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. 2004 Dec;13(12):3151–3160. doi: 10.1110/ps.04745004

Possible role of region 152–156 in the structural duality of a peptide fragment from sheep prion protein

Simon Megy 1, Gildas Bertho 1, Sergey A Kozin 2, Pascale Debey 2, Gaston Hui Bon Hoa 3, Jean-Pierre Girault 1
PMCID: PMC2287313  PMID: 15537751

Abstract

The conformational conversion of the nonpathogenic “cellular” prion isoform into a pathogenic “scrapie” protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a β-like structure. A peptide spanning helix H1 and β-strand S2 (residues 142–166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a β-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152–156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.

Keywords: NMR, structural duality, TFE, salt bridges, prion protein, peptide, helix H1, region 152–156

The nonpathogenic “cellular” isoform of the prion protein (PrPC) is a strongly conserved cell surface glycoprotein expressed in all mammalian species studied so far (Bendheim et al. 1992). Its conformational conversion into a pathogenic “scrapie” isoform (PrPSc) is the fundamental event in the pathogenicity of transmissible spongiform encephalopathies (TSE) (Prusiner 1998). The major structural feature of prion conversion manifests itself as an increase of the β-sheet content in PrPSc (Griffith 1967; Prusiner 1991; Pan et al. 1993). Transgenic studies argue that infectious PrPSc acts as a template (Prusiner et al. 1990; Telling et al. 1995) upon which the normal PrPC is refolded into a pathogenic isoform through a process facilitated by an unknown as yet factor “X” (Kaneko et al. 1997).

The mammalian PrPC contains 209 amino acid residues, from 23 to 231 in human numbering. The minimal prion fragment required for infectious propagation was mapped to residues 90–231 (Prusiner 1998). NMR studies of the recombinant mouse, hamster, bovine, and human prion proteins (Riek et al. 1997, 1998) showed that all these molecules have very similar 3D structures, including a flexible unstructured “tail” composed of residues 23–120 and a mostly α-helical globular core part 121–231. This globular PrP is composed of two short antiparallel β-strands (S1 and S2) and three α-helices (H1–H3) (Riek et al. 1996). This globular domain can further be divided into two subdomains (Jamin et al. 2002), one long hairpin subdomain (helix H1 and the β-sheet) and one purely α-helical subdomain (helices H2 and H3).

Within the globular domain of the molecule, the region containing helix H1 is known to be one of the most flexible of the prion proteins (Viles et al. 2001). The fragment containing helix H1 and strand S2 is the most probable site for conformational conversion of PrPC (Riek et al. 1996; Prusiner 2001). The question of how helix H1 in particular can undergo such a major structural rearrangement from helical conformation to a structure involving a significant amount of β-sheet remains unsolved. The deletion of this region (helix H1 and strand S2) has been recently shown to inhibit formation of the PrPSc (Vorberg et al. 2001). A recent study indicates a bipartite function of helix H1 in the maturation and aggregation of PrP (Winklhofer et al. 2003). As helices H2 and H3 are stabilized by a disulfide bond and form the C-terminal scaffold, they probably have nearly the same conformation in PrPSc and PrPC (Muramoto et al. 1996). However, structural studies of PrPSc have been limited because of its aggregated state (Prusiner et al. 1983; Caughey et al. 1991; Gasset et al. 1993; Safar et al. 1993). The exact role of helix H1 and strand S2 in the conformational conversion process remains to be clearly elucidated.

Many prion-derived peptides were analyzed (Gasset et al. 1992; Come et al. 1993; Tagliavini et al. 1993; De Gioia et al. 1994; Nguyen et al. 1995; Zhang et al. 1995; Heller et al. 1996; Inouye and Kirschner 1997; Pillot et al. 1997; Ragg et al. 1999) in an attempt to clarify the molecular basis that might be involved in promoting the PrPC to PrPSc conformational transition. Most of them belong to the 90–145 region, and have intrinsic propensity to produce insoluble intermolecular aggregates of an extended β-like structure.

We investigated by CD and NMR spectroscopies the solution structure of a linear 26-mer peptide (hereafter referred to as peptide n3) (Kozin et al. 2000, 2001). Its sequence GNDYE5DRYYR10ENMYR15YPNQV20YYRPV25C contains 25 residues corresponding to the domain 145–169 of sheep prion protein (Goldmann et al. 1990) (142–166 in human prion protein numbering) and a C-terminal cysteine (the bold letters represent the segments corresponding to helix H1 [residues 144–154] and β-strand S2 [residues 161–164], respectively). In contrast to the prion-derived peptides studied earlier, peptide n3 (1) remains soluble in aqueous solution and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a β-hairpin like conformation at neutral pH.

This peptide has also been recently studied by fluorescence measurements, and has been suggested to act as a potential inhibitor that could prevent the formation of PrPSc (Pato et al. 2004).

The experimental results obtained in the present work show that this peptide can also fold into the helix H1 conformation when dissolved in a TFE/PB mixture. This conversion from a β-like to helix structure was studied by CD and NMR. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures, and evidenced a higher mobility of the residues following the end of helix H1. This structural duality of the peptide is reminiscent of the overall conformational transition of PrP from helix to β-sheet. We propose that the potential nucleation site for the molecular rearrangement of the prion protein may be localized within this peptide.

Results

Secondary structure analysis

The peptide secondary structure was analyzed in different experimental conditions by far-UV CD spectroscopy. Early study (Kozin et al. 2000) in aqueous buffered solution showed that the peptide produced a broad negative Cotton effect at 208 nm, with a shoulder at 216 nm, and two smaller signals, namely, a positive one at 233 nm and a negative one at 241 nm, which are characteristic of nonrandom coil conformation (Venyaminov and Yang 1996). We showed that the increase of TFE concentration results in a transformation of the CD signal (Fig. 1 ▶). Indeed, in the range of 20–99.5% 2,2,2-trifluoroethanol (TFE), CD spectra of the peptide showed two negative peaks at 208 nm and 218 nm and a positive peak at 195 nm, typical of α-helix structure. An isodichroic point at 205 nm indicates equilibrium between two distinct conformations.

Figure 1.

Figure 1.

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Far-UV CD spectra of the n3 peptide obtained in mixed PB–TFE solutions at 298 K. TFE concentrations expressed v/v: 0% (black), 10% (blue), 20% (cyan), 30% (magenta), 40% (green), 80% (yellow), and 96% (red). The inset demonstrates the changes in the mean residue weight ellipticity, Θ, at 222 nm.

