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PrP charge structure encodes interdomain interactions.

Martínez J, Sánchez R, Castellanos M, Makarava N, Aguzzi A, Baskakov IV, Gasset M - Sci Rep (2015)

Bottom Line: We found that charges contain the information for interdomain interactions.Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency.These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

View Article: PubMed Central - PubMed

Affiliation: Instituto Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain.

ABSTRACT
Almost all proteins contain charged residues, and their chain distribution is tailored to fulfill essential ionic interactions for folding, binding and catalysis. Among proteins, the hinged two-domain chain of the cellular prion protein (PrP(C)) exhibits a peculiar charge structure with unclear consequences in its structural malleability. To decipher the charge design role, we generated charge-reverted mutants for each domain and analyzed their effect on conformational and metabolic features. We found that charges contain the information for interdomain interactions. Use of dynamic light scattering and thermal denaturation experiments delineates the compaction of the α-fold by an electrostatic compensation between the polybasic 23-30 region and the α3 electronegative surface. This interaction increases stability and disfavors fibrillation. Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency. In the fibrillar state, use of circular dichroism, atomic-force and fluorescence microscopies reveal that the N-terminal positive clusters and the α3 electronegative surface dictate the secondary structure, the assembly hierarchy and the growth length of the fibril state. These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

No MeSH data available.


Related in: MedlinePlus

Properties of the PrP wt and mutant fibrils.(a) Specific ThT binding of the PrP wt and mutant fibrils. Typically 50 μM of fibrils in 10 mM ammonium acetate pH 5 were incubated for 10 min with ThT (15 μM) before fluorescence determination. ThT fluorescence intensities were corrected for the background (absence of fibrils) and divided by the protein amount in the pellet of a 12000 rpm 20 min centrifugation. The depicted data represent the average of two independent experiments performed in duplicate (*p < 0.01). (b) Far-UV CD spectra of the PrP wt and mutant assemblies in 10 mM ammonium acetate pH 5. The fibrils were formed in 50 mM MES pH 6.5 containing 2 M GdnCl at 37 °C under continuous rotation at 24 rpm, and then dialyzed against 10 mM ammonium acetate pH 5.
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f5: Properties of the PrP wt and mutant fibrils.(a) Specific ThT binding of the PrP wt and mutant fibrils. Typically 50 μM of fibrils in 10 mM ammonium acetate pH 5 were incubated for 10 min with ThT (15 μM) before fluorescence determination. ThT fluorescence intensities were corrected for the background (absence of fibrils) and divided by the protein amount in the pellet of a 12000 rpm 20 min centrifugation. The depicted data represent the average of two independent experiments performed in duplicate (*p < 0.01). (b) Far-UV CD spectra of the PrP wt and mutant assemblies in 10 mM ammonium acetate pH 5. The fibrils were formed in 50 mM MES pH 6.5 containing 2 M GdnCl at 37 °C under continuous rotation at 24 rpm, and then dialyzed against 10 mM ammonium acetate pH 5.

Mentions: Similar to the α-folded wt PrP chain, variants with altered charge structure form fibrils that differ in their shape and proteinase-resistant core2135. To gain insight into the effects of the charged design on the fibril structure and its properties, we used the reaction conditions yielding PrP wt S-fibrils with resolution for imaging analysis. S-fibrils, produced under slow orbital agitation as opposed to the fast rotation leading to the R-polymorph, are featured by a β-sheet-like far-UV CD spectrum along with a thin and curvy fibril topology that expose the 90–102 region while partially shield the POM17 epitope66 (Figs 5, 6, 7). It must be stressed that this fibrillation reaction was performed in the presence of 2 M GndCl, which abrogates the charge effects on the native α-fold and permits the assignation of the deviations from the wt behavior to differences in the assembly process. Fibrils formed by all PrP mutants displayed a reduced specific ThT binding, suggesting major structural differences (Fig. 5a).


