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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-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 recMoPrPc23–231 (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).
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pone-0098753-g004: 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 recMoPrPc23–231 (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).

Mentions: In addition to CD analysis of ShPrP 90–232 and MoPrP 90–231 oligomers, the FTIR of the amide I band was used to characterize MoPrP 23–231 oligomers. The full-length construct was used so that we could focus on the characterization of the more physiologically relevant full-length recMoPrP 23–231 construct. The FTIR spectrum is shown for an oligomer sample from 0.4 mg/mL recMoPrP 23–231 shaken at 250 rpm and 37°C for 3 days (Fig. 4). The FTIR spectrum along with its second derivative of the oligomer sample shows the presence of different peaks compared to those found for the predominantly α-helical recMoPrPc23–231. Spectral deconvolution was performed on the FTIR absorbance spectra to determine the secondary structure composition of these oligomers (Fig. 4B). The areas of the resulting Gaussian peaks seen in Fig. 4B were used to determine the structural content (Table 1). The FTIR data shows that the recMoPrP 23–231 sample transitioned from 33% α-helix in the recPrPc sample to only 16% α-helix in the oligomeric sample. In its place the structure of the oligomers has transitioned to 18% β-sheet with significant turn and loop peaks (∼1662 and 1679 cm−1).


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

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

pone-0098753-g004: 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 recMoPrPc23–231 (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).
Mentions: In addition to CD analysis of ShPrP 90–232 and MoPrP 90–231 oligomers, the FTIR of the amide I band was used to characterize MoPrP 23–231 oligomers. The full-length construct was used so that we could focus on the characterization of the more physiologically relevant full-length recMoPrP 23–231 construct. The FTIR spectrum is shown for an oligomer sample from 0.4 mg/mL recMoPrP 23–231 shaken at 250 rpm and 37°C for 3 days (Fig. 4). The FTIR spectrum along with its second derivative of the oligomer sample shows the presence of different peaks compared to those found for the predominantly α-helical recMoPrPc23–231. Spectral deconvolution was performed on the FTIR absorbance spectra to determine the secondary structure composition of these oligomers (Fig. 4B). The areas of the resulting Gaussian peaks seen in Fig. 4B were used to determine the structural content (Table 1). The FTIR data shows that the recMoPrP 23–231 sample transitioned from 33% α-helix in the recPrPc sample to only 16% α-helix in the oligomeric sample. In its place the structure of the oligomers has transitioned to 18% β-sheet with significant turn and loop peaks (∼1662 and 1679 cm−1).

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