Abstract:
Proteins exist as ensembles of multiple states within a free energy landscape, where partially unfolded forms (PUFs) are present alongside the stable, folded native state (N). These PUFs arise through protein dynamics and play an important role in protein misfolding—a process associated with multiple diseases. However, their characterization is very challenging due to their low abundance and transient nature. This study focuses on the role of high-energy PUFs in initiating misfolding of the mammalian prion protein (PrP), which is associated with fatal neurodegenerative diseases. Using hydrogen-deuterium exchange and mass spectrometry (HDX-MS), the study shows that the N state of PrP is in equilibrium with multiple aggregation- prone PUFs that can initiate misfolding. Two sets of conserved residues—proline and aromatic residues—have been identified as ‘gatekeepers’ against misfolding into toxic β-rich oligomers. Mutations of these residues destabilize the N state and lower the energy barrier to PUFs, accelerating misfolding by up to 1000-fold due to increased PUF populations. It is also shown that increasing the rigidity of the β2-α2 loop significantly impacts the dynamics and misfolding pathways of PrP. Additionally, filling cavities present in the hydrophobic core of the protein results in a significant reduction in the equilibrium population of PUFs, impeding misfolding. The study also identifies a molten globule-like conformation of the protein, which misfolds into a β-rich dimer. Importantly, the structure of the dimer is similar to that of the oligomers, suggesting that misfolding to the oligomer is induced at the level of the dimeric unit by monomer-monomer association, and that the oligomer grows by the accretion of misfolded dimeric units.
Additionally, the prion protein undergoes spontaneous phase separation into highly dynamic liquid droplets under physiological conditions. The study further discusses HDX-MS findings of these droplets, revealing the key molecular events involved in the phase separation of the prion protein and the transition from liquid-like droplets to solid-like states.
