NR ATFO
AU Wille,H.; Govaerts,C.; Latawiec,D.; Cohen,F.E.; Prusiner,S.B.
TI Probing the structure of PrPsc via heavy metal complexes and electron crystallography
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Oral sessions ORAL-19
PT Konferenz-Vortrag
AB
The insolubility of the scrapie prion protein (PrPsc) has hindered attempts to solve its structure by X-ray crystallography or NMR spectroscopy. Using electron crystallography of 2D crystals, we gained first insights into the molecular architecture of N-terminally truncated PrPsc (PrP 27-30) and a miniprion (PrPsc106) (Wille et al., PNAS 99:3536, 2002; Govaerts et al., PNAS 101:8342, 2004).
We employed different heavy metal negative stains to probe the structure of PrP 27-30 and PrPsc106. The interactions of the heavy metals with the crystal lattice are governed by secondary, tertiary and quaternary structure elements of the protein and the charge, size and stability constants of the heavy metal complex. The differences in heavy metal binding can be visualized even at low resolution and enabled us to localize the internal deletion of PrPsc106 at the center of the trimer that forms the unit cell. The results obtained by heavy metal binding together with higher resolution images constrain molecular modeling and argue for a left-handed, parallel beta-helical architecture in PrPsc. In order to proceed to low-dose cryoelectron microscopy, the quantity and quality (i.e., size and degree of order) of the 2D crystals must be improved. Our current research focuses on determining the parameters that are crucial for 2D crystallization. We have produced greater quantities of sufficiently well ordered crystals and are working to improve the crystal size. After growing 2D crystals suitable for low-dose cryoelectron crystallography, we will focus on collecting high-resolution data, with the goal of generating a three-dimensional reconstruction of PrPsc.
We acknowledge generous support from the Sherman Fairchild Foundation and the National Institute on Aging (Grants AG02132 and AG10770).
AD Holger Wille, Cédric Govaerts, Diane Latawiec, Fred E. Cohen, Stanley B. Prusiner, University of California San Francisco, USA
SP englisch
PO Deutschland