SV-analysis of Aβ42 at pH 10.

<p>40 μM Aβ42 had been centrifuged at 40,000 rpm and 20°C. By <i>c</i>(<i>s</i>) analysis the most prominent species was detected at s = 0.6 S (A); GA-MC analysis revealed the dominant species at <i>s</i> = 0.67S with <i>f/f</i>_<i>0</i> = 1.35 (B). Results are presented in a pseudo-3D plot with a color coded third dimension indicating the species fraction. In both evaluations a small amount of near spherical aggregates appeared at 5 S. The minor species detected below 1 S are artifacts due to a base line deconvolution problem.</p

Kinetics of amyloid formation.

<p>ThT-fluorescence kinetics of 40 μM Aβ42 in 10 mM NaP_i at 37°C (black) and 20°C (blue). The maximum RFUs had been normalized to 1. Determined lag times were ~8 h at 37°C and ~80 h at 20°C (A). CD spectra of 37 μM Aβ42 incubated at 20°C for 0 (black), 29 (magenta), 49 (cyan) and 74 h (blue). The spectrum of Aβ42 in NaP_i, pH 7.4 changes within the indicated times from a predominantly random coil spectrum to a β-sheet rich spectrum. Superimposed spectra possess an isosbestic point at 209 nm, indicating a two-state transition (B).</p

Solvent parameters for 10 mM NaPi, pH 7.4 and v- of Aβ42 for the temperatures used in the SV experiments.

<p>Calculations were performed with Sednterp or UltraScan II/III.</p

Observed <i>s</i>-value species of Aβ42.

<p>The oligomeric state was determined for a globular particle with <i>f</i>/<i>f</i>₀ = 1.1. The error was calculated as standard deviation of the mean of <i>s</i>-values obtained by applying the c(s) routine to the given number of different data sets. Different data sets correspond to independent sample preparations.</p

Seeded fibrillation of huPrP.

<p>Different combinations of seeds from NaPTA precipitated brain homogenates of either diseased or control animals and 3 µM huPrP as substrate within ThT amyloid seeding assay. Time dependent ThT fluorescence is shown for 3 µM huPrP in combination with seeds from CJD-affected human brain (<b>A</b>), scrapie-affected sheep brain (<b>B</b>), BSE-affected bovine brain (<b>C</b>), and CWD-affected deer brain (<b>D</b>) (closed circles). The controls (open rectangles) were prepared from the respective non-diseased brain material from human (A) and animals (<b>B; C and D</b>).</p

<i>De novo</i> fibrillation of huPrP – ThT kinetics and TIRF micrographs.

<p><i>De novo</i> fibrillation is shown in (<b>A</b>), an amyloid specific increase in ThT fluorescence is only observable at 0.02% SDS and 9 µM huPrP. (<b>B</b>) <i>De novo</i> generated amyloid fibrils (plateau phase) have been stained by ThT and visualised by TIRF microscopy. (<b>C</b>) Ultrastructure of negative-stained <i>de novo</i> huPrP fibrils obtained by transmission electron microscopy. Bar equals 100 nm.</p

Pre-amyloid and amyloid state of cerPrP.

<p>CD spectra (<b>A</b>) of cerPrP with different concentrations of SDS (0.02–0.2%) have been recorded directly after adapting the SDS buffer conditions. CD spectra show a shift from α-random dominated towards β-sheet rich secondary structure with decreasing concentration of SDS. (<b>B</b>) <i>De novo</i> fibrillation of cerPrP, an amyloid specific increase in ThT fluorescence is observable at 0.02% SDS and 9 µM cerPrP. (<b>C</b>) cerPrP (3 µM) in combination with seeds from CWD brain or control brain.</p

Pre-amyloid state of huPrP – CD spectroscopy and sedimentation velocity analysis of huPrP.

<p>CD spectra (<b>A</b>) of huPrP with varying concentrations of SDS (0.02–0.2%) have been recorded directly after adapting the SDS buffer conditions. CD spectra show a shift from α-random dominated towards β-sheet rich secondary structure with decreasing concentration of SDS. (<b>B and C</b>) A sedimentation velocity experiment at 20°C and 40.000 rpm was performed to determine the state of oligomerisation of huPrP. Fig. 1 <b>B</b> shows the successive radial absorbance scans of 5 µM huPrP, which have been corrected for time and radially invariant noise. For clarity only every 3^rd scan was plotted. Fig. 1 <b>C</b> shows the deviation of measured data points from the fitted sedimentation boundary after the fitting procedure using Ultrascan 3. The absorption difference at 230 nm is plotted in relation to the distance from the rotor axis. The RMSD of the fit was 0.00322 A₂₃₀.</p

Seeding activity of PrP^Sc seeds of different species in huPrP.

<p>The box plot shows seeding activity of seeds from the species human, cattle, sheep and deer in huPrP obtained from identical replicates of single seeding experiments. Seeding activity was calculated by dividing the average of relative fluorescence of samples with PrP^Sc positive seeds and PrP^Sc negative seeds within 80-100 h (plateau phase). For seeds from CJD (N = 7) or BSE (N = 8) material a high seeding activity is observable. For seeds from scrapie (N = 10) or CWD (N = 8) a strong species barrier is apparent.</p

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p