NR AOYG
AU Riesner,D.; Leffers,K.W.; Wille,H.; Jansen,K.; Schell,J.; Tatzelt,J.; Prusiner,S.B.
TI Dimers, oligomers, multimers, and fibrils: Induction and characterization of different intermediates of PrP refolding
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Oral sessions OS-08
PT Konferenz-Vortrag
AB A series of conformational states of PrP can be established by systematic variation of low concentrations of sodium dodecyl sulphate (< 0.1%) or by the interaction with the bacterial chaperone GroEL or GroEL/ES. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were also obtained with full length recombinant PrP, hamster PrP 27-30, PrPc isolated either from hamster brain or from transgenic, non-infected CHO-cells. Varying the incubation conditions (SDS-concentration, sodium chloride concentration, incubation time, always neutral pH and room temperature) the following conformations were established and characterized in respect to secondary structure by CD-spectroscopy, to molecular mass by fluorescence spectroscopy and analytical ultracentrifugation, to aggregate structure by electron microscopy: alpha-helical dimers, beta-sheeted, still soluble oligomers, insoluble multimers with some but not much highly ordered structure and large multimers with regular fibril structure. The influence of secondary components of prions which were analyzed as specific lipids and a polysaccharide scaffold on conformational transitions was investigated. When multimers were formed from PrPc from CHO-cells not only a partial PK-resistance was found as with PrP from other sources, but also the specific cut characteristic for N-terminal truncation of PrP was identified. The number of SDS-molecules bound to PrP in different conformations were determined, and it is discussed that the strongest binding sites for SDS are identical or similar to the sites of interaction with GroEL thereby inducing identical or very similar transitions. The fibrils formed from PrPc or denatured PrP 27-30 are at present in bioassays for testing the infectivity. However after 200 days of incubation, no symptoms could be seen.
AD D. Riesner, K.-W. Leffers, K. Jansen, J. Schell, Heinrich-Heine-Universität Düsseldorf, Germany; H. Wille, S.B. Prusiner, UCSF, USA; J. Tatzelt, Max Planck Institut für Biochemie, Germany
SP englisch
PO Deutschland