NR AOTY
AU Hepp,A.K.; Bieschke,J.G.; Giese,A.; Bertsch,U.; Kretzschmar,H.A.
TI Binding dynamics of PrPc to raft-like liposome membranes
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-86
PT Konferenz-Poster
AB
The physiological form of the prion protein (PrPc) is bound to the membrane by a glycosylphosphatidylinositol (GPI)-anchor on its C-terminal side. It seems to be located in cholesterol- and sphingolipid-rich membrane domains, called membrane rafts. Additionally the existence of a second, GPI-anchor independent way of membrane association of PrPc, mediated by its hydrophobic domains is being discussed.
To investigate membrane binding modes of PrP, we use as a model system raft-like small unilamellar liposomes labelled with fluorescent lipid analogs and fluorescently labelled recombinant PrP in a dual-color fluorescence correlation spectroscopy (FCS) setup. We performed kinetic studies of the GPI-anchor independent binding process and found a formation of PrPc-liposome-aggregates after 90 minutes incubation time. Subsequently, rPrP aggregation was induced in solution by SDS-treatment. Aggregated PrPc seems to have the same binding kinetics as untreated PrPc.
The liposome model combined with the FCS technique can now be used for further investigations including the influence of copper concentration, on different rPrP constructs and especially interactions between the misfolded isoform PrPsc and the physiological PrPc. In order to get insights in the interactions between GPI-anchored PrPc and PrPsc we create a fusion Protein of PrP residues 23 to 213 with an intein domain, that can autocatalytically be cut off. The remaining PrP23-213 is then activated for a fusion reaction to the synthetic peptide with amino acids 214-231 of the PrPc which is already linked to a fatty acid anchor.
AD A.K. Hepp, J.G. Bieschke, A. Giese, U. Bertsch, H.A. Kretzschmar, Institute for Neuropathology, LMU Munich, Marchioninistr. 17, 81377 München, Germany
SP englisch
PO Deutschland