ThT fluorescence detection of amyloid formation during spontaneous and PrP^Sc-seeded fibrillation. (A) Amyloid formation of 100 ng μl^‑1 ovrecPrP(25‑233) in volumes of 150 μl was analyzed in 96-well plates in absence or presence of PrP^Sc-seeds obtained by PTA-precipitation of scrapie sheep brain homogenate. As controls for PrP^Sc-seeded fibrillation, PTA-precipitated scrapie sheep brain homogenate in absence of ovrecPrP(25-233) (PrP^Sc seed only) and PTA-precipitated normal sheep brain homogenate in presence of ovrecPrP(25-233) (neg. seed + recPrP) demonstrate the specificity of the fibrillation assay. (B) Amyloid formation of 1000 ng μl⁻¹ ovrecPrP(25‑233) in NMR-sufficient volumes of 10 ml was analyzed. Note that seeded fibril growth (PrP^Sc seed + recPrP) is completed before spontaneous conversion (recPrP only) starts. The decrease in ThT-fluorescence intensity after long times is attributed to lateral fibril association and blockage of ThT-binding sites. Values shown are means ± standard errors for up to 10 distinct experiments. The gradually increasing error bars reflect the fibril size distribution in a heterogeneous system.

Immunoblots of samples inoculated into transgenic mice and of brain material from transgenic mice. (A) PrP^Sc in brains of tgshpXI (ARQ) and tg338 (VRQ) mice, inoculated with scrapie PrP^Sc, after spontaneous fibrillation of ovrecPrP(25‑233) (SP), or with non-fibrillated ovrecPrP(25‑233) (NON), respectively, with (+) or without (−) digestion with 50 μg/ml PK for 2 h at 37°C. Only mice inoculated with fibrillated ovrecPrP(25‑233) show PK-resistant PrP27‑30 identical to scrapie PrP27‑30. (B) Spontaneously fibrillated ovrecPrP(25‑233) after digestion with increasing concentrations of PK for 1 h at 37°C does not show any PK-resistance. (C) For comparison, PrP^Sc-seeds are depicted as characterized by a partial PK-resistance even at high PK-concentrations. (D) PrP^Sc-seeded ovrecPrP(25‑233) after digestion with increasing concentrations of PK for 1 h at 37°C does not show any PK-resistance. In light of the 300-fold excess of ovrecPrP(25‑233) during seeded fibrillation (see Materials and Methods for details), the undigested PrP^Sc-specific bands are too weak to show any PK-resistance after digestion. Any atypical PrP-bands were not observed. Apparent molecular masses based on migration of protein standards are indicated in kDa.

Secondary structural properties. As revealed by CD-spectroscopy, the secondary structural transition of monomeric ovrecPrP(25 233) due to spontaneous and PrP^Sc-seeded fibrillation results in spectral minima at 218 nm typical for β-sheets. Zero crossings at 204 nm and spectral maxima at 193 nm are probable to indicate additional α-helical/random coil elements. For comparison, a spectrum of α‑helical monomeric ovrecPrP(25-233) is also presented. All given values are mean residue molar ellipticities ([θ]_MRW).

Ultrastructural properties. Low magnification amplitude AFM-images (A and B) and corresponding fibril width distributions (C and D) obtained after (A and C) spontaneous or (B and D) PrP^Sc-seeded fibrillation of ovrecPrP(25-233) display variability in fibrillar topology. The black curves in (C and D) are fits with a Gaussian function illustrating that spontaneous and PrP^Sc-seeded ovrecPrP-fibrils have identical average widths within the margin of error. However, spontaneously generated ovrecPrP-fibrils have only a length of up 400 nm (A), whereas PrP^Sc-fibrils in scrapie-infected sheep brain homogenate (E) and PrP^Sc-seeded fibrils (B) are several μm long. In (A), an inset of higher magnification shows the morphology of spontaneously generated ovrecPrP-fibrils to higher details. Immediately before AFM-measurement, all samples were subjected to mild sonication for 10 sec. Thus, (E) depicts PrP^Sc-fibrils as they were used for seeding of ovrecPrP(25‑233).

2D-(¹³C‑¹³C)- and (¹⁵N‑¹³C)-solid-state NMR correlation spectra. For identification of spin systems, homo- and heteronuclear correlation spectra were analyzed jointly. The aliphatic regions of (A) (¹³C‑¹³C)- and (B) (¹⁵N‑¹³C)-correlation spectra after spontaneous fibrillation of ovrecPrP(25‑233) are depicted, acquired with (A) PDSD-mixing for 20 ms at 11 kHz MAS and (B) DREAM-mixing at 23 kHz MAS, to obtain intraresidue correlations. All cross peaks are labeled according to Tables S1, S2, and S3. Note that in (B) several tentative assignments are present more than one time, reflecting the ambiguity as summarized in Table S3.

Secondary chemical shifts. A comparison of secondary chemical shifts of ovrecPrP(25‑233) after spontaneous (black) or PrP^Sc-seeded (red) conversion suggests that both amyloid types share a similar overall arrangement of secondary structure elements. All amino acid residues are listed in alphabetic order.

Structure and dynamics details. Solid-state NMR comparison of ovrecPrP(25‑233) after spontaneous (black) or PrP^Sc-seeded (red) conversion reveals an increased flexibility after PrP^Sc-seeding. (A) Overlay of 2D-(¹³C‑¹³C)-correlation spectra acquired with the identical number of transients and increments and processed identically. DREAM mixing at 23 kHz was performed to obtain only direct correlations. Spin systems after PrP^Sc-seeded conversion for which cross peaks are weakened up to almost completely vanished on both sides of the diagonal are indicated by arrows. (B) For intensity analysis, Cα‑Cβ/Cβ‑Cα-peak volumes in α‑helical and β‑sheet chemical shift ranges of 2D-(¹³C-¹³C)-DREAM spectra were calculated. Peak volumes were normalized 2-fold: (1) To account for deviating protein amounts in the samples, all values were standardized to the peak volume of the corresponding spectrum diagonal. (2) The highest peak volume of each amino acid type (α‑helical black or β‑sheet black or α‑helical red or β‑sheet red) was set to 100 %. Note that α‑helical and β‑sheet spin systems are not present for all amino acid types. Whereas all threonines are located in β‑strands, all arginines and leucines are characterized by α‑helical secondary structure signatures. Prolines are not known to be present in any secondary structure. Valines are located in α‑helical as well as β‑strand conformations. (C–E) High magnification overlays of some exemplary regions of (C) leucine, (D) proline and valine, and (E) alanine residues show that cross peak intensities for prolines (all N‑terminal of position 168), α‑helical valines, α‑helical leucines (at positions 128, 133, and 141), and α‑helical alanines (clustered between positions 116 and 136) are reduced or even completely vanished, whereas β‑strand alanines and β‑strand valines do not display a decrease in cross peak intensities.