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Biochem. J. (2007) 401 (533–540) (Printed in Great Britain)

NMR characterization of the pH 4 b-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility

Denis B. D. O'SULLIVAN*, Christopher E. JONES*1, Salama R. ABDELRAHEIM†, Andrew R. THOMPSETT†, Marcus W. BRAZIER†, Harold TOMS*, David R. BROWN† and John H. VILES*2

*School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, U.K., and †Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K.

Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely a-helical isoform to a b-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an a-helical protein into one that contains a high proportion of b-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. ¹⁵N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(23–231) show that a total of 150 dispersed amide signals are resolved in the native form, whereas only 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. Three-dimensional ¹⁵N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23–118. ¹⁵N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112–231), which shows only 16 ¹⁵N-HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten-globule state.

Key words: CD spectroscopy, folding intermediate, prion protein (PrP), secondary structure, translational diffusion, T₂ relaxation.

Abbreviations used: CJD, Creutzfeldt–Jacob disease; HSQC, heteronuclear single-quantum correlation; NOE, nuclear Overhauser effect; PrP, prion protein; huPrP, human PrP; mPrP, mouse PrP; PrP^C, cellular isoform of PrP; PrP^Sc, scrapie isoform of PrP; STE, stimulated echo.

¹Present address: Center for Metals in Biology, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

²To whom correspondence should be addressed (email j.viles@qmul.ac.uk).

INTRODUCTION

CJD (Creutzfeldt–Jacob disease) in humans, BSE (bovine spongiform encephalopathy) in cattle and scrapie in sheep are fatal neurodegenerative diseases. The infectious agent of these diseases is devoid of nucleic acid and is almost entirely composed of PrP^Sc [scrapie isoform of PrP (prion protein)], which is a misfolded form of PrP^C (cellular isoform of PrP) [1]. The misfolding and folding intermediates of PrP are therefore of significant interest to those studying the mechanism of prion replication. Recently, it has been shown that it is possible to take synthetic recombinant PrP^C and cause it to misfold so that it can act as a seed for subsequent prion propagation after inoculation in transgenic mice overexpressing PrP^C [2].

The mature form of the benign mammalian PrP is a 208-residue glycoprotein that possesses two N-glycosylation sites and one disulfide bridge. After transportation through the secretory pathway, PrP^C is tethered to the cell surface via a glycosylphosphatidylinositol anchor at the C-terminus [1]. Three-dimensional NMR solution structures of PrP^C from a number of mammalian species have been reported [3–6] which show that PrP^C consists of two structurally distinct domains. The N-terminal domain (residues 23–125) is highly disordered [7] and is notable for its ability to bind Cu²⁺ ions at pH 6 and above [8–12]. The N-terminus contains a highly conserved octa-repeating sequence, PHGGGWGQ, between residues 57 and 90. The C-terminal domain (residues 126–231) is predominantly helical, containing three a-helices (residues 144–154, 175–193 and 200–219) as well as two short anti-parallel b-strands (residues 128–131 and 161–164).

The conversion of native PrP^C into its misfolded isoform PrP^Sc is accompanied by a number of biophysical changes. PrP^C is monomeric and readily susceptible to proteolysis with proteinase K but PrP^Sc forms insoluble aggregates that are resistant to proteinase K digestion [13–15]. Furthermore, IR spectroscopy show that while PrP^C is predominately a-helical, PrP^Sc possesses a considerable amount of b-sheet structure [16,17].

An early CD study on the unfolding behaviour of mPrP (mouse PrP)-(121–231) indicated that the structural domain of PrP undergoes a transition from the native folded form (at neutral pH values) to a stable soluble conformational intermediate rich in b-sheet at pH 4 under mildly denaturing conditions (3.5 M urea), whereas at pH 2 only the unfolded form was present [18]. Similarly, an unfolding study performed on huPrP (human PrP)-(90–231) using guanidinium chloride as denaturant showed the existence of a stable b-intermediate between pH 3.5 and 4.0 [19]. Subsequent studies confirmed the presence of the huPrP-(90–231) b-intermediate and also highlighted the necessity of having NaCl present for the formation of this b-intermediate in urea [20,21].

Studies by Baskakov and others have shown that PrP (with its disulfide bond intact) can fold in one of three forms [20–23]. Firstly, the a-helix-rich, soluble monomeric form, PrP^C, is rapidly formed under near neutral non-denaturing conditions [3–6]. Secondly, a high-molecular-mass multimer that forms amyloid fibres is favoured under neutral pH, moderately denaturing conditions and agitation [22]. The third form is a soluble species favoured at low pH (~4) under moderately denaturing conditions with a high salt content [20,21,23]. This latter soluble species has a high b-sheet content as indicated by CD [18–21,23]. Size-exclusion chromatography suggests that this soluble oligomer is octameric [22], while light scattering experiments suggest that this oligomer contains 8–15 monomers [24] or higher molecular mass species [21,25].

