Photo-induced crosslinking of prion protein oligomers and prions

Area of Science:

• Biochemistry
• Structural Biology
• Neuroscience

Background:

• Prion diseases stem from misfolded prion proteins (PrPSc), which form insoluble aggregates.
• These aggregates resist traditional structural analysis methods like X-ray crystallography and NMR.
• Stabilizing these elusive oligomers is crucial for understanding their structure and function.

Purpose of the Study:

• To investigate photo-induced crosslinking (PICUP) as a method to stabilize prion protein aggregates.
• To analyze the architecture of prion aggregates and identify intermolecular contact sites.
• To assess the impact of crosslinking on prion infectivity and aggregation-inducing capacity.

Main Methods:

• Utilized photo-induced crosslinking (PICUP) to covalently link interacting amino acid side chains in prion protein aggregates.
• Employed varying light intensities to control the degree of crosslinking.
• Monitored crosslinking efficiency using fluorescence correlation spectroscopy.
• Performed functional studies on crosslinked recombinant PrP (rPrP) and PrPSc in brain homogenates.

Main Results:

• PICUP successfully crosslinked recombinant PrP (rPrP) and disease-associated PrPSc aggregates.
• The degree of crosslinking was controllable via light intensity and monitored by fluorescence correlation spectroscopy.
• Specific intermolecular crosslinking of PrPSc was achieved even in crude brain homogenates.
• Stabilized rPrP aggregates retained their ability to induce further protein aggregation.
• Crosslinking of PrPSc did not significantly alter prion infectivity levels.

Conclusions:

• Photo-induced crosslinking is an effective method for stabilizing elusive prion protein aggregates.
• This technique allows for the analysis of aggregate architecture without compromising prion infectivity.
• PICUP offers a valuable tool for studying the structural basis of prion propagation and developing therapeutic strategies.