NR AWQM
AU Wille,H.; Latawiec,D.; Serban,A.; Murugesu,M.; Deering,C.; Prusiner,S.B.; Long,J.R.; Safar,J.G.
TI Growing 2D prion crystals from polyoxometalate complexes
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions S-30
PT Konferenz-Poster
AB Determining the structure of infectious prions at near-atomic resolution via electron crystallography has been constrained by the limited availability and quality of 2D crystals. Thus far, all 2D crystals of infectious, N-terminally truncated PrPsc (PrP 27-30) have been prepared from prion-infected brains by a well established density gradient purification procedure (Prusiner et al., Biochemistry 21:6942, 1982). We determined that in this procedure the 2D crystals form at the same time as the more commonly seen prion rods, during the limited digestion with proteinase K in the presence of sarkosyl. While we were able to increase the number of 2D crystals in our preparations by optimizing a range of buffer and protease digestion parameters, we failed to improve their size and order. We attribute this failure to the fact that, at this stage of the purification procedure, PrP 27-30 is only moderately enriched and numerous contaminating proteins and peptides interfere with crystallization. In order to overcome this problem, we decided to change our purification procedure completely and instead use sodium phosphotungstate (PTA), a polyoxometalate (POM) that selectively precipitates PrPsc and PrP 27-30 (Safar et al., Nature Med. 4:1157, 1998). This procedure allows rapid purification of PrP 27-30 as well as other protease-resistant and protease-sensitive conformers of PrPsc. Large and relatively well ordered 2D crystals could be found in some of these preparations, particularly after removal of PTA via dialysis. By employing various POMs (Lee et al., JACS 127:13802, 2005), we observed differences in the distributions between fibrillar prion rods and 2D crystals. Some POMs seem to favor the polymerization of PrP 27-30 into 2D crystals over the formation of prion rods. We may be able to identify conditions that allow the polymerization of PrP 27-30 into even larger, well ordered 2D crystals, suitable for high-resolution, cryo low-dose electron crystallography. We acknowledge generous support from the Sherman Fairchild Foundation and the National Institute on Aging (Grants AG02132 and AG10770).
AD H. Wille, G. Safar: Institute for Neurodegenerative Diseases, Department of Neurology, University of California, San Francisco, CA 94143, USA; D. Latawiec, A. Serban, C. Deering: Institute for Neurodegenerative Diseases, University of California, San Francisco, CA 94143, USA; M. Murugesu, J.R. Long: Department of Chemistry, University of California, Berkeley, CA 94720, USA; S.B. Prusiner: Institute for Neurodegenerative Diseases, Departments of Neurology, and Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA E-mail: hwille@ind.ucsf.edu
SP englisch
PO Italien