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Progress towards structural understanding of infectious sheep PrP-amyloid.

Müller H, Brener O, Andreoletti O, Piechatzek T, Willbold D, Legname G, Heise H - Prion (2014)

Bottom Line: Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-sheet core C-terminal of residue ∼155.These findings are not in agreement with all current models for PrP-amyloid.We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region.

View Article: PubMed Central - PubMed

Affiliation: a Institute of Complex Systems; ICS-6: Structural Biochemistry; Forschungszentrum Jülich (FZJ) ; Jülich , Germany.

ABSTRACT
The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-sheet core C-terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.

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Related in: MedlinePlus

Structure and dynamics details. Solid-state NMR comparison of ovrecPrP(25‑233) after spontaneous (black) or PrPSc-seeded (red) conversion reveals an increased flexibility after PrPSc-seeding. (A) Overlay of 2D-(13C‑13C)-correlation spectra acquired with the identical number of transients and increments and processed identically. DREAM mixing at 23 kHz was performed to obtain only direct correlations. Spin systems after PrPSc-seeded conversion for which cross peaks are weakened up to almost completely vanished on both sides of the diagonal are indicated by arrows. (B) For intensity analysis, Cα‑Cβ/Cβ‑Cα-peak volumes in α‑helical and β‑sheet chemical shift ranges of 2D-(13C-13C)-DREAM spectra were calculated. Peak volumes were normalized 2-fold: (1) To account for deviating protein amounts in the samples, all values were standardized to the peak volume of the corresponding spectrum diagonal. (2) The highest peak volume of each amino acid type (α‑helical black or β‑sheet black or α‑helical red or β‑sheet red) was set to 100 %. Note that α‑helical and β‑sheet spin systems are not present for all amino acid types. Whereas all threonines are located in β‑strands, all arginines and leucines are characterized by α‑helical secondary structure signatures. Prolines are not known to be present in any secondary structure. Valines are located in α‑helical as well as β‑strand conformations. (C–E) High magnification overlays of some exemplary regions of (C) leucine, (D) proline and valine, and (E) alanine residues show that cross peak intensities for prolines (all N‑terminal of position 168), α‑helical valines, α‑helical leucines (at positions 128, 133, and 141), and α‑helical alanines (clustered between positions 116 and 136) are reduced or even completely vanished, whereas β‑strand alanines and β‑strand valines do not display a decrease in cross peak intensities.
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f0007: Structure and dynamics details. Solid-state NMR comparison of ovrecPrP(25‑233) after spontaneous (black) or PrPSc-seeded (red) conversion reveals an increased flexibility after PrPSc-seeding. (A) Overlay of 2D-(13C‑13C)-correlation spectra acquired with the identical number of transients and increments and processed identically. DREAM mixing at 23 kHz was performed to obtain only direct correlations. Spin systems after PrPSc-seeded conversion for which cross peaks are weakened up to almost completely vanished on both sides of the diagonal are indicated by arrows. (B) For intensity analysis, Cα‑Cβ/Cβ‑Cα-peak volumes in α‑helical and β‑sheet chemical shift ranges of 2D-(13C-13C)-DREAM spectra were calculated. Peak volumes were normalized 2-fold: (1) To account for deviating protein amounts in the samples, all values were standardized to the peak volume of the corresponding spectrum diagonal. (2) The highest peak volume of each amino acid type (α‑helical black or β‑sheet black or α‑helical red or β‑sheet red) was set to 100 %. Note that α‑helical and β‑sheet spin systems are not present for all amino acid types. Whereas all threonines are located in β‑strands, all arginines and leucines are characterized by α‑helical secondary structure signatures. Prolines are not known to be present in any secondary structure. Valines are located in α‑helical as well as β‑strand conformations. (C–E) High magnification overlays of some exemplary regions of (C) leucine, (D) proline and valine, and (E) alanine residues show that cross peak intensities for prolines (all N‑terminal of position 168), α‑helical valines, α‑helical leucines (at positions 128, 133, and 141), and α‑helical alanines (clustered between positions 116 and 136) are reduced or even completely vanished, whereas β‑strand alanines and β‑strand valines do not display a decrease in cross peak intensities.

