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A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S - PLoS Pathog. (2015)

Bottom Line: Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163.Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers.Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

No MeSH data available.


Related in: MedlinePlus

Raman spectroscopy of cofactor and protein-only PrPSc, focusing on spectral regions assigned to tyrosine side chains.The primary sequence of the region examined by DXMS is shown, with residues 144–163 boxed and tyrosine residues highlighted in green. Raman shifts corresponding to the ν(C = C) ring mode (~1620 cm-1) and the tyrosine Fermi-doublet (~850 and 830 cm-1) are shown.
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ppat.1005017.g004: Raman spectroscopy of cofactor and protein-only PrPSc, focusing on spectral regions assigned to tyrosine side chains.The primary sequence of the region examined by DXMS is shown, with residues 144–163 boxed and tyrosine residues highlighted in green. Raman shifts corresponding to the ν(C = C) ring mode (~1620 cm-1) and the tyrosine Fermi-doublet (~850 and 830 cm-1) are shown.

Mentions: We further sought to confirm a conformational difference between cofactor and protein-only PrPSc in the α1-β2 domain using Raman spectroscopy (Fig 4). Analysis of the Raman spectra acquired from these two conformers identified multiple Raman shifts that could be assigned to tyrosine residues, which are plentiful in the PrP C-terminus and specifically enriched in the α1-β2 domain (6 of 11 total C-terminal tyrosines). By Raman spectroscopy, protein-only PrPSc appears to contain more exposed tyrosine residues than cofactor PrPSc as evidenced from the increased ring ν(C = C) intensity at ~1620 cm-1 (Fig 4, left panel), the 850 cm-1/830 cm-1 ratio being greater than 1 (Fig 4, right panel) [22], and the increased ring ν(CH) intensity at ~3075 cm-1 (S8 Fig, left panel) [23,24], consistent with our DXMS results. In addition, and also consistent with our DXMS data, protein-only PrPSc appears to contain more exposed CNH groups than cofactor PrPSc as indicated by the increased intensity in the 1530–1580 cm-1 Amide II region, corresponding to ν(CN) and δ(CNH) Raman shifts (S8 Fig, right panel), as well as an increased ν(CN) intensity at ~3300 cm-1 (S8 Fig, left panel). These exposed CNH groups likely originate from the 4 exposed arginine (R) and single exposed glutamine (Q), asparagine (N) and tryptophan (W) residues in the α1-β2 domain, or from CNH-containing side chains in the N-terminal portion of the PK-resistant PrPSc core (residues ~91–115).


A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S - PLoS Pathog. (2015)

Raman spectroscopy of cofactor and protein-only PrPSc, focusing on spectral regions assigned to tyrosine side chains.The primary sequence of the region examined by DXMS is shown, with residues 144–163 boxed and tyrosine residues highlighted in green. Raman shifts corresponding to the ν(C = C) ring mode (~1620 cm-1) and the tyrosine Fermi-doublet (~850 and 830 cm-1) are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488359&req=5

ppat.1005017.g004: Raman spectroscopy of cofactor and protein-only PrPSc, focusing on spectral regions assigned to tyrosine side chains.The primary sequence of the region examined by DXMS is shown, with residues 144–163 boxed and tyrosine residues highlighted in green. Raman shifts corresponding to the ν(C = C) ring mode (~1620 cm-1) and the tyrosine Fermi-doublet (~850 and 830 cm-1) are shown.
Mentions: We further sought to confirm a conformational difference between cofactor and protein-only PrPSc in the α1-β2 domain using Raman spectroscopy (Fig 4). Analysis of the Raman spectra acquired from these two conformers identified multiple Raman shifts that could be assigned to tyrosine residues, which are plentiful in the PrP C-terminus and specifically enriched in the α1-β2 domain (6 of 11 total C-terminal tyrosines). By Raman spectroscopy, protein-only PrPSc appears to contain more exposed tyrosine residues than cofactor PrPSc as evidenced from the increased ring ν(C = C) intensity at ~1620 cm-1 (Fig 4, left panel), the 850 cm-1/830 cm-1 ratio being greater than 1 (Fig 4, right panel) [22], and the increased ring ν(CH) intensity at ~3075 cm-1 (S8 Fig, left panel) [23,24], consistent with our DXMS results. In addition, and also consistent with our DXMS data, protein-only PrPSc appears to contain more exposed CNH groups than cofactor PrPSc as indicated by the increased intensity in the 1530–1580 cm-1 Amide II region, corresponding to ν(CN) and δ(CNH) Raman shifts (S8 Fig, right panel), as well as an increased ν(CN) intensity at ~3300 cm-1 (S8 Fig, left panel). These exposed CNH groups likely originate from the 4 exposed arginine (R) and single exposed glutamine (Q), asparagine (N) and tryptophan (W) residues in the α1-β2 domain, or from CNH-containing side chains in the N-terminal portion of the PK-resistant PrPSc core (residues ~91–115).

Bottom Line: Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163.Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers.Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

No MeSH data available.


Related in: MedlinePlus