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Shaking alone induces de novo conversion of recombinant prion proteins to β-sheet rich oligomers and fibrils.

Ladner-Keay CL, Griffith BJ, Wishart DS - PLoS ONE (2014)

Bottom Line: This conversion does not require any denaturant, detergent, or any other chemical cofactor.Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample.These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

View Article: PubMed Central - PubMed

Affiliation: Department of Computing Science, University of Alberta, Edmonton, Alberta, Canada; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada; National Institute for Nanotechnology, Edmonton, Alberta, Canada.

ABSTRACT
The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

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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 recMoPrPc23–231(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).
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pone-0098753-g006: 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 recMoPrPc23–231(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).

Mentions: FTIR spectroscopy was also used to characterize the fully converted, shaking-induced fibrils. The extent of their conversion and fibril content was confirmed by RENAGE. Figure 6A shows the FTIR absorbance spectra and second derivative of both the full-length, native recMoPrPc23–231 and the same protein fully converted to fibrils via shaking. The negative peaks seen in the second derivative spectra were used to assign the secondary structure components based on previously assigned PrPsc FTIR spectra [29] and de novo PrP fibril FTIR spectra [30]. For the shaking-induced PrP fibrils, the prominent peaks are a 1627 cm−1 peak assigned to intermolecular hydrogen bonds characteristic of β-sheets and a 1634 cm−1 peak assigned to β-pleated sheets. In comparison, in the recMoPrPc23–231 spectrum the predominant peak is 1652 cm−1, which is the characteristic absorbance of α-helices (Fig. 6A). FTIR spectral deconvolution was used to determine the percentage of secondary structure components from the shaking-induced fibril FTIR spectrum (Fig. 6B). Table 2 shows the full peak assignment and secondary structure percentages determined from Gaussian deconvolution of the FTIR spectrum. From this spectral deconvolution analysis it was determined that shaking-induced fibrils contained 38% β-sheet and 5% α-helix (Table 2). In addition, we compared the FTIR spectrum obtained for shaking-induced fibrils with those from fibrils generated by shaking PrPc in 2 M guanidine HCl (GdnHCl) and 1 M urea, pH 7 as described by Baskakov and colleagues [4], [30] (Fig. S2A). Differences between the fibrils formed by the two conversion methods are very slight, but there appears to be small differences in the amount of intermolecular hydrogen bonding (1627 cm−1) and intramolecular β-pleated sheet (1634 to 1637 cm−1). In particular the GdnHCl/urea-formed fibrils exhibit a slight increase in the amount of the intermolecular hydrogen bonding (1627 cm−1) compared to the shaking-induced fibrils. (Fig. S2 and Table S1).


Shaking alone induces de novo conversion of recombinant prion proteins to β-sheet rich oligomers and fibrils.

Ladner-Keay CL, Griffith BJ, Wishart DS - PLoS ONE (2014)

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 recMoPrPc23–231(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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043794&req=5

pone-0098753-g006: 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 recMoPrPc23–231(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).
Mentions: FTIR spectroscopy was also used to characterize the fully converted, shaking-induced fibrils. The extent of their conversion and fibril content was confirmed by RENAGE. Figure 6A shows the FTIR absorbance spectra and second derivative of both the full-length, native recMoPrPc23–231 and the same protein fully converted to fibrils via shaking. The negative peaks seen in the second derivative spectra were used to assign the secondary structure components based on previously assigned PrPsc FTIR spectra [29] and de novo PrP fibril FTIR spectra [30]. For the shaking-induced PrP fibrils, the prominent peaks are a 1627 cm−1 peak assigned to intermolecular hydrogen bonds characteristic of β-sheets and a 1634 cm−1 peak assigned to β-pleated sheets. In comparison, in the recMoPrPc23–231 spectrum the predominant peak is 1652 cm−1, which is the characteristic absorbance of α-helices (Fig. 6A). FTIR spectral deconvolution was used to determine the percentage of secondary structure components from the shaking-induced fibril FTIR spectrum (Fig. 6B). Table 2 shows the full peak assignment and secondary structure percentages determined from Gaussian deconvolution of the FTIR spectrum. From this spectral deconvolution analysis it was determined that shaking-induced fibrils contained 38% β-sheet and 5% α-helix (Table 2). In addition, we compared the FTIR spectrum obtained for shaking-induced fibrils with those from fibrils generated by shaking PrPc in 2 M guanidine HCl (GdnHCl) and 1 M urea, pH 7 as described by Baskakov and colleagues [4], [30] (Fig. S2A). Differences between the fibrils formed by the two conversion methods are very slight, but there appears to be small differences in the amount of intermolecular hydrogen bonding (1627 cm−1) and intramolecular β-pleated sheet (1634 to 1637 cm−1). In particular the GdnHCl/urea-formed fibrils exhibit a slight increase in the amount of the intermolecular hydrogen bonding (1627 cm−1) compared to the shaking-induced fibrils. (Fig. S2 and Table S1).

Bottom Line: This conversion does not require any denaturant, detergent, or any other chemical cofactor.Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample.These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

View Article: PubMed Central - PubMed

Affiliation: Department of Computing Science, University of Alberta, Edmonton, Alberta, Canada; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada; National Institute for Nanotechnology, Edmonton, Alberta, Canada.

ABSTRACT
The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

Show MeSH
Related in: MedlinePlus