# Unfolding Mechanism and Fibril Formation Propensity of Human Prion Protein in the Presence of Molecular Crowding Agents

## TL;DR

This paper studies how human prion protein unfolds and forms harmful fibrils in cell-like conditions, revealing how molecular crowding affects these processes.

## Contribution

The study reveals how molecular crowding affects the unfolding and fibril formation of human prion protein.

## Key findings

- HuPrP(90–231) thermal unfolding involves two transitions in cell-mimicking conditions.
- Molecular crowding delays prion fibril formation despite promoting an intermediate state.
- Crowding alters both equilibrium states and kinetic pathways of prion protein.

## Abstract

The pathological process of prion diseases implicates that the normal physiological cellular prion protein (PrPC) converts into misfolded abnormal scrapie prion (PrPSc) through post-translational modifications that increase β-sheet conformation. We recently demonstrated that HuPrP(90–231) thermal unfolding is partially irreversible and characterized by an intermediate state (β-PrPI), which has been revealed to be involved in the initial stages of PrPC fibrillation, with a seeding activity comparable to that of human infectious prions. In this study, we report the thermal unfolding characterization, in cell-mimicking conditions, of the truncated (HuPrP(90–231)) and full-length (HuPrP(23–231)) human prion protein by means of CD and NMR spectroscopy, revealing that HuPrP(90–231) thermal unfolding is characterized by two successive transitions, as in buffer solution. The amyloidogenic propensity of HuPrP(90–231) under crowded conditions has also been investigated. Our findings show that although the prion intermediate, structurally very similar to β-PrPI, forms at a lower temperature compared to when it is dissolved in buffer solution, in cell-mimicking conditions, the formation of prion fibrils requires a longer incubation time, outlining how molecular crowding influences both the equilibrium states of PrP and its kinetic pathways of folding and aggregation.