Mentions: Higher mean residue molar ellipticity intensities after PrPSc-seeding indicated an increased proportion of ovrecPrP-conformers amenable to CD analysis which implies higher flexibility of at least some segments of PrPSc-seeded conformations (Fig. 3). In 2D-(13C‑13C)-solid-state NMR spectra using homonuclear transfer based on dipolar recoupling, cross peak intensities of specific amino acid residues, e.g. of proline and leucine residues, are weakened when samples are generated by PrPSc-seeding. To account for possibly deviating hydration states, samples were rehydrated by adding 10 μl of H2O each. No change in the respective intensities or line widths of cross peaks was observed. A comparison of identically recorded and processed 2D-(13C‑13C)-DREAM spectra of identically prepared (except for the presence or absence of PrPSc-seeds during fibrillation) 10‑mg samples of both amyloid types confirmed an increased flexibility after PrPSc-seeding. An overlay of the aliphatic regions of these spectra is depicted in Figure 7A, whereas Figure 7C–E display some regions of interest in higher magnification. DREAM spectra display cross peaks only for correlations between directly bound 13C atoms and thus allowed for quantitative intensity analysis. Since only some cross peaks of lysine, glutamine, glutamate, aspartate, histidine, isoleucine, asparagine, serine, and tyrosine residues differed in intensity, a quantitative conclusion could hardly be drawn for them without heteronuclear 3D-spectra reducing the number of overlapping cross peaks. However, we determined the volumes of all Cα‑Cβ‑/Cβ‑Cα‑cross peaks with α‑helical or β‑sheet chemical shift signatures, respectively, for the spectrally separated proline, valine, arginine, leucine, alanine, and threonine residues.(Fig. 7B). For threonine residues (not present between positions 115–155; 9 of 11 threonine residues are C‑terminal of position 185), no difference in the normalized cross peak intensities was observed comparing spontaneously generated and PrPSc-seeded samples. Cross peak intensities of β‑sheet alanine and β‑sheet valine residues also did not differ within an error margin of 10%. In contrast, all proline cross peaks almost completely vanished in the spectra of PrPSc-seeded samples. The most C-terminal of 15 ovrecPrP(25‑233) proline residues is located at sequence position 168. The substantially reduced proline intensities therefore suggest that PrPSc-seeding results in enhanced flexibility of the N‑terminal fibril segment. The cross peaks of α‑helical alanine, α‑helical leucine, α‑helical arginine, and α‑helical valine residues are also considerably weakened after PrPSc-seeding (Fig. 7). Seven of 9 alanine residues are clustered between positions 116 and 136. All 3 leucine residues are at positions 128, 133, and 141. Eight of 11 arginine residues are located N‑terminal of position 168. Valine residues with α‑helical chemical shift signatures are believed to be located between residues 115 to 155 (see above). Taken together, only cross peak intensities of residues N‑terminal of Pro168 seem to be affected indicating again that PrPSc-seeding induces a more flexible fibril region N-terminal of Pro168.Figure 7.


Progress towards structural understanding of infectious sheep PrP-amyloid.

Müller H, Brener O, Andreoletti O, Piechatzek T, Willbold D, Legname G, Heise H - Prion (2014)

Structure and dynamics details. Solid-state NMR comparison of ovrecPrP(25‑233) after spontaneous (black) or PrPSc-seeded (red) conversion reveals an increased flexibility after PrPSc-seeding. (A) Overlay of 2D-(13C‑13C)-correlation spectra acquired with the identical number of transients and increments and processed identically. DREAM mixing at 23 kHz was performed to obtain only direct correlations. Spin systems after PrPSc-seeded conversion for which cross peaks are weakened up to almost completely vanished on both sides of the diagonal are indicated by arrows. (B) For intensity analysis, Cα‑Cβ/Cβ‑Cα-peak volumes in α‑helical and β‑sheet chemical shift ranges of 2D-(13C-13C)-DREAM spectra were calculated. Peak volumes were normalized 2-fold: (1) To account for deviating protein amounts in the samples, all values were standardized to the peak volume of the corresponding spectrum diagonal. (2) The highest peak volume of each amino acid type (α‑helical black or β‑sheet black or α‑helical red or β‑sheet red) was set to 100 %. Note that α‑helical and β‑sheet spin systems are not present for all amino acid types. Whereas all threonines are located in β‑strands, all arginines and leucines are characterized by α‑helical secondary structure signatures. Prolines are not known to be present in any secondary structure. Valines are located in α‑helical as well as β‑strand conformations. (C–E) High magnification overlays of some exemplary regions of (C) leucine, (D) proline and valine, and (E) alanine residues show that cross peak intensities for prolines (all N‑terminal of position 168), α‑helical valines, α‑helical leucines (at positions 128, 133, and 141), and α‑helical alanines (clustered between positions 116 and 136) are reduced or even completely vanished, whereas β‑strand alanines and β‑strand valines do not display a decrease in cross peak intensities.
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Related In: Results  -  Collection