NMR solution structure

The absence of time-dependent aggregation of the n3 peptide at millimolar concentrations at neutral pH allowed us to study conformational features of the peptide by NMR spectroscopy. All the NMR experiments were carried out at 500.13 MHz, using a 4-mM peptide concentration in an 87/13 (v/v) TFE/“PB” buffer, (10 mM sodium phosphate buffer [pH 6.5]). Spin systems were determined using two-dimensional TOCSY spectra with 35–70-msec mixing times. Assignments were confirmed by NOESY (Fig. 2 ▶) and ROESY experiments.

Figure 2.

Figure 2.

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Expansion of the fingerprint region of a 500-MHz two-dimensional [1H, 1H]-NOESY NMR spectrum of the n3 peptide in 87/13 (v/v) TFE/PB buffer at 310 K. Assignments of cross-peaks are denoted with the sequence number. The sequential assignment is shown from residues 142–157 (2–16 in peptide numbering). The unambiguous medium-range NOEs αN (i, i + 3), characteristic of a helical conformation, are pointed with arrows.

The sequential assignment of all backbone amide resonances was carried out by using the standard protocol developed by Wüthrich (1986). All experiments were performed at three different temperatures (278, 293, and 310 K) to solve assignment ambiguities resulting from signal overlaps. All 13Cα and 13Cβ resonances were assigned, and most of 13C′ resonances were found.

Secondary structures were identified using the CSI protocol, involving 1Hα, 13C′, 13Cα, and 13Cβ chemical shifts (Tables 1,2) (Wishart and Sykes 1994). We applied the criteria for secondary structure and the differences between observed and random-coil 1Hα and 13Cβ chemical shifts from Asp 3 to Tyr 14 showed negative values, consistent with an α-helical arrangement. Also for these residues, the same calculation for 13C′ and 13Cα results in positive values, confirming their helical propensity (Fig. 3 ▶).

Table 1.

Proton chemical shifts assignments [δ in ppm from Sodium 3-triméthylsilyl (2,2,3,3-2H4) propionate, 278 K (pH 6.5), in an 87/13 (v/v) TFE/PB solution], amide signal shift temperature coefficients (Δ (δ NH)/ΔT, in ppb K−1), and3J-HαHN scalar couplings constants (in Hz) of the n3 peptide

Residue NH CαH CβH Other Δ (δ NH)/ΔT 3JHαHN
1 Gly (G142) 3.92
2 Asn (N143) 8.33 4.86 2.94 NγH 7.26, 6.45 −2.8 7.4
3 Asp (D144) 8.38 4.65 2.87 −5.3 3.9
4 Tyr (Y145) 7.94 4.32 3.13 Ar2, 6H 7.12; Ar3,5H 6.87 −2.8 3.4
5 Glu (E146) 8.16 4.12 2.18 CγH 2.56 −6.1 3.1
6 Asp (D147) 8.11 4.62 3.00, 2.95 −6.9 3.4
7 Arg (R148) 7.83 4.07 1.89 CγH 1.68, 1.61; CδH 3.16; NɛH 7.05; NηH 6.51 −3.7 3.1
8 Tyr (Y149) 7.97 4.24 3.09, 2.98 Ar2,6H 6.99; Ar3,5H 6.82 −7.9 3.1
9 Tyr (Y150) 8.15 4.31 3.20 Ar2,6H 7.18; Ar3,5H 6.88 −10.0 a
10 Arg (R151) 8.06 4.08 1.99, 1.89 CγH 1.74; CδH 3.26; NɛH 7.16; NηH 6.55 −8.2 2.8
11 Glu (E152) 8.17 4.22 2.18 CγH 2.62, 2.58 −8.1 3.7
12 Asn (N153) 7.75 4.61 2.67 NγH 6.98, 6.02 −1.6 4.9
13 Met (M154) 7.82 4.25 1.96 CγH 2.40; CɛH 2.03 −3.3 4.3
14 Tyr (Y155) 7.57 4.53 3.01 Ar2,6H 7.13; Ar3,5H 6.84 −4.1 4.9
15 Arg (R156) 7.48 4.30 1.70 CγH 1.48; CδH 3.15; NɛH 6.98; NηH 6.51 −2.3 7.4
16 Tyr (Y157) 7.68 4.92 3.15, 2.96 Ar2,6H 7.17; Ar3,5H 6.85 −4.8 5.9
17 Pro (P158) 4.89 2.31, 2.07 CγH 2.05; CδH 3.84, 3.62
18 Asn (N159) 7.91 4.69 2.88 NγH 7.22, 6.42 −6.4 4.9
19 Gln (Q160) 8.19 4.28 2.13 CγH 2.42; NɛH 7.17, 6.42 −5.7 4.3
20 Val (V161) 7.66 3.98 2.05 CγH 0.89, 0.80 −5.0 6.5
21 Tyr (Y162) 7.46 4.54 3.06, 2.89 Ar2,6H 7.06; Ar3,5H 6.83 −5.5 5.6
22 Tyr (Y163) 7.52 4.56 3.03 Ar2,6H 7.10; Ar3,5H 6.83 −5.6 5.6
23 Arg (R164) 7.39 4.68 1.86, 1.75 CγH 1.67; CδH 3.26, 3.20; NɛH 7.04; NηH 6.55 −4.4 6.8
24 Pro (P165) 4.49 2.31, 2.07 CγH 2.05; CδH 3.64
25 Val (V166) 7.48 4.22 2.17 CγH 1.01 −6.2 7.7
26 Cys 7.68 4.65 3.02, 2.10 −6.6 7.1
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a Not determined.

Table 2.

