J. Mol. Biol. (1995) 252, 412–422 X-ray Diffraction of Scrapie Prion Rods and PrP Peptides Jack T. Nguyen 1 , Hideyo Inouye 2 , Michael A. Baldwin 1 Robert J. Fletterick 3,4 , Fred E. Cohen 3,4,5 , Stanley B. Prusiner 1,3 and Daniel A. Kirschner 2 * 1 Department of Neurology Certain neurodegenerative diseases in humans and animals are caused by small proteinaceous infectious particles called prions. Limited proteolysis University of California and detergent extraction of the prions containing PrP Sc generate prion rods San Francisco, CA 94143 USA that are composed of a polypeptide having an apparent molecular mass of 27 to 30 kDa. This polypeptide, termed prion protein PrP 27–30, has a ragged 2 Neurology Research-Enders 2 N terminus that begins at about residue 90, but retains scrapie infectivity. Children’s Hospital, 300 Moreover, the findings in a patient having an inherited prion disease of a Longwood Avenue, Boston truncated PrP with its C terminus at residue 145 suggest that the residues MA 02115, USA 90 to 145 may be of particular importance in the pathogenesis of prion diseases. To determine the three-dimensional organization of prion rods and 3 Department of Biochemistry to identify the core region involved in amyloid formation, we recorded X-ray and Biophysics, University of diffraction patterns from rods purified from scrapie-infected Syrian hamster California, San Francisco (SHa) brains which contain PrP 27–30, and from synthetic SHaPrP peptides. CA 94143, USA Three peptides were studied corresponding to residues 113 to 120 (peptide 4 Department of A8A, an octamer composed of glycines and alanines), 109 to 122 (H1, the first Pharmaceutical Chemistry predicted -helical region of PrP C ), and 90 to 145 (a 56 residue peptide University of California, San containing both H1 and the second predicted -helical region of PrP C , H2). Francisco, CA 94143, USA Electron microscopy, carried out in parallel with the X-ray measurements, revealed that all the samples formed linear polymers which were 60 to 5 Department of Medicine 200 Å wide, with fibrillar or ribbon-like morphology. Gels and dried University of California, San preparations of prion rods gave X-ray patterns that indicated a -sheet Francisco, CA 94143, USA conformation, in which the hydrogen bond distance was 4.72 Å and the intersheet distance was 8.82 Å. For the three PrP peptides, the intersheet spacings varied widely, owing to the side-chains of the residues involved in the formation of the -sheet interactions, i.e., 5.13 Å for A8A, 5.91 Å for lyophilized H1, 7.99 Å from solubilized and dried H1 and 9.15 Å for the peptide SHa 90–145. The intersheet distance of PrP 27–30 was thus within the observed range for the peptides, and suggests that the amyloidogenic core of PrP is closely modeled by the peptide SHa 90–145.  1995 Academic Press Limited Keywords: prion diseases; beta-amyloid; beta-pleated sheet; electron microscopy; synthetic peptides *Corresponding author Present address for H. Inouye and D. A. Kirschner: Department of Biological Sciences, University of Massachusetts Lowell, One University Avenue-Olsen 6, Lowell, MA 01854, USA. Abbreviations used: PrP, prion protein; PrP Sc , scrapie (infectious) isoform of PrP; PrP C , cellular (non-infec- tious) isoform of PrP; PrP 27–30, the polypeptide generated by digesting PrP Sc with proteinase K, which hydrolyzes amino acids from its N-terminus; SHa, Syrian hamster; SHa 90–145, residues 90–145 of PrP; H1, the first predicted -helical region of PrP C , consisting of residues 109–122; H2, the second predicted -helical region of PrP C , consisting of residues 129–141; Introduction In scrapie and other prion diseases, the scrapie isoform of the prion protein (PrP Sc ) is an essential, if A8A, residues 113–120 of PrP; TFA, trifluoroacetic acid; AcN, acetonitrile; HFIP, 1,1,1,3,3,3-hexafluoro-2-prop- anol; CJD, Creutzfeldt-Jakob disease; GSS, Gerstmann- Stra ¨ussler-Scheinker syndrome; LF, low frequency; FTIR, Fourier transform infrared spectroscopy; CD, circular dichroism; H3, the third predicted -helical region of PrP C ; H4, the fourth predicted -helical region of PrP C . 0022–2836/95/390412–10 $12.00/0  1995 Academic Press Limited