NR AOVH
AU Kunze,S.; Gnoth,S.; Metze,J.; Bodemer,W.
TI Immobilization of prion protein on mica
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-17
PT Konferenz-Poster
AB
Pathogenic processes and pathology of Transmissible Spongiform Encephalopathies (TSE) of man and animal are characterized by amyloidal deposits of beta-sheet rich prion protein (PrP) isoforms. The formation of such amyloidal aggregates depends on intra- as well as intermolecular binding forces. Ligand molecules ablating the formation of pathological prion protein amyloids by inhibition of conformational transition and formation of aggregates or rendering prion protein aggregates susceptible to proteolytic degradation are candidates for therapeutic measures.
Our experimental approach aims at determining direct interaction forces of single PrP molecules and one of its suspected ligands by use of single molecule force spectroscopy, combined with single molecule imaging using the Atomic Force Microscope (AFM).
For this purpose it is essential to immobilize the protein onto a solid support in a submonolayer quantity. According to a test of different materials mica seems to be best suited as solid support for PrP immobilization.
The result of different immobilization protocols was checked by imaging the mica surface with AFM. To be as near as possible to the natural environment these measurements should be performed in liquid. Therefore AFM measurements were done in a fluid cell in intermittent-contact mode.
The immobilization of prion protein was examined for various solution parameters, e.g. without additives on the base of unspecific interactions. Nevertheless, the immobilization of PrP on mica could be enhanced by addition of divalent copper salts to the PrP solution, resulting in a monolayer coverage of the surface.
For a detailed analysis of bound prion protein samples were dried and measured in intermittent-contact mode on air. Several PrP precipitates were analyzed to generate a distribution of their geometrical parameters.
Next steps of our work will include ligand immobilization onto the AFM cantilever and force measurements between PrP and ligands.
AD S. Kunze, S. Gnoth, J. Metze, Institute for Bioprocessing and Analytical Measurement Techniques, Heilbad Heiligenstadt, Germany; W. Bodemer, German Primate Center, Göttingen, Germany
SP englisch
PO Deutschland