13C chemical shifts assignments of the n3 peptide (δ in ppm from DSS and TSPD4) using1H-13C chemical shift correlation, 310 K (pH 6.5), in an 87/13 (v/v) TFE/PB solution

Residue 13C′ 13Cα 13Cβ Other
1 Gly (G142) a 44.2
2 Asn (N143) 173.2 53.8 39.4
3 Asp (D144) 174.6 54.8 39.2
4 Tyr (Y145) 175.6 57.5 39.0 Cδ 133.6, Cɛ 118.9
5 Glu (E146) a 59.2 29.0 Cγ 33.6, Cδa
6 Asp (D147) 175.3 56.0 39.1
7 Arg (R148) 176.1 59.8 31.2 Cγ 27.5, Cδ 44.2, Cζ 133.7
8 Tyr (Y149) 174.4 62.4 39.1 Cδ 133.7, Cɛ 118.9
9 Tyr (Y150) 179.9 61.4 39.1 Cδ 133.8, Cɛ 118.9
10 Arg (R151) 176.4 59.4 30.8 Cγ 31.7, Cδ 44.2, Cζ 133.8
11 Glu (E152) 176.3 58.6 29.1 Cγ 33.6, Cδa
12 Asn (N153) a 55.5 40.2 Cγ 133.7
13 Met (M154) a 57.9 33.4 Cγ 133.7, Cɛ 17.0
14 Tyr (Y155) 174.6 58.9 39.2 Cδ 133.6, Cɛ 118.9
15 Arg (R156) a 57.7 31.7 Cγ 27.9, Cδ 44.0, Cζ 133.7
16 Tyr (Y157) a 56.9 39.1 Cδ 133.8, Cɛ 118.9
17 Pro (P158) 173.8 64.5 32.3 Cγ 27.8, Cδ 51.1
18 Asn (N159) 173.7 54.6 39.1 Cγa
19 Gln (Q160) 173.5 57.9 29.6 Cγ 34.7, Cδ 133.8
20 Val (V161) 174.3 64.7 32.8 Cγ 21.1
21 Tyr (Y162) 173.8 58.9 39.3 Cδ 133.6, Cɛ 118.9
22 Tyr (Y163) 173.1 59.0 39.2 Cδ 133.7, Cɛ 118.9
23 Arg (R164) 171.9 54.7 31.5 Cγ 27.4, Cδ 44.2, Cζ 133.7
24 Pro (P165) 174.7 64.5 32.4 Cγ 27.9, Cδ 51.1
25 Val (V166) a 62.8 34.0 Cγ 21.1
26 Cys a 58.2 28.7
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a Not determined.

Figure 3.

Figure 3.

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Deviation of chemical shift values (Δδ, in ppm), determined for the peptide in TFE, from those characteristic of the random coil, derived (A) from 1Hα chemical shifts, and (B) from 13Cα (black), 13Cβ (gray), and 13C′ (white) chemical shifts. Sequence-dependent corrections were added. Any group of three or more consequent residues is considered to form a helical structure, if for each residue within the group corresponding, Δδ 1Hα and Δδ 13Cβ are negative, whereas 13Cα and 13C′ are positive. The significant values indicative of a helix conformation are shown within the shadowed box.

Moreover, the 3J-HαHN scalar coupling constants indicated a helical structure for this region of the peptide (Fig. 4 ▶). Indeed, these values are weak (less than 5 Hz), which means that these residues tend to populate α-helical φ angles.

Figure 4.

Figure 4.

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(A) The observed sequential and medium-range NOE connectivities are indicated by lines connecting the two residues that are related by the NOE. Thick and medium bars indicate strong and medium NOE intensities, respectively. Thin bars indicate the weak and very weak NOE intensities. (B) The 3J-HαHN couplings are indicated by squares. Filled squares identify residues with 3J-HαHN <4 Hz, and 2/3 filled squares correspond to 3J-HαHN between 4 Hz and 5 Hz. This indicates a local α-type conformation from residues 144–155 (3–14 in peptide numbering). The 1/3 filled squares identify residues with 3J-HαHN between 5 Hz and 6 Hz, and the open squares correspond to 3J-HαHN >6 Hz. (C) The Δ (δ NH)/ΔT are represented by circles. The filled circles identify residues with Δ (δ NH)/ ΔT >-5 ppb/K. The half-filled circles represent residues with Δ (δ NH)/ ΔT between -5 and -8 ppb/K. The open circles correspond to Δ (δ NH)/ ΔT <-8 ppb/K.

The amide signal shift temperature coefficients (Δ [δ NH]/ΔT, in ppb K−1) were derived for all backbone amide protons of the peptide (Fig. 4 ▶). For the residues involved in intramolecular hydrogen-bonding network and/or hidden in a structural core protected from solvent, one can expect low absolute values of this coefficient compared to the range of values measured for random-coil peptides (Blanco et al. 1994). In our study, only three out of 23 measured residues show values >-8 ppb K−1, which indicates a good structuration of the peptide in TFE and suggests the presence of secondary structures protecting the amide protons from external water molecules. However, these values are globally higher than those typically found in helical structures protected from solvent. These results are consistent with the fact that helix H1 has been shown to undergo hydrogen-deuterium exchange more easily than the central portions of helices H2 and H3 (Liu et al. 1999b), suggesting a looser, less compact structure that might unfold more readily.

All distance restraints used in the structure calculation were derived from NOEs observed at 278 K in TFE/H2O buffer during NOESY experiments at a 250-msec mixing time. The NOE pattern observed was typical of a helical structure (Fig. 4A ▶). Extensive unambiguous medium-range NOEs αN(i,i + 3) and NOEs α β (i,i + 3) (Fig. 2 ▶) were observed for residues 3–14 (residues 144–155 in human numbering) and indicated helix conformation for this region.

Structure description

A 3D structure of the n3 peptide was obtained following the molecular dynamic protocol described in the Materials and Methods section using a final set of 252 NOE-derived distance constraints (153 sequential, 97 medium-range, and two long-range (i - j ≥ 5) residues, Table 3) at 278 K. No intraresidual distance constraints or hydrogen bonds were included in the calculations. Thirteen dihedral angle constraints, deduced from 3J-HαHN coupling constants as described in the Materials and Methods section, were imposed and coupling constants were directly used as constraints.

Table 3.

