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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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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.
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pone-0098753-g002: 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.

Mentions: CD was used to analyze oligomers that were formed by shaking recShPrP 90–232 and recMoPrP 90–231 and confirmed by RENAGE. This showed that ShPrP 90–232 oligomers have a β-sheet structure (Fig. 2). An aliquot of the same sample used for CD was analyzed by RENAGE and exhibited a bimodal size distribution with smaller oligomers (octamers to dodecamers) and larger oligomers (>16-mers), but no or little fibril band. Secondary structure content of these shaking-induced oligomers was found to be 25% β-sheet and 12% α-helix for recShPrP 90–231 as calculated by CDPro. A similar CD spectrum was acquired for a shaken recMoPrP 90-231 sample with approximately equal abundance of oligomers and fibrils (by RENAGE). These MoPrP90–231 oligomers were also found to be β-sheet rich (24% β-sheet from CDPro). In contrast, monomeric recShPrPc90–232 and recMoPrPc90–231 contained 42% α-helix and 10% β-sheet. This result is consistent with previous findings that the appearance of discreet oligomer bands seen by RENAGE match with having a β-sheet structure [22].


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098753-g002: 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.
Mentions: CD was used to analyze oligomers that were formed by shaking recShPrP 90–232 and recMoPrP 90–231 and confirmed by RENAGE. This showed that ShPrP 90–232 oligomers have a β-sheet structure (Fig. 2). An aliquot of the same sample used for CD was analyzed by RENAGE and exhibited a bimodal size distribution with smaller oligomers (octamers to dodecamers) and larger oligomers (>16-mers), but no or little fibril band. Secondary structure content of these shaking-induced oligomers was found to be 25% β-sheet and 12% α-helix for recShPrP 90–231 as calculated by CDPro. A similar CD spectrum was acquired for a shaken recMoPrP 90-231 sample with approximately equal abundance of oligomers and fibrils (by RENAGE). These MoPrP90–231 oligomers were also found to be β-sheet rich (24% β-sheet from CDPro). In contrast, monomeric recShPrPc90–232 and recMoPrPc90–231 contained 42% α-helix and 10% β-sheet. This result is consistent with previous findings that the appearance of discreet oligomer bands seen by RENAGE match with having a β-sheet structure [22].

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