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Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

Narayanan SP, Nair DG, Schaal D, Barbosa de Aguiar M, Wenzel S, Kremer W, Schwarzinger S, Kalbitzer HR - Sci Rep (2016)

Bottom Line: Xenon bound PrP was modelled by restraint molecular dynamics.As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain.This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics and Physical Biochemistry and Centre of Magnetic Resonance in Chemistry and Biomedicine (CMRCB), University of Regensburg, 93040 Regensburg, Germany.

ABSTRACT
Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

No MeSH data available.


Related in: MedlinePlus

Paramagnetic effects in spin labelled full-length prion protein.The relative change −ΔVmax/V0 = (V0 − Vmax)/V0 of cross peak volumes as a function of the sequence position j in [1H, 15N]-HSQC spectra of 15N-enriched huPrP(23–231) variants G93C (a) and T107C (b) labeled with methanesulfonothioate and maleimide based nitroxyl spin label, respectively. Solid and broken lines represent standard deviations σ0 and 2σ0. Proline residues are marked with P. Additionally invisible and unassignable residues in the spectra are marked with X. V0 corresponds to the cross peak volume after reduction of the spin label and Vmax the cross peak volume after labeling (see Materials and Methods).
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f6: Paramagnetic effects in spin labelled full-length prion protein.The relative change −ΔVmax/V0 = (V0 − Vmax)/V0 of cross peak volumes as a function of the sequence position j in [1H, 15N]-HSQC spectra of 15N-enriched huPrP(23–231) variants G93C (a) and T107C (b) labeled with methanesulfonothioate and maleimide based nitroxyl spin label, respectively. Solid and broken lines represent standard deviations σ0 and 2σ0. Proline residues are marked with P. Additionally invisible and unassignable residues in the spectra are marked with X. V0 corresponds to the cross peak volume after reduction of the spin label and Vmax the cross peak volume after labeling (see Materials and Methods).

Mentions: For obtaining information about transient interactions of the N-terminus with the globular domain, which also lead to formation of the transient cavity D, full-length prion protein variants with amino acid substitutions G93C and T107C were labelled with compounds containing unpaired electrons. In particular, the two different spin labels (1-oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl methanethiosulfonate and 1-oxyl-3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidine) were applied in this study. Residues spatially close to the active spin label should show a reduction of their cross peak volumes due to enhanced T2- relaxation caused by strong electron-nucleus dipole-dipole coupling when compared to the reduced diamagnetic sample. Since the labeling reaction itself was not quantitative and since for the N-terminal residues no unique structure is to be expected, the analysis of the paramagnetic enhancement (PRE) was only done in a qualitatively manner by comparing differences in cross peak volumes of the paramagnetic and diamagnetic sample (after reduction of the spin label). The relative cross peak volume changes −ΔV/V0 = (V0 − V)/V0 were plotted as a function of the sequence position with the cross peak volumes V and V0 of active and reduced spin label, respectively. Solid and broken lines represent standard deviation σ0 and 2σ0 to zero of −ΔV/V0 the V/V0 (Fig. 6). The residues with significant cross peak volume changes are summarized in Table S2. They are assumed to be spatially close to the spin label in the time average but the averaging is non-uniform because of the r−6-dependence of the PRE effect.


Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

Narayanan SP, Nair DG, Schaal D, Barbosa de Aguiar M, Wenzel S, Kremer W, Schwarzinger S, Kalbitzer HR - Sci Rep (2016)

Paramagnetic effects in spin labelled full-length prion protein.The relative change −ΔVmax/V0 = (V0 − Vmax)/V0 of cross peak volumes as a function of the sequence position j in [1H, 15N]-HSQC spectra of 15N-enriched huPrP(23–231) variants G93C (a) and T107C (b) labeled with methanesulfonothioate and maleimide based nitroxyl spin label, respectively. Solid and broken lines represent standard deviations σ0 and 2σ0. Proline residues are marked with P. Additionally invisible and unassignable residues in the spectra are marked with X. V0 corresponds to the cross peak volume after reduction of the spin label and Vmax the cross peak volume after labeling (see Materials and Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920026&req=5

f6: Paramagnetic effects in spin labelled full-length prion protein.The relative change −ΔVmax/V0 = (V0 − Vmax)/V0 of cross peak volumes as a function of the sequence position j in [1H, 15N]-HSQC spectra of 15N-enriched huPrP(23–231) variants G93C (a) and T107C (b) labeled with methanesulfonothioate and maleimide based nitroxyl spin label, respectively. Solid and broken lines represent standard deviations σ0 and 2σ0. Proline residues are marked with P. Additionally invisible and unassignable residues in the spectra are marked with X. V0 corresponds to the cross peak volume after reduction of the spin label and Vmax the cross peak volume after labeling (see Materials and Methods).
Mentions: For obtaining information about transient interactions of the N-terminus with the globular domain, which also lead to formation of the transient cavity D, full-length prion protein variants with amino acid substitutions G93C and T107C were labelled with compounds containing unpaired electrons. In particular, the two different spin labels (1-oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl methanethiosulfonate and 1-oxyl-3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidine) were applied in this study. Residues spatially close to the active spin label should show a reduction of their cross peak volumes due to enhanced T2- relaxation caused by strong electron-nucleus dipole-dipole coupling when compared to the reduced diamagnetic sample. Since the labeling reaction itself was not quantitative and since for the N-terminal residues no unique structure is to be expected, the analysis of the paramagnetic enhancement (PRE) was only done in a qualitatively manner by comparing differences in cross peak volumes of the paramagnetic and diamagnetic sample (after reduction of the spin label). The relative cross peak volume changes −ΔV/V0 = (V0 − V)/V0 were plotted as a function of the sequence position with the cross peak volumes V and V0 of active and reduced spin label, respectively. Solid and broken lines represent standard deviation σ0 and 2σ0 to zero of −ΔV/V0 the V/V0 (Fig. 6). The residues with significant cross peak volume changes are summarized in Table S2. They are assumed to be spatially close to the spin label in the time average but the averaging is non-uniform because of the r−6-dependence of the PRE effect.

Bottom Line: Xenon bound PrP was modelled by restraint molecular dynamics.As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain.This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics and Physical Biochemistry and Centre of Magnetic Resonance in Chemistry and Biomedicine (CMRCB), University of Regensburg, 93040 Regensburg, Germany.

ABSTRACT
Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

No MeSH data available.


Related in: MedlinePlus