PrP charge structure encodes interdomain interactions.

Martínez J, Sánchez R, Castellanos M, Makarava N, Aguzzi A, Baskakov IV, Gasset M - Sci Rep (2015)

Properties of the PrP wt and mutant fibrils.(a) Specific ThT binding of the PrP wt and mutant fibrils. Typically 50 μM of fibrils in 10 mM ammonium acetate pH 5 were incubated for 10 min with ThT (15 μM) before fluorescence determination. ThT fluorescence intensities were corrected for the background (absence of fibrils) and divided by the protein amount in the pellet of a 12000 rpm 20 min centrifugation. The depicted data represent the average of two independent experiments performed in duplicate (*p < 0.01). (b) Far-UV CD spectra of the PrP wt and mutant assemblies in 10 mM ammonium acetate pH 5. The fibrils were formed in 50 mM MES pH 6.5 containing 2 M GdnCl at 37 °C under continuous rotation at 24 rpm, and then dialyzed against 10 mM ammonium acetate pH 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555102&req=5

f5: Properties of the PrP wt and mutant fibrils.(a) Specific ThT binding of the PrP wt and mutant fibrils. Typically 50 μM of fibrils in 10 mM ammonium acetate pH 5 were incubated for 10 min with ThT (15 μM) before fluorescence determination. ThT fluorescence intensities were corrected for the background (absence of fibrils) and divided by the protein amount in the pellet of a 12000 rpm 20 min centrifugation. The depicted data represent the average of two independent experiments performed in duplicate (*p < 0.01). (b) Far-UV CD spectra of the PrP wt and mutant assemblies in 10 mM ammonium acetate pH 5. The fibrils were formed in 50 mM MES pH 6.5 containing 2 M GdnCl at 37 °C under continuous rotation at 24 rpm, and then dialyzed against 10 mM ammonium acetate pH 5.
Mentions: Similar to the α-folded wt PrP chain, variants with altered charge structure form fibrils that differ in their shape and proteinase-resistant core2135. To gain insight into the effects of the charged design on the fibril structure and its properties, we used the reaction conditions yielding PrP wt S-fibrils with resolution for imaging analysis. S-fibrils, produced under slow orbital agitation as opposed to the fast rotation leading to the R-polymorph, are featured by a β-sheet-like far-UV CD spectrum along with a thin and curvy fibril topology that expose the 90–102 region while partially shield the POM17 epitope66 (Figs 5, 6, 7). It must be stressed that this fibrillation reaction was performed in the presence of 2 M GndCl, which abrogates the charge effects on the native α-fold and permits the assignation of the deviations from the wt behavior to differences in the assembly process. Fibrils formed by all PrP mutants displayed a reduced specific ThT binding, suggesting major structural differences (Fig. 5a).

Bottom Line: We found that charges contain the information for interdomain interactions.Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency.These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

View Article: PubMed Central - PubMed

Affiliation: Instituto Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain.

ABSTRACT
Almost all proteins contain charged residues, and their chain distribution is tailored to fulfill essential ionic interactions for folding, binding and catalysis. Among proteins, the hinged two-domain chain of the cellular prion protein (PrP(C)) exhibits a peculiar charge structure with unclear consequences in its structural malleability. To decipher the charge design role, we generated charge-reverted mutants for each domain and analyzed their effect on conformational and metabolic features. We found that charges contain the information for interdomain interactions. Use of dynamic light scattering and thermal denaturation experiments delineates the compaction of the α-fold by an electrostatic compensation between the polybasic 23-30 region and the α3 electronegative surface. This interaction increases stability and disfavors fibrillation. Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency. In the fibrillar state, use of circular dichroism, atomic-force and fluorescence microscopies reveal that the N-terminal positive clusters and the α3 electronegative surface dictate the secondary structure, the assembly hierarchy and the growth length of the fibril state. These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

No MeSH data available.


Related in: MedlinePlus