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f0007: Structure and dynamics details. Solid-state NMR comparison of ovrecPrP(25‑233) after spontaneous (black) or PrPSc-seeded (red) conversion reveals an increased flexibility after PrPSc-seeding. (A) Overlay of 2D-(13C‑13C)-correlation spectra acquired with the identical number of transients and increments and processed identically. DREAM mixing at 23 kHz was performed to obtain only direct correlations. Spin systems after PrPSc-seeded conversion for which cross peaks are weakened up to almost completely vanished on both sides of the diagonal are indicated by arrows. (B) For intensity analysis, Cα‑Cβ/Cβ‑Cα-peak volumes in α‑helical and β‑sheet chemical shift ranges of 2D-(13C-13C)-DREAM spectra were calculated. Peak volumes were normalized 2-fold: (1) To account for deviating protein amounts in the samples, all values were standardized to the peak volume of the corresponding spectrum diagonal. (2) The highest peak volume of each amino acid type (α‑helical black or β‑sheet black or α‑helical red or β‑sheet red) was set to 100 %. Note that α‑helical and β‑sheet spin systems are not present for all amino acid types. Whereas all threonines are located in β‑strands, all arginines and leucines are characterized by α‑helical secondary structure signatures. Prolines are not known to be present in any secondary structure. Valines are located in α‑helical as well as β‑strand conformations. (C–E) High magnification overlays of some exemplary regions of (C) leucine, (D) proline and valine, and (E) alanine residues show that cross peak intensities for prolines (all N‑terminal of position 168), α‑helical valines, α‑helical leucines (at positions 128, 133, and 141), and α‑helical alanines (clustered between positions 116 and 136) are reduced or even completely vanished, whereas β‑strand alanines and β‑strand valines do not display a decrease in cross peak intensities.
Mentions: Higher mean residue molar ellipticity intensities after PrPSc-seeding indicated an increased proportion of ovrecPrP-conformers amenable to CD analysis which implies higher flexibility of at least some segments of PrPSc-seeded conformations (Fig. 3). In 2D-(13C‑13C)-solid-state NMR spectra using homonuclear transfer based on dipolar recoupling, cross peak intensities of specific amino acid residues, e.g. of proline and leucine residues, are weakened when samples are generated by PrPSc-seeding. To account for possibly deviating hydration states, samples were rehydrated by adding 10 μl of H2O each. No change in the respective intensities or line widths of cross peaks was observed. A comparison of identically recorded and processed 2D-(13C‑13C)-DREAM spectra of identically prepared (except for the presence or absence of PrPSc-seeds during fibrillation) 10‑mg samples of both amyloid types confirmed an increased flexibility after PrPSc-seeding. An overlay of the aliphatic regions of these spectra is depicted in Figure 7A, whereas Figure 7C–E display some regions of interest in higher magnification. DREAM spectra display cross peaks only for correlations between directly bound 13C atoms and thus allowed for quantitative intensity analysis. Since only some cross peaks of lysine, glutamine, glutamate, aspartate, histidine, isoleucine, asparagine, serine, and tyrosine residues differed in intensity, a quantitative conclusion could hardly be drawn for them without heteronuclear 3D-spectra reducing the number of overlapping cross peaks. However, we determined the volumes of all Cα‑Cβ‑/Cβ‑Cα‑cross peaks with α‑helical or β‑sheet chemical shift signatures, respectively, for the spectrally separated proline, valine, arginine, leucine, alanine, and threonine residues.(Fig. 7B). For threonine residues (not present between positions 115–155; 9 of 11 threonine residues are C‑terminal of position 185), no difference in the normalized cross peak intensities was observed comparing spontaneously generated and PrPSc-seeded samples. Cross peak intensities of β‑sheet alanine and β‑sheet valine residues also did not differ within an error margin of 10%. In contrast, all proline cross peaks almost completely vanished in the spectra of PrPSc-seeded samples. The most C-terminal of 15 ovrecPrP(25‑233) proline residues is located at sequence position 168. The substantially reduced proline intensities therefore suggest that PrPSc-seeding results in enhanced flexibility of the N‑terminal fibril segment. The cross peaks of α‑helical alanine, α‑helical leucine, α‑helical arginine, and α‑helical valine residues are also considerably weakened after PrPSc-seeding (Fig. 7). Seven of 9 alanine residues are clustered between positions 116 and 136. All 3 leucine residues are at positions 128, 133, and 141. Eight of 11 arginine residues are located N‑terminal of position 168. Valine residues with α‑helical chemical shift signatures are believed to be located between residues 115 to 155 (see above). Taken together, only cross peak intensities of residues N‑terminal of Pro168 seem to be affected indicating again that PrPSc-seeding induces a more flexible fibril region N-terminal of Pro168.Figure 7.

Bottom Line: Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-sheet core C-terminal of residue ∼155.These findings are not in agreement with all current models for PrP-amyloid.We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region.

View Article: PubMed Central - PubMed

Affiliation: a Institute of Complex Systems; ICS-6: Structural Biochemistry; Forschungszentrum Jülich (FZJ) ; Jülich , Germany.

ABSTRACT
The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-sheet core C-terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.

Show MeSH
Related in: MedlinePlus