NMR restraints and structural statistics for the 20 top-ranked peptide n3 conformers (obtained by simulated annealing) in an 87/13 (v/v) TFE/PB solution

NOE restraints
    Total restraints 252
    Sequential 153
    Medium range (i − j < 5) 97
    Long range (i − j ≥ 5) 2
    NOE constraints per residue 9.7
NOE violations
    Maximum individual violation (Å) 0.551
    Average number of violations (≥0.5 Å) per structure 0.5
Other restraints
    Number of 3J HαHN coupling constants 22
    Number of Hα CSI constraints 25
    Number of Cα and Cβ CSI constraints 25
Ramachandran analysis
    Residues in most favored regions (%) 63.2
    Residues in additional allowed regions (%) 16.1
    Residues in generously allowed regions (%) 15.7
    Residues in disallowed regions (%) 5.0
Global rmsd to a mean structure
    All backbone atoms (Å) 1.5 ± 0.6
    All nonhydrogen atoms (Å) 2.2 ± 0.8
Local rmsd to a mean structure (res. 144–156 and 159–165, respectively)
    All backbone atoms (Å) 0.3 ± 0.1 0.2 ± 0.1
    All nonhydrogen atoms (Å) 1.3 ± 0.3 0.9 ± 0.3
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After minimization, 20 structures based on low residual distance and dihedral angle violations and lower overall energies were selected to compute the solution structure of the n3 peptide. A ribbon model of the mean structure is shown in Figure 5A ▶, whereas the 20 final structures superimposed for the minimum backbone deviations between residues 3 and 15 are displayed in Figure 5B ▶.

Figure 5.

Figure 5.

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Ribbon models of the n3 peptide structure in 87/13 (v/v) TFE/ PB buffer. (A) Ribbon model of the mean molecule calculated from the 20 best structures. (B) Superimposition of the 20 best structures calculated. The helical part of the backbone is shown in red. The corresponding side chains are shown in blue.

Structural statistics are presented in Table 3. The structural models fit the NMR data well, with less than one violation of NOE constraints per structure, and a maximal violation of 0.55 Å. The Ramachandran plot can be considered as satisfactory, with only 5% of residues outside of the allowed regions.

The total number of NOE constraints observed per residue is illustrated in Figure 6A ▶. The reduced number of interresidue NOEs observed between P158 (residue 17) and Q160 (residue 19) suggests a break in structure between two well-structured regions. This is consistent with the values observed for the average local root-mean-square deviation (RMSD) per residue of the n3 peptide (backbone), shown in Figure 6B ▶. This graphic highlights a higher mobility of residues 157 and 158 (16 and 17 in peptide numbering), according to the reduced NOEs number.

Figure 6.

Figure 6.

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(A) Number of NOE restraints per residue (intraresidual NOEs were neglected). (B) Backbone heavy atom local rmsd values per residue for the family of 20 structures relative to the average structure. (C) The position of the helix is indicated at the bottom as a filled bar.

The RMSD to the mean structure for all backbone atoms is 1.5 ± 0.6 Å. This value drops to 0.3 ± 0.1 and 0.2 ± 0.1 if one only considers the residues of the 144–156 and 157–165 regions (3–15 and 18–24 in peptide numbering, respectively) (Fig. 7 ▶). This demonstrates that the structure of the n3 peptide is well defined for the 144–156 and the 157–165 regions, separated by a more flexible hinge, mainly corresponding to residues 157 and 158. The overall structure of the n3 peptide in TFE shows the absence of intermolecular contacts between segments H1 and S2. Superimposition of the structure of the n3 peptide in 87/13 (v/v) TFE/PB buffer and of the corresponding fragment from the entire bovine protein (Fig. 8 ▶) shows how very similar the two structures of the residues implicated in helix H1 are.

Figure 7.

Figure 7.

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Views of the backbone (N, Cα, C′) atoms of the 20 best structures of the n3 peptide and superimpositions of structures for best fit on backbone atoms. Fit (A) for all residues; (B) for residues 144–156 (3–15 in peptide numbering); (C) for residues 159–165 (18–24 in peptide numbering).

Figure 8.

Figure 8.

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Superimposition of the structure of the n3 peptide in 87/13 (v/v) TFE/PB buffer (in green) and of the corresponding fragment from the entire bovine protein (in gray and blue). The extended C-terminal part of the bovine protein is strand S2 (stabilized by strand S1 in the globular domain of the PrPC). The two structures are fitted from residue 142 to residue 155.

Discussion

For the mean structure and the majority of the 20 best conformers, the Molmol software (Koradi et al. 1996) identifies an α-helix from residues 144 to 151, followed by a 310 helix, from residues 152 to 155. The same secondary structures are obtained for these residues in the bovine prion protein (PDB code 1DX1; Lopez Garcia et al. 2000) and in the human prion protein at pH 7.0 (PDB code 1HJN; Calzolai and Zahn 2003). Interestingly, previous studies (Sharman et al. 1998; Ziegler et al. 2003) have shown that several synthetic prion peptides encompassing helix H1 and β-strand S2 were soluble in water solution only under acidic condition and precipitated at (±) neutral pH. Those peptides differ mostly from peptide n3 in the total electrostatic charge in the pH 6.5–7.5 range. The n3 peptide is highly soluble at aforementioned pH range, for which the total net charge of peptide n3 is 0.

Structural similarity of helix H1 segment in peptide n3 and in PrPC

Analysis of the structures obtained made it possible to identify the hydrogen bonds that appear to stabilize the peptide conformation. The H-bonding network was determined by using the CNS software (Brünger et al. 1998). Five backbone amide-carboxyl hydrogen bonds were found between the following amino acids: 143–147, 144–148, 146–150, 147–151, and 148–152 in the α-helix structure. Four were detected in the 310 helix: 150–153, 151–154, 152–155, and 153–156. Therefore, the H-bonding network in the α-helical fragment of the n3 peptide is similar to that observed in helix H1 of native prion proteins. The C-terminal fragment of the peptide possesses an explicit but irregular conformation, which is characterized by two H-bonds between residues 154–159 and 159–162.

The particular role of charged residues in the stabilization of helix H1 in both the peptide fragment in TFE and the whole prion protein PrPC is also to be elucidated. As helix H1 is the most hydrophilic helix in all the known protein structures, such hydrophilicity implies that intermolecular electrostatic interactions play a significant role in stabilizing the structure of helix H1 (Morrissey and Shakhnovich 1999). Interestingly, another common structural feature of the α-helical region found in the peptide fragment studied in TFE and the known structures of prion protein PrPC is the relative distance between charged groups of selected side chains for residue pairs: 147–151, 148–152, and 152–156 (Table 4). Such positions provide one with the possibility of producing a network of salt bridges in helix H1, as was previously assumed (Morrissey and Shakhnovich 1999).

Table 4.

