NR AREA
AU Kunze,S.; Metze,J.; Bodemer,W.
TI Imaging of single prion protein molecules by Atomic Force Microscopy
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Poster GL-09
PT Konferenz-Poster
AB
The immobilization of prion protein (PrP) on a solid support is an essential step to investigate direct interaction forces between PrP molecules and their suspected ligands by use of single molecule force spectroscopy. Ligand molecules should bind to cellular PrP in order to inhibit its conformational transition and thus to prevent the formation of pathological amyloidal aggregates, making themselves candidates for therapeutic measures in prion diseases.
For this study we have used recombinant prion protein comprising amino acids 23-208, which was synthesized in bacteria. Its quality was repeatedly monitored by Western Blot and two-dimensional gel electrophoresis. PrP was immobilized on mica. Different immobilization protocols were compared by imaging the mica surface by Atomic Force Microscopy (AFM) in liquid in order to determine their efficiency and suitability. AFM is a most useful technique to visualize protein structures. The protein of interest can be studied in a native state, under well controlled experimental conditions like pH, ion concentration, detergents or other additives. The method is non-destructive to the proteins and has an imminent high resolution in the nanometer range.
Initial experiments showed that PrP molecules can be immobilized on a mica surface without additives on the base of unspecific interactions. However, directed interactions can be utilized in terms of binding to the octapeptide repeat region. Thus, the addition of copper(II) to the PrP solution resulted in an enhanced PrP immobilization, whereas nickel(II) seems to decrease the amount of bound prion protein.
Apart from measuring in liquid, dried PrP preparations were also imaged in air. This high resolution data provided for the first time a distribution of geometrical parameters of the PrP precipitates. Our findings point not only to immobilization of recombinant PrP molecules but also allowed to perform determination of single molecule particle volumes.
AD S. Kunze, J. Metze, Institute for Bioprocessing and Analytical Measurement Techniques e.V., Rosenhof, D-37308 Heilbad Heiligenstadt; W. Bodemer, German Primate Center, Department of Infectionpathology, Kellnerweg 4, D-37077 Göttingen
SP englisch
PO Deutschland
OR Tagungsband