Prions are composed largely, if not entirely, of prion protein (PrPsc in the case of scrapie). Although the formation of PrPs from the cellular prion protein (PrPc) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPsc synthesis. To assess this possibility, we purified both PrPC and PrPsc by using nondenaturing procedures and determined the secondary structure ofeach. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high a-helix content (42%) and no (3sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the -sheet content of PrPSc was 43% and the a-helix

By comparing the amino acid sequences of 11 mammalian and 1 avian prion proteins (PrP), structural analyses predicted four a-helical regions. Peptides corresponding to these regions of Syrian hamster PrP were synthesized, and, contrary to predictions, three ofthe four spontaneously formed amyloids as shown by electron microscopy and Congo red staining. By IR spectroscopy, these amyloid peptides exhibited secondary structures composed largely of 13-sheets. The first of the predicted helices is the 14-amino acid peptide corresponding to residues 109-122; this peptide and the overlapping 15-residue sequence 113-127 both form amyloid. The most highly amyloidogenic peptide is AGAAAAGA, which corresponds to Syrian hamster PrP residues 113-120 and is conserved across all species for which the PrP sequence has been determined. Two other predicted a-helices corresponding to residues 178-191 and 202-218 form amyloids and exhibit considerable 13-sheet structure when synthesized as peptides. These findings suggest the possibility that the conversion of the cellular isoform of PrP to the scrapie isoform of PrP Involves the transition of one or more putative PrP a-helices into 13-sheets and that prion diseases are disorders of protein conformation. 27-30 (19, 20).In the study reported here, we synthesized peptides homologous to portions of the Syrian hamster (SHa) PrP sequence and examined their physical characteristics by attenuated total reflection (ATR) Fourier transform IR spectroscopy (FTIR) (21). The particular peptides were chosen based on structural predictions where the PrP amino acid sequences from 11 mammalian and 1 avian sources were compared (22). Those analyses suggested the possibility that PrP might fold into a monomeric molecule with four a-helices (ref. 23; J.-M.G., F.C., R.F., and S.B.P., unpublished results). Unexpectedly, synthetic peptides corresponding to three of the four putative a-helices exhibit extensive 13-sheet structure as well as the ultrastructural and tinctorial properties of amyloid.MATERIALS AND METHODS All PrP peptides (Table 1) were assembled by using the 9-fluorenylmethoxycarbonyl variant of the Merrifield solidphase method on an Applied Biosystems model 432A Synergy peptide synthesizer. Peptides with side-chain protection were cleaved from the resins by using thioanisole/ethanedithiol/ trifluoroacetic acid (TFA) and precipitated with tert-butyl methyl ether. They were pelleted by centrifugation, suspended in ether, washed three times, repelleted, dissolved in water/ isopropanol, and filtered. Peptides without side-chain protection were cleaved with 90%o (vol/vol) TFA. The solutions were lyophilized, and the peptides were analyzed by reversed-phase HPLC and plasma desorption mass spectrometry. The majority ofpeptides were >80%o pure and were used without further purification. The initial product for peptide H4 was =20%6 pure, and it was purified by preparative HPLC and reanalyzed. The synthesis of SHaPrP-(113-120) gave 40%o Ala6Gly2 (the desired product) and 60% Ala5Gly2, which ...

Limited proteolysis of the scrapie prion protein (PrPsc) generates PrP 27-30, which polymerizes into amyloid. By attenuated total reflection-Fourier tranform infrared spectroscopy, PrP 27-30 polymers contained 54% 1-sheet, 25% a-helix, 10% turns, and 11% random coil; dispersion into detergent-ipid-protein-complexes preserved infectivity and secondary structure. Almost 60% of the 1-sheet was low-frequency infrared-absorbing, reflecting intermolecular aggregation. Decreased low-frequency 1-sheet and in-

LytA amidase is the best known bacterial autolysin. It breaks down the N-acetylmuramoyl-L-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae and requires the presence of choline residues in the cell-wall teichoic acids for activity. Genetic experiments have supported the hypothesis that its 36-kDa chain has evolved by the fusion of two independent modules: the NH2-terminal module, responsible for the catalytic activity, and the COOH-terminal module, involved in the attachment to the cell wall. The structural organization of LytA amidase and of its isolated COOH-terminal module (C-LytA) and the variations induced by choline binding have been examined by differential scanning calorimetry and analytical ultracentrifugation. Deconvolution of calorimetric curves have revealed a folding of the polypeptide chain in several independent or quasi-independent cooperative domains. Elementary transitions in C-LytA are close but not identical to those assigned to the COOH-terminal module in the complete amidase, particularly in the absence of choline. These results indicate that the NH2-terminal region of the protein is important for attaining the native tertiary fold of the COOH terminus. Analytical ultracentrifugation studies have shown that LytA exhibits a monomer <--> dimer association equilibrium, through the COOH-terminal part of the molecule. Dimerization is regulated by choline interaction and involves the preferential binding of two molecules of choline per dimer. Sedimentation velocity experiments give frictional ratios of 1.1 for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that deviated from that of globular rigid particles. When considered together, present results give evidence that LytA amidase might be described as an elongated molecule consisting of at least four domains per subunit (two per module) designated here in as N1, N2, C1, and C2. Intersubunit cooperative interactions through the C2 domain in LytA dimer occur under all experimental conditions, while C-LytA requires the saturation of low affinity choline binding sites. The relevance of the structural features deduced here for LytA amidase is examined in connection with its biological function.