Comparison of distances (Å) between charged groups of selected side chains inside peptide segment corresponding to 142–166 human prion protein sequence

Peptide n3
in TFE (mostly helical conformation) in PB buffer (mostly β-hairpin-like conformation) PrPCa
Residues
    R148–E152 (Arg7–Glu11) 6.5 13.4 5.7
    E152–R156 (Glu11–Arg15) 9.5 2.7 9.6
    D147–R151 (Asp6–Arg10) 6.5 3.3 6.8
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a Example given for the bovine PrPC (Lopez Garcia et al. 2000).

Effect of solvent on peptide conformation

The peptide conformations in TFE and in phosphate buffer solution are very different. The main difference concerns the structure of segment H1, which, in water solution, adopts an extended conformation stabilized by specific intramolecular contacts with amino acid residues of region S2. These interactions were shown to be very specific and stable (Kozin et al. 2001). However, they are disrupted in the presence of TFE. The way in which the medium surrounding a polypeptide chain affects its structure is still poorly understood (Chitra and Smith 2001). However, TFE has a lower dielectric constant than water, suggesting that electrostatic interactions will be strengthened in a TFE/water mixture. In this study, it appears that, contrary to phosphate buffer, the TFE medium simulates the protein environment in which the segment corresponding to helix H1 adopts its native conformation. Hence, the conformational behavior of the n3 peptide is reminiscent of the α → β prion structural conversion.

Structural duality of peptide n3

Earlier (Liu et al. 1999a), it was found that the synthetic hexadecapeptide mPrP(143–158) encompassing prion segment H1 showed significant intrinsic helical propensity in both H2O and a 1:1 mixture of H2O and TFE. Our results are in keeping with the fact that, in the absence of contacts with other prion segments like L3 or S2, the H1 segment always adopts its native-like helical conformation.

Wüthrich (Riek et al. 1996; Korth et al. 1997) and Prusiner (2001) have proposed prion conversion models in which helix H1 underwent transconformation into extended β-sheet structure upon intra- or/and intermolecular interactions with a preexisting β-sheet (strands S1 and S2). The conformation behavior of peptide n3 reported here and in our previous study (Kozin et al. 2001) suggests that peptide n3 could be a useful model system to work for a better understanding of the α → βconformational transition occurring in the prion protein.

Implication of the role of the 152–156 region in peptide structural rearrangement

A particular feature of the n3 peptide is the salt bridge E152–R156 found in water. The interaction between E152 and R156, which could thus increase both the length and the stability of helix H1, does not exist in PrP and in peptide n3 in TFE. Further inspection of the structure of the n3 peptide in TFE shows that the aromatic ring of Y155 resides at the end of helix H1, between the side chains of E152 and R156 (Fig. 9 ▶). These data suggest that the tyrosine at position 155 may act as a barrier to the E152–R156 salt bridge (Table 4). However, this ionic interaction has only been observed in the structure of the n3 peptide in PB buffer solution (Kozin et al. 2001).

Figure 9.

Figure 9.

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Schematic model of the ionic interaction (gray arrows) involved in the structure of peptide n3: On the left, peptide in TFE showing Y155 located at the end of helix H1, between the side chains of E152 and R156 and probably acting as a barrier to the E152–R156 salt bridge. On the right, ionic interactions observed in PB buffer solution. The gray arrows indicate distances <3.5 Å as shown in Table 4. Thus, the nature of the residue located in position 155 could play a role in the stability of helix H1.

The absence of the E152–R156 salt bridge is also observed in all the known structures of PrP (Riek et al. 1996; Liu et al. 1999b; Lopez Garcia et al. 2000; Zahn et al. 2000). The nature of the residue at position 155, which is a tyrosine for sheep and mice, an asparagine for the Syrian hamster, and a histidine for humans and bovine, could play a particular role relative to the possible formation of the E152–R156 salt bridge in PrPSc.

Interestingly, Priola (Priola et al. 2001) has found that mutation at position 155 strongly influences the rate of conversion from PrPC to PrPSc. In the hamster sequence, introduction of a tyrosine instead of an asparagine at position 155 (154 in hamster numbering) significantly reduced the formation of protease-resistant PrP. This is consistent with the fact that Y155 may block the possibility of a stabilization of helix H1 by the E152–R156 salt bridge (Fig. 9 ▶).

Moreover, in human prion protein, the protonation of H155 and H187 presumably contributes to these structural changes (Calzolai and Zahn 2003). In view of our results, histidine 155 could act as a possible “pH-dependent switch” to prion conversion.

Thus, the nature of the residue located at position 155 may play a key role in stabilizing helix H1. Interestingly, this residue may be involved in the TSE species barrier (Billeter et al. 1997; Priola et al. 2001).

Several mutations of residues implicated in putative salt bridges thought to stabilize helix H1 have been reported (Speare et al. 2003; Ziegler et al. 2003). Only a small local destabilization of helix H1 in the D147A and E152A mutants of human prion was observed, which implies the existence of a helix-stabilizing interaction in the wild-type peptide (Ziegler et al. 2003).

In conclusion, this study highlights the structural duality of the n3 peptide, which had been previously assumed (Kozin et al. 2001). Peptide n3 adopts in TFE/PB mixture a well-defined helical conformation, which is stabilized by a specific network of H-bonds. The measured distances between the charged groups of residues such as D147–R151, R148–E152, and E152–R156 are also very different from those observed in PB buffer.

Another potential application of the n3 peptide has been recently suggested. The peptide n3 could serve as a template to develop an inhibitor to the formation of PrSc (Pato et al. 2004).

According to these results, other mutants in the 152–156 region, particularly at position 155 could now be considered.

Materials and methods

Peptide synthesis and purification

The n3 peptide was synthesized and purified as previously described (Kozin et al. 2001). Mass spectrometry and amino acid analysis were performed to check the peptide purity and verify its sequence. The purity exceeded 99%.

Circular dichroism (CD) spectroscopy

CD spectra of the 150 μM n3 peptide for PB buffer and various concentrations of TFE samples (0%, 10%, 20%, 30%, 40%, 80%, and 96% expressed in v/v) were measured at 298 K using a JASCO J-710 instrument. Samples were studied in quartz cells with path lengths of 0.5 mm or 1 mm, following the protocol previously described (Kozin et al. 2001).

