NR AREE
AU Stoehr,J.; Kaimann,T.K.; Metzger,S.; Riesner,D.
TI Dimers, oligomers and fibrils in prion conversion
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Poster GL-13
PT Konferenz-Poster
AB
The conversion of the soluble, alpha-helical, protease sensitive and not infectious form of the prion protein, PrPc, into an insoluble, protease resistant, predominantly ß-structured, and infectious form, PrPsc, plays a key role in prion disease.
This structural transition can be induced in vitro by slight changes in solvent conditions i. e. low concentrations (<0.1 %) of the anionic detergent SDS (sodium dodecyl sulfate): At 0.1 % SDS recombinant PrP 90-231 is monomeric and has a mainly alpha-helical structure. Removal of the SDS by mere dilution leads to a structural transition to a form rich in ß-sheet, accompanied by multimerization and aggregation.
In order to separate the structural transition from the oligomerization process, equilibrium properties of recombinant PrP at intermediate SDS concentrations were analyzed. A non-covalent dimer of alpha-helical PrP was found at SDS-concentrations of 0.055-0.07 % and was discussed as a potential first step in the conversion process1. The structural properties of the dimer were analyzed by chemical cross-linking, using EDC as a zero-length cross-linking reagent. Two sequence segments, PrP90-106 and PrP195-204, are potentially involved in forming the chemical cross-link and are therefore candidates for contact sites within the dimer.
Further dilution of SDS and addition of physiological concentrations of NaCl led to a fibrillar aggregation state after a prolonged incubation time. The amyloid character of this aggregation state was analyzed by electron microscopy (EM) and Thiofalvin T (ThT) binding to these structures. EM studies reveal typical insoluble fibrillary structures with a diameter of 8 to 10 nm whereas ThT binding to these fibrils resulted in an immense change in their fluorescence emission spectrum.
1. Jansen et al. (2001) Biological Chemistry, 382, 683-691
AD Jan Stoehr, Tina Kaimann, Sabine Metzger & Detlev Riesner, Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, Universitätsstraße 1, 40225 Düsseldorf
SP englisch
PO Deutschland
OR Tagungsband