Photo-induced crosslinking of prion protein oligomers and prions.
Prion diseases are caused by a unique type of infectious agent, which is thought to consist of a misfolded beta-sheeted form of the alpha-helical cellular prion protein (PrPC). This misfolded isoform (PrPSc) tends to form insoluble amyloid-like aggregates, impeding classical structural analysis by X-ray crystallography or NMR. Intermolecular crosslinking may provide a means of stabilizing notoriously elusive oligomers for further analysis and may be used for analyzing aggregate architecture by characterising intermolecular contact sites. Using a photo-induced crosslinking method (PICUP), aggregates of recombinant PrP (rPrP) and PrPSc were linked at interacting surfaces via amino acid side chains. The degree of crosslinking within PrP aggregates was adjustable using varying light intensities and could efficiently be monitored by fluorescence correlation spectroscopy. Specific intermolecular crosslinking of PrPSc molecules was achieved even in crude brain homogenate. Functional studies showed that stabilized aggregates of rPrP did not loose their capacity to induce further protein aggregation and crosslinking of PrPSc did not alter significantly the level of infectivity, indicating that photo-induced covalent linkage of PrPSc does not destruct surfaces important for prion propagation.
Piening
Niklas
N
Zentrum für Neuropathologie und Prionforschung, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 23, 81377 München, Germany.
Weber
Petra
P
Högen
Tobias
T
Beekes
Michael
M
Kretzschmar
Hans
H
Giese
Armin
A
D000682:Amyloid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry; D006801:Humans; D051379:Mice; D010777:Photochemistry / Q000379:methods; D018621:PrPC Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018620:PrPSc Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017096:Prion Diseases / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D000682:Amyloid; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011994:Recombinant Proteins