Nuclear magnetic resonance (NMR) spectroscopy

NMR samples were dissolved in TFE-d2OH/“PB” buffer (10 mM sodium phosphate buffer [pH 6.5]) 87:13 (v/v). A crystal of TSPD4, 3-(trimethylsilyl)[2,2,3,3-d4] propionic acid, sodium salt, was used as internal reference for the proton shifts. The experiments were run at 500.13 MHz for 1H on a Bruker AMX 500 spectrometer equipped with a Silicon Graphics workstation. The WATERGATE method (Piotto et al. 1992) was used in all experiments to eliminate the water signal rather than the presaturation method. 1D, 2D-TOCSY, 2D-ROESY, and 2D-NOESY spectra were recorded at several temperatures within the 278–310 K range. Mixing times of 35–70 msec were used for 2D-TOCSY. For 2D-ROESY experiments, a spin-lock of 200–400 msec was used. 2D phase-sensitive NOESY experiments were carried out using the States-TPPI method with a mixing time in the 50–800 msec range. The 3J-HαHN scalar coupling constants were measured in 1D spectrum and extracted from 2D-TOCSY. The 13C NMR chemical shifts were carefully calibrated using DSS (4,4-dimethyl 4-silapentane sodium sulfonate) and TSPD4. The assignments of 13C were made at 310 K using two 1H-13C Chemical Shift Correlation spectra: PFG-HSQC (Pulse Field Gradient–Heteronuclear Single Quantum Correlation) phase-sensitive using sensitive enhancement (Hurd and John 1991), and PFG-HMBC (Pulse Field Gradient–Heteronuclear Multiple Bond Correlation) (Bax and Summers 1986).

Structural calculations and data deposition

Model structures were calculated by simulated annealing (SA) using torsion angle dynamics as implemented in the program CNS (Brünger et al. 1998). Calculations were performed on a Silicon Graphics Indigo2 workstation. Distance constraints were derived from cross-peaks in NOESY spectra recorded at 500 MHz and 278 K (solvent: 87% TFE, 13% PB) with a mixing time of 250 msec. The NOE cross-peaks classified as strong, medium, weak, and very weak were converted into 252 interresidual distance restraints of 1.8–2.5 Å, 1.8–3.5 Å, 1.8–4.5 Å, and 1.8–5.5 Å, respectively. Appropriate pseudoatom corrections were applied to non-stereo-specifically assigned protons (Wüthrich 1986). Several rounds of structure calculations and assignments were performed to resolve ambiguities. 3J-HαHN coupling constants were used directly as constraints. Backbone dihedral restraints for φ angle were used as −60 ± 30° for the 13 residues presenting a 3J-HαHN value less than 5 Hz. Chemical shift index of Hα, Cα, and Cβ, were calculated and modified for all residues but the N terminus Gly, applying some sequence-dependent corrections (Schwarzinger et al. 2001) and used directly as constraints.

Finally, the 20 best-minimized models with the lowest overall energies obtained with the standard CNS simulated annealing protocol were retained for analysis. Structures were displayed with the Molmol program (Koradi et al. 1996) and evaluated using Pro-check-NMR (Laskowski et al. 1996). The atomic coordinates have been deposited in the Protein Data Bank (available at http://www.rcsb.org) (PDB code 1M25). Proton chemical shifts table and the 3J-HαHN scalar coupling constants of the n3 peptide have been deposited with the BioMagResBank (http://www.bmrb.wisc.edu) (code BMRB–5405).

Acknowledgments

S.A.K. was supported by an INRA fellowship. We thank Dr. Hanitra Rabesona and Dr. Thomas Haertlé for providing the peptide. We thank Béatrice Berna (Centre for Technical Languages, Université René Descartes-Paris V) for his critical reading of this manuscript.

Abbreviations

  • PrP, prion protein

  • PrPC, “cellular” isoform

  • PrPSc, “scrapie” isoform

  • TFE, trifluoroethanol

  • PB buffer, 10 mM sodium phosphate buffer (pH 6.5)

  • MD, molecular dynamics

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04745004.

