Figure 1.
RENAGE indicates that shaking recPrPc generates oligomers.
A PrP PICUP ladder (lane 1) is used to size the oligomers formed by shaking recMoPrP 90–231 in pH 5.5 buffer at 350 rpm and 37°C for 1 day (lane 2). Shaking-induced oligomers are predominantly a distribution of 8-mers to 13-mers. In comparison oligomers formed in urea and salt exhibit a bimodal size distribution of 7-mers to 12-mers (lane 3). Longer periods of shaking recMoPrP 90–231 (shaking at 350 rpm, 37°C for 2 days) will also generate a fibril band and bimodal distribution of 8-mers to 12-mers and larger oligomers (>16-mers) (lane 4).
Figure 2.
Circular dichroism (CD) indicates that shaking-induced oligomers contain significant quantities of β-sheet.
Shaking recShPrP 90–232 at 350 rpm (in pH 5.5 water and 150 mM NaCl) induces conversion from an α-helical protein (red line) to a β-sheet rich structure (blue line). The inset, on the right, shows the corresponding RENAGE gel of the same sample, indicating a preponderance of oligomers. CDPro analysis for native PrPc gives 43% α-helix and 10% β-sheet, and for oligomers it yields 16% α-helix and 24% β-sheet.
Figure 3.
RENAGE of shaking-converted prions under various conditions.
A) ShPrP 90–232 oligomers are formed by shaking in pH 5.5, 6.2 and 7.4 buffers, at 350 rpm and 37°C. B) RENAGE after shaking at 350 rpm for 1 day with full length recMoPrP 23–231 (lane 1), truncated recMoPrP 90–231 (lane 2) and C-terminal domain recMoPrP 120–231 (lane 3) and after 2 days with recMoPrP 23–231 (lane 4), truncated recMoPrP 90–231 (lane 5) and C-terminal domain recMoPrP 120–231 (lane 6). Samples in panel B were shaken at 350 rpm and 37°C in pH 6.2 buffer. C) Shaking a 0.6 mL solution of recShPrP90–232 in a 0.6 mL centrifuge tube without any air or bubbles for two weeks (lane 1) as compared to the same sample of 0.4 mL in a 1.5 mL centrifuge tube (i.e. with air), shaken for one week (lane 2).
Figure 4.
Fourier transform infrared spectroscopy shows that shaking-induces conversion to oligomers with increased β-sheet structure, dominated by turns and loops.
A) FTIR of oligomers formed by shaking-induced conversion (at 250 rpm and 37°C) of recMoPrP 23–231 (black line) is drastically different from monomeric recMoPrPc 23–231 (grey line). The absorbance spectra are shown in solid lines and the corresponding 2nd derivative spectra are shown in dashed lines. B) Spectral deconvolution and component analysis of the fibril FTIR spectrum (solid line) is fit with Gaussian peaks to a deconvoluted spectrum (dashed line).
Table 1.
Secondary structure composition of shaking-induced oligomers as determined from deconvolution and curve fitting of the FTIR amide I band.
Figure 5.
Electron microscopy confirms the formation of oligomers and fibrils seen in RENAGE.
Negative stain EM of shaking-induced prion oligomers (panel A) and fibrils (panel B). The oligomers shown here were formed from shaking recMoPrP 90–231 at 350 rpm at room temperature for 1 day. The fibril sample was formed by shaking recShPrP 90–232 at 350 rpm at 37°C for 5 days. The corresponding RENAGE analysis of the same sample is shown alongside the micrograph. The indicated scale bar = 100 nm.
Figure 6.
Fourier transform infrared spectroscopy shows that shaking-induced fibrils are rich in β-sheet.
A) FTIR of fibrils formed by shaking-induced conversion of recMoPrP 23–231 (at 250 rpm and 37°C) shows they are rich in β-sheet structure (black line), as compared to monomeric recMoPrPc 23–231(grey line). The absorbance FTIR spectra are shown in solid lines and the corresponding 2nd derivative spectra are shown in dashed lines. B) Spectral deconvolution and component analysis of the fibril FTIR spectrum (black) generated by fitting Gaussian peaks to a deconvoluted spectrum (brown line).
Table 2.
Secondary structure composition of shaking-induced fibrils as determined from deconvolution and curve fitting of the FTIR amide I band.
Figure 7.
Time course of the formation of prion oligomers and fibrils.
A) RENAGE gel of shaking converted recMoPrP 23–231 at 250 rpm and 37°C shows a time dependent loss of monomer, formation of oligomers and subsequent formation of fibrils. The chromatogram profile of each gel lane was acquired to determine the fibril content. A representative profile after 27 hours of shaking is shown. B) Plot of the time dependent thioflavin T (ThT) fluorescence (open diamonds; black line) and RENAGE fibril peak area (purple filled squares; purple line) shows a sigmoidal growth in both ThT fluorescence enhancement and fibril formation.
Figure 8.
Growth of shaking-induced fibrils with seeding is exponential.
A) RENAGE gel of the time course of shaking induced conversion of recMoPrP 23–231 at 250 rpm and 37°C. B) RENAGE gel of shaking induced conversion of the same recMoPrP (same batch) under the same conditions except with seeding using 5% MoPrP 23–231 fibrils into fresh recMoPrPc. C) The chromatogram profile of each gel lane was acquired to determine the fibril content. Time dependent fibril content growth is shown plotted against time and has a sigmoidal dependence when starting with only fresh recMoPrPc 23–231 (open squares, black line). Upon seeding with 5% PrP fibrils the fibril content grows logarithmically (grey circles, grey line).
Figure 9.
Shaking-induced fibrils have Proteinase K resistance.
SDS-PAGE of recMoPrPc 23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.
Figure 10.
Sonication of PrP generates oligomers.
RENAGE of recMoPrP 90–231 sonicated for 8 cycles of 10 seconds each, show that oligomers are generated (lane 1). Furthermore sonication of recMoPrP 23–231 in a single PMCA-like round generates oligomers (lane 2). The formation of oligomers in a PMCA-like round is enhanced by placing the probe inside the solution (lane 3).