Analysis of rPrP physical properties under solvent conditions used for PMCA.

<p>(A) Analysis of hydrodynamic radius of rPrP at concentration 1 μM, 3 μM or 5 μM as indicate in 10 mM Na-acetate buffer, pH 5 (buffer condition # 1), 10 mM Na-acetate buffer, pH 5, 135 mM NaCl, 2 mM EDTA (condition # 2), or 20 mM Tris-HCl buffer, pH 7.5, 135 mM NaCl, 2 mM EDTA (condition # 3). Error bars represent percentage polydispersity for each sample (B) Far UV spectra of rPrP (5 μM) collected in 10 mM Na-acetate buffer, pH 5 (buffer condition # 1, open triangles), 20 mM Tris-HCl buffer, pH 7.5 in the absence (solid circle) or presence of 135 mM NaCl (open circle). (C) Temperature-induced unfolding of rPrP monitored by a circular dichroism at 222 nm and conducted in buffer condition # 1 (square), # 2 (circle), or # 3 (triangle). The solid lines represent the nonlinear least-square fit of the data to a two-state unfolding model. (D) The apparent melting temperatures (Tm) of rPrP denaturation measured in three buffer conditions specified in the panel A. (D) (E) Dependence of apparent melting temperature (Tm) on rPrP concentration. The temperature-induced denaturation of rPrP was conducted in 20mM Tris-HCl buffer, pH 7.5, 135 mM NaCl, 2mM EDTA and monitored by a circular dichroism at 222 nm.</p

Effect of PE and POPG on conformation and stability of rPrP.

<p>(A) Far UV CD spectra of rPrP (5μM) alone (black squares) or in the presence of 10 μM PE (circle) or POPG (triangle). (B) Temperature-induced unfolding of rPrP in the absence of lipids (solid square) or presence of PE at following concentrations: 50 μM (black circles), 150 μM (black triangle), 250 μM (white triangels) or 500 μM (white diamonds). The data were normalized and the solid lines represent the nonlinear least-square fit of the data to a two-state unfolding model. (C) Temperature-induced unfolding of rPrP (20 μM) in the absence of lipids (square) or presence of 50 μM POPG (triangle). All experiments were performed in 20 mM Tris-HCl buffer, pH 7.5, 135 mM NaCl, 2 mM EDTA and 0.05% Triton. Temperature-induced unfolding was monitored by a circular dichroism at 222 nm.</p

Analysis of tryptophan emission for assessing solvent environment.

<p>Fluorescent emission spectra collected for 10 μM rPrP alone (upper panel) or in the presence of 20 μM PE (middle panel) or 20 μM POPG (lower panel). In upper and middle panel, single Gaussian components are represented by dotted lines. In lower panel, individual Gaussian components are represented by dashed lines and their sum is represented by dotted line. All experiments were performed in 20 mM Tris-HCl buffer, pH 7.5, 135 mM NaCl, 2 mM EDTA and 0.05% Triton.</p

Solvent conditions used in two PMCA protocols for converting rPrP into PrP^Sc.

<p>Solvent conditions used in two PMCA protocols for converting rPrP into PrP^Sc.</p

Melting temperature of rPrP denaturation in the presence of PE.

<p>Melting temperature of rPrP denaturation in the presence of PE.</p

Analysis of growth rate of PMCAb-derived PrP^Sc.

<p>PrP^Sc amplification yield plotted as a function of PMCAb cycle number (left panels) or cumulative incubation interval length (right panels) obtained in PMCAb reactions consisting of incubation intervals with lengths of 30 min (▴), 10 min (□) or 5 min (•) between sonications and seeded with PMCAb-derived HY (A, B), 263K (C, D), SSLOW (E, F) or LOTSS (G, H). Three independent experiments for each condition were performed and average ± SD is shown. To produce PMCAb-derived PrP^Sc, sPMCAb were seeded with brain-derived PrP^Sc, then six standard sPMCAb rounds were conducted.</p

Assessment of Strain-Specific PrP^Sc Elongation Rates Revealed a Transformation of PrP^Sc Properties during Protein Misfolding Cyclic Amplification

<div><p>Prion replication is believed to consist of two components, a growth or elongation of infectious isoform of the prion protein (PrP^Sc) particles and their fragmentation, a process that provides new replication centers. The current study introduced an experimental approach that employs Protein Misfolding Cyclic Amplification with beads (PMCAb) and relies on a series of kinetic experiments for assessing elongation rates of PrP^Sc particles. Four prion strains including two strains with short incubation times to disease (263K and Hyper) and two strains with very long incubation times (SSLOW and LOTSS) were tested. The elongation rate of brain-derived PrP^Sc was found to be strain-specific. Strains with short incubation times had higher rates than strains with long incubation times. Surprisingly, the strain-specific elongation rates increased substantially for all four strains after they were subjected to six rounds of serial PMCAb. In parallel to an increase in elongation rates, the percentages of diglycosylated PrP glycoforms increased in PMCAb-derived PrP^Sc comparing to those of brain-derived PrP^Sc. These results suggest that PMCAb selects the same molecular features regardless of strain initial characteristics and that convergent evolution of PrP^Sc properties occurred during <em>in vitro</em> amplification. These results are consistent with the hypothesis that each prion strain is comprised of a variety of conformers or ‘quasi-species’ and that change in the prion replication environment gives selective advantage to those conformers that replicate most effectively under specific environment.</p> </div

Schematic diagrams illustrating two alternative hypotheses on origin of prion mutations.

<p>(<b>A</b>) The “cloud” hypothesis proposes that prion isolates are intrinsically heterogeneous and consists of major (red) and minor (various colors) PrP^Sc variants. Changes in the replication environment might provide selective advantages for replication of a minor variant leading to transformation of the PrP^Sc population. (<b>B</b>) The deformed templating model postulates that diverse structural variants are generated as a result of changes in replication environment via numerous PrP^Sc-dependent trial-and-error seeding events. A newly generated variant that fits better than parent PrP^Sc to the altered environment replaces the original PrP^Sc variant.</p

Statistical analysis of brain-derived and PMCAb-derived amplification yields.

<p>Amplification yields of brain-derived (A, C, E, D) and PMCAb-derived (B, D, F, H) PrP^Sc for HY (A, B), 263K (C, D), SSLOW (E, F) and LOTSS (G, H) measured at 48 cycles. Three independent experiments for each condition were performed and average ± SD is shown. Statistical analyses were performed using Student's t-test. *P<0.05; **P<0.01; ***P<0.001; NS, non-significant.</p

Assessing glycoform content in brain-derived and PMCAb-derived PrP^Sc.

<p>(A) Western blot of brain-derived and PMCAb-derived PrP^Sc of 263K, HY, SSLOW and LOTSS. Arrow indicates diglycosylated PrP^Sc, while arrowhead points at monoglycosylated PrP^Sc glycoforms. To produce PMCAb-derived PrP^Sc, sPMCAb reactions were seeded with scrapie brain material and subjected to six standard sPMCAb rounds. Undigested NBH is provided as a reference. (B) PK resistance profiles for brain derived (top panel) and PMCAb-derived (bottom panel) PrP^Sc of 263K (blue), HY (green), SSLOW (red) and LOTSS (brown).</p