References

  1. Bax, A. and Summers, M.F. 1986. 1H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 108 2093–2094. [Google Scholar]
  2. Bendheim, P.E., Brown, H.R., Rudelli, R.D., Scala, L.J., Goller, N.L., Wen, G.Y., Kascsak, R.J., Cashman, N.R., and Bolton, D.C. 1992. Nearly ubiquitous tissue distribution of the scrapie agent precursor protein. Neurology 42 149–156. [DOI] [PubMed] [Google Scholar]
  3. Billeter, M., Riek, R., Wider, G., Hornemann, S., Glockshuber, R., and Wuthrich, K. 1997. Prion protein NMR structure and species barrier for prion diseases. Proc. Natl. Acad. Sci. 94 7281–7285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blanco, F.J., Rivas, G., and Serrano, L. 1994. A short linear peptide that folds into a native stable β-hairpin in aqueous solution. Nat. Struct. Biol. 1 584–590. [DOI] [PubMed] [Google Scholar]
  5. Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography & NMR system: A new software suite for macro-molecular structure determination. Acta Crystallogr. D 54 905–921. [DOI] [PubMed] [Google Scholar]
  6. Calzolai, L. and Zahn, R. 2003. Influence of pH on NMR structure and stability of the human prion protein globular domain. J. Biol. Chem. 278 35592–35596. [DOI] [PubMed] [Google Scholar]
  7. Caughey, B.W., Dong, A., Bhat, K.S., Ernst, D., Hayes, S.F., and Caughey, W.S. 1991. Secondary structure analysis of the scrapie-associated protein PrP 27–30 in water by infrared spectroscopy. Biochemistry 30 7672–7680. [DOI] [PubMed] [Google Scholar]
  8. Chitra, R. and Smith, P.E. 2001. Properties of 2,2,2-trifluoroethanol and water mixtures. J. Chem. Phys. 114 426–435. [Google Scholar]
  9. Come, J.H., Fraser, P.E., and Lansbury Jr., P.T. 1993. A kinetic model for amyloid formation in the prion diseases: Importance of seeding. Proc. Natl. Acad. Sci. 90 5959–5963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Gioia, L., Selvaggini, C., Ghibaudi, E., Diomede, L., Bugiani, O., Forloni, G., Tagliavini, F., and Salmona, M. 1994. Conformational polymorphism of the amyloidogenic and neurotoxic peptide homologous to residues 106–126 of the prion protein. J. Biol. Chem. 269 7859–7862. [PubMed] [Google Scholar]
  11. Gasset, M., Baldwin, M.A., Lloyd, D.H., Gabriel, J.M., Holtzman, D.M., Cohen, F., Fletterick, R., and Prusiner, S.B. 1992. Predicted α-helical regions of the prion protein when synthesized as peptides form amyloid. Proc. Natl. Acad. Sci. 89 10940–10944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gasset, M., Baldwin, M.A., Fletterick, R.J., and Prusiner, S.B. 1993. Perturbation of the secondary structure of the scrapie prion protein under conditions that alter infectivity. Proc. Natl. Acad. Sci. 90 1–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goldmann, W., Hunter, N., Foster, J.D., Salbaum, J.M., Beyreuther, K., and Hope, J. 1990. Two alleles of a neural protein gene linked to scrapie in sheep. Proc. Natl. Acad. Sci. 87 2476–2480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Griffith, J.S. 1967. Self-replication and scrapie. Nature 215 1043–1044. [DOI] [PubMed] [Google Scholar]
  15. Heller, J., Kolbert, A.C., Larsen, R., Ernst, M., Bekker, T., Baldwin, M., Prusiner, S.B., Pines, A., and Wemmer, D.E. 1996. Solid-state NMR studies of the prion protein H1 fragment. Protein Sci. 5 1655–1661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hurd, R.E. and John, B.K. 1991. Gradient-enhancement proton-detected heteronuclear multiple-quantum coherence spectroscopy. J. Magn. Reson. 91 648–653. [Google Scholar]
  17. Inouye, H. and Kirschner, D.A. 1997. X-ray diffraction analysis of scrapie prion: Intermediate and folded structures in a peptide containing two putative α-helices. J. Mol. Biol. 268 375–389. [DOI] [PubMed] [Google Scholar]
  18. Jamin, N., Coic, Y.M., Landon, C., Ovtracht, L., Baleux, F., Neumann, J.M., and Sanson, A. 2002. Most of the structural elements of the globular domain of murine prion protein form fibrils with predominant β-sheet structure. FEBS Lett. 529 256–260. [DOI] [PubMed] [Google Scholar]
  19. Kaneko, K., Zulianello, L., Scott, M., Cooper, C.M., Wallace, A.C., and James, T.L. 1997. Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc. Natl. Acad. Sci. 94 10069–10074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Koradi, R., Billeter, M., and Wüthrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graph. 14 51–55. [DOI] [PubMed] [Google Scholar]
  21. Korth, C., Stierli, B., Streit, P., Moser, M., Schaller, O., Fischer, R., Shulz-Schaeffer, W., Kretzschmar, H., Raeber, A., Braun, U., et al. 1997. Prion (PrPSc)-specific epitope defined by a monoclonal antibody. Nature 390 74–77. [DOI] [PubMed] [Google Scholar]
  22. Kozin, S.A., Bertho, G., Mazur, A.K., Rabesona, H., Girault, J.-P., Haertlé, T., Takahashi, M., Debey, P., and Hui Bon Hoa, G. 2000. Prion peptide folds into water-stable monomeric β-sheet : Possible molecular trigger of prion disease. In Twenty-sixth European Peptide Symposium (eds. J. Martinez and J.A. Fehrentz), pp. 495–496. EDK, Montpellier, France.
  23. Kozin, S.A., Bertho, G., Mazur, A.K., Rabesona, H., Girault, J.P., Haertle, T., Takahashi, M., Debey, P., and Hoa, G.H. 2001. Sheep prion protein synthetic peptide spanning helix 1 and β-strand 2 (residues 142–166) shows β-hairpin structure in solution. J. Biol. Chem. 276 46364–46370. [DOI] [PubMed] [Google Scholar]
  24. Laskowski, R.A., Rullmann, J.A., MacArthur, M.W., Kaptein, R., and Thornton, J.M. 1996. AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8 477–486. [DOI] [PubMed] [Google Scholar]
  25. Liu, A., Riek, R., Zahn, R., Hornemann, S., Glockshuber, R., and Wuthrich, K. 1999a. Peptides and proteins in neurodegenerative disease: Helix propensity of a polypeptide containing helix 1 of the mouse prion protein studied by NMR and CD spectroscopy. Biopolymers 51 145–152. [DOI] [PubMed] [Google Scholar]
  26. Liu, H., Farr-Jones, S., Ulyanov, N.B., Llinas, M., Marqusee, S., Groth, D., Cohen, F.E., Prusiner, S.B., and James, T.L. 1999b. Solution structure of Syrian hamster prion protein rPrP(90–231). Biochemistry 38 5362–5377. [DOI] [PubMed] [Google Scholar]
  27. Lopez Garcia, F., Zahn, R., Riek, R., and Wuthrich, K. 2000. NMR structure of the bovine prion protein. Proc. Natl. Acad. Sci. 97 8334–8339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morrissey, M.P. and Shakhnovich, E.I. 1999. Evidence for the role of PrP(C) helix 1 in the hydrophilic seeding of prion aggregates. Proc. Natl. Acad. Sci. 96 11293–11298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Muramoto, T., Scott, M., Cohen, F.E., and Prusiner, S.B. 1996. Recombinant scrapie-like prion protein of 106 amino acids is soluble. Proc. Natl. Acad. Sci. 93 15457–15462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nguyen, J., Baldwin, M.A., Cohen, F.E., and Prusiner, S.B. 1995. Prion protein peptides induce α-helix to β-sheet conformational transitions. Biochemistry 34 4186–4192. [DOI] [PubMed] [Google Scholar]
