism where the A-state is a late folding intermediate. Combined with previous evidence linking A-state accumulation to aggregation, the results indicate that this native-like state serves as a branching point between the folding and aggregation pathways.

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions

“…Such a structural perturbation also appears to have occurred in a molten globule-like kinetic intermediate that is also populated at equilibrium under highly acidic conditions, which has been implicated as a key precursor to oligomerization (68,69). Molecular dynamics studies on the prion protein (34,70) residues in the protein.…”

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

“…An interesting finding in the research on folding intermediates is the presence of the molten globule, wherein secondary structure elements are conserved in their native conformation, but the native side chain packing is perturbed in the tertiary structure . However, apart from molten globules, several intermediates can exist in the folding pathway of a protein with different amounts of secondary and tertiary structure . Direct detection of these higher energy intermediates is often impossible because they are unstable and transient .…”

“…Direct detection of these higher energy intermediates is often impossible because they are unstable and transient . Structural insights of higher intermediates can be obtained from NMR relaxation dispersion experiments, native state hydrogen exchange and thiol labeling experiments . Molecular dynamics (MD) simulations can also potentially hint towards the existence of higher energy intermediates .…”

“…4,5 However, apart from molten globules, several intermediates can exist in the folding pathway of a protein with different amounts of secondary and tertiary structure. [6][7][8] Direct detection of these higher energy intermediates is often impossible because they are unstable and transient. 9 Structural insights of higher intermediates can be obtained from NMR relaxation dispersion experiments, native state hydrogen exchange and thiol labeling experiments.…”

Thermodynamic characterization for the denatured state of bovine prion protein and the BSE Associated variant E211K

“…Early efforts in the understanding of genetic prion disease found little evidence to support a change in the global stability as a means to explain enhanced misfolding in the disease associated variants [21]. Subsequent study then focused on the identification of folding intermediates using a variety of approaches that all showed that the C-terminal portion of the prion protein folded more rapidly [22,23]. Investigation into pre-oligomer structures also showed the presence of an acid-induced molten globule state where the C-terminal region remained folded but under physiologic conditions an equilibrium intermediate is not detected [24].…”