  31. Pan, K.M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlborn, I., Huang, Z., Fletterick, R.J., Cohen, F.E., et al. 1993. Conversion of α-helices into β-sheet features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. 90 10962–10966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pato, C., Celier, C., Rezaei, H., Grosclaude, J., and Marden, M.C. 2004. Heme as an optical probe of a conformational transition of ovine recPrP. Protein Sci 13 1100–1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pillot, T., Lins, L., Goethals, M., Vanloo, B., Baert, J., Vandekerckhove, J., Rosseneu, M., and Brasseur, R. 1997. The 118–135 peptide of the human prion protein forms amyloid fibrils and induces liposome fusion. J. Mol. Biol. 274 381–393. [DOI] [PubMed] [Google Scholar]
  34. Piotto, M., Saudek, V., and Sklenar, V. 1992. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2 661–665. [DOI] [PubMed] [Google Scholar]
  35. Priola, S.A., Chabry, J., and Chan, K. 2001. Efficient conversion of normal prion protein (PrP) by abnormal hamster PrP is determined by homology at amino acid residue 155. J. Virol. 75 4673–4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Prusiner, S.B. 1991. Molecular biology of prion diseases. Science 252 1515–1522. [DOI] [PubMed] [Google Scholar]
  37. ———. 1998. Prions. Proc. Natl. Acad. Sci 95 13363–13383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. ———. 2001. Shattuck lecture—Neurodegenerative diseases and prions. N. Engl. J. Med. 344 1516–1526. [DOI] [PubMed] [Google Scholar]
  39. Prusiner, S.B., McKinley, M.P., Bowman, K.A., Bolton, D.C., Bendheim, P.E., Groth, D.F., and Glenner, G.G. 1983. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35 349–358. [DOI] [PubMed] [Google Scholar]
  40. Prusiner, S.B., Scott, M., Foster, D., Pan, K.M., Groth, D., Mirenda, C., Torchia, M., Yang, S.L., Serban, D., Carlson, G.A., et al. 1990. Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 63 673–686. [DOI] [PubMed] [Google Scholar]
  41. Ragg, E., Tagliavini, F., Malesani, P., Monticelli, L., Bugiani, O., Forloni, G., and Salmona, M. 1999. Determination of solution conformations of PrP106–126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics. Eur. J. Biochem. 266 1192–1201. [DOI] [PubMed] [Google Scholar]
  42. Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wuthrich, K. 1996. NMR structure of the mouse prion protein domain PrP(121–321). Nature 382 180–182. [DOI] [PubMed] [Google Scholar]
  43. Riek, R., Hornemann, S., Wider, G., Glockshuber, R., and Wuthrich, K. 1997. NMR characterization of the full-length recombinant murine prion protein, mPrP(23–231). FEBS Lett. 413 282–288. [DOI] [PubMed] [Google Scholar]
  44. Riek, R., Wider, G., Billeter, M., Hornemann, S., Glockshuber, R., and Wuthrich, K. 1998. Prion protein NMR structure and familial human spongiform encephalopathies. Proc. Natl. Acad. Sci. 95 11667–11672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Safar, J., Roller, P.P., Gajdusek, D.C., and Gibbs Jr., C.J. 1993. Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J. Biol. Chem. 268 20276–20284. [PubMed] [Google Scholar]
  46. Schwarzinger, S., Kroon, G.J.A., Foss, T.R., Chung, J., Wright, P.E., and Dyson, H.J. 2001. Sequence-dependent correction of random coil NMR chemical shifts. J. Am. Chem. Soc. 123 2970–2978. [DOI] [PubMed] [Google Scholar]
  47. Sharman, G.J., Kenward, N., Williams, H.E., Landon, M., Mayer, R.J., and Searle, M.S. 1998. Prion protein fragments spanning helix 1 and both strands of β sheet (residues 125–170) show evidence for predominantly helical propensity by CD and NMR. Fold. Des. 3 313–320. [DOI] [PubMed] [Google Scholar]
  48. Speare, J.O., Rush III, T.S., Bloom, M.E., and Caughey, B. 2003. The role of helix 1 aspartates and salt bridges in the stability and conversion of prion protein. J. Biol. Chem. 278 12522–12529. [DOI] [PubMed] [Google Scholar]
  49. Tagliavini, F., Prelli, F., Verga, L., Giaccone, G., Sarma, R., Gorevic, P., Ghetti, B., Passerini, F., Ghibaudi, E., Forloni, G., et al. 1993. Synthetic peptides homologous to prion protein residues 106–147 form amyloid-like fibrils in vitro. Proc. Natl. Acad. Sci. 90 9678–9682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Telling, G.C., Scott, M., Mastrianni, J., Gabizon, R., Torchia, M., Cohen, F.E., DeArmond, S.J., and Prusiner, S.B. 1995. Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83 79–90. [DOI] [PubMed] [Google Scholar]
  51. Venyaminov, S.Y. and Yang, J.T. 1996. Circular dichroism and the conformational analysis of biomolecules, pp. 69–104. Plenum Press, New York.
  52. Viles, J.H., Donne, D., Kroon, G., Prusiner, S.B., Cohen, F.E., Dyson, H.J., and Wright, P.E. 2001. Local structural plasticity of the prion protein. Analysis of NMR relaxation dynamics. Biochemistry 40 2743–2753. [DOI] [PubMed] [Google Scholar]
  53. Vorberg, I., Chan, K., and Priola, S.A. 2001. Deletion of β-strand and α-helix secondary structure in normal prion protein inhibits formation of its protease-resistant isoform. J. Virol. 75 10024–10032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Winklhofer, K.F., Heske, J., Heller, U., Reintjes, A., Muranyi, W., Moarefi, I., and Tatzelt, J. 2003. Determinants of the in vivo folding of the prion protein. A bipartite function of helix 1 in folding and aggregation. J. Biol. Chem. 278 14961–14970. [DOI] [PubMed] [Google Scholar]
  55. Wishart, D.S. and Sykes, B.D. 1994. The 13C chemical-shift index: A simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4 171–180. [DOI] [PubMed] [Google Scholar]
  56. Wüthrich, K. 1986. NMR of proteins and nucleic acids. John Wiley & Sons, New York.
  57. Zahn, R., Liu, A., Luhrs, T., Riek, R., von Schroetter, C., Lopez Garcia, F., Billeter, M., Calzolai, L., Wider, G., and Wuthrich, K. 2000. NMR solution structure of the human prion protein. Proc. Natl. Acad. Sci. 97 145–150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhang, H., Kaneko, K., Nguyen, J.T., Livshits, T.L., Baldwin, M.A., Cohen, F.E., James, T.L., and Prusiner, S.B. 1995. Conformational transitions in peptides containing two putative α-helices of the prion protein. J. Mol. Biol. 250 514–526. [DOI] [PubMed] [Google Scholar]
  59. Ziegler, J., Sticht, H., Marx, U.C., Muller, W., Rosch, P., and Schwarzinger, S. 2003. CD and NMR studies of prion protein (PrP) helix 1. Novel implications for its role in the PrPC → PrPSc conversion process. J. Biol. Chem. 278 50175–50181. [DOI] [PubMed] [Google Scholar]

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