Polymorphism at residue 129 is a key determinant of susceptibility to sporadic and acquired prion diseases. Recent studies have shown that stability of the native a-helical PrP is unaffected by polymorphism at 129 (Met¹²⁹ or Val¹²⁹) [26]. However, the presence of valine at position 129 accelerates fibril formation at pH 7 [27], while conversely Met¹²⁹ favours the formation of the pH 4 soluble intermediate [28]. Interestingly, only individuals homozygous for methionine at 129 (Met/Met) have contracted variant CJD [29]. Prion propagation in transgenic mice is triggered by preparations containing fibrils [2], although the toxic form is likely to be smaller protofibrils [30].

Structural details, on a per-residue basis, are lacking for the pH 4 folding intermediate, despite its importance in influencing the susceptibilities to prion propagation. We therefore aim to characterize the pH 4 intermediate using NMR methods. Chemical shift dispersion in three-dimensional ¹⁵N-HSQC (heteronuclear single-quantum correlation)-TOCSY experiments will indicate the extent of folding of PrP. ¹⁵N transverse relaxation measurements will provide information on the flexibility of the pH 4 intermediate main chain and ¹H translational diffusion will indicate its molecular size.

EXPERIMENTAL

Expression and purification of recombinant mPrP-(23–231) and mPrP-(112–231)

Tag-free and C-terminally His-tagged full-length mPrP-(23–231) were cloned into a pET-23 vector as previously described [31]. In addition, a truncated PrP fragment, PrP-(112–231), was expressed which lacks the natively unstructured N-terminal domain. The proteins were expressed in 2-litre flasks of Escherichia coli BL21(DE3) cells in ¹⁵N-labelled minimal medium containing (1 g/l) ¹⁵NH₄Cl (Cambridge Isotope Laboratories). Cultures were grown at 37 °C to a D₆₀₀ of 0.9 and the protein expression was induced by the addition of isopropyl-b-D-thiogalactopyranoside to a final concentration of 1 mM. Bacterial pellets were harvested after 4 h and sonicated in Buffer A containing 8 M urea, 200 mM NaCl and 50 mM Tris (pH 7.6). The resulting solution was cleared of bacterial debris as previously described [31]. The protein (tag-free or His-tagged) was absorbed on a copper-charged metal affinity column made from chelating Sepharose (Amersham Biosciences). The proteins were washed with at least 20 column volumes of Buffer A and eluted from the column using Buffer A supplemented with 300 mM imidazole. The protein was found to be greater than 95% pure by PAGE and Coomassie staining. The protein was refolded by successive rounds of dilution in deionized water and concentrated in a Vivaspin centrifugal concentrator (Vivascience) with a 10000 Da molecular-mass cutoff. The refolded protein was dialysed twice at 4 °C against deionized water to remove residual urea. The concentration of mPrP-(23–231) was measured by its absorbance at 280 nm with a theoretical molar absorption coefficient (e) of 62280 M^-1·cm^-1 [32] and confirmed by BCA (bicinchoninic acid) assay (Sigma).

Preparation of samples

NMR samples of full-length mPrP-(23–231) were prepared in four different buffer systems. Samples of native His-tagged and tag-free mPrP-(23–231) were prepared in 20 mM sodium acetate at pH 5.2. A b-intermediate sample of mPrP-(23–231) was generated by preparing the protein in 3.5 M urea, 150 mM NaCl and 20 mM sodium acetate at pH ~3.9 (between pH 3.7 and 4.4). An acid denatured sample of mPrP-(23–231) was made by preparing the protein in 3.5 M urea, 150 mM NaCl and 20 mM sodium acetate at pH 1.82. The fourth condition was using tag-free mPrP-(23–231) and mPrP-(112–231) at pH ~3.9 with a raised temperature of 37 °C in 20 mM sodium acetate instead of denaturant. The NMR samples contained 90% (v/v) water/10% (v/v) ²H₂O and 0.005% (w/v) sodium azide to inhibit bacterial growth. Protein concentrations for NMR experiments were approx. 5 mg/ml.

NMR spectroscopy

All NMR spectra were acquired at 30 °C (or 37 °C when stated) on a Bruker Avance spectrometer operating at 600.1 MHz for ¹H nuclei using a 5 mm inverse detection triple-resonance z-gradient probe. Phase-sensitive two-dimensional ¹⁵N-HSQC spectra were acquired using Echo/antiecho gradient selection. ¹H acquisition parameters were 0.122 s acquisition time, 1 s fixed delay and 2048 complex (t₂) points, while 256 complex points were collected for the ¹⁵N dimension. Thirty-two transients were recorded for each t₁ interval. The ¹H and ¹⁵N dimensions possessed spectral widths of 14 and 25 p.p.m. respectively. The ¹⁵N dimension was zero filled to 256 data points before squared cosine apodization and Fourier transformation.

Resonance assignments of the pH 4 intermediate were obtained using phase-sensitive three-dimensional ¹⁵N-HSQC-NOESY [33] and ¹⁵N-HSQC-TOCSY spectra [34] using Echo/antiecho gradient selection. The three-dimensional ¹⁵N-HSQC-TOCSY and three-dimensional ¹⁵N-HSQC-NOESY spectra were obtained using a mixing time of 60 and 150 ms respectively. For both experiments, 2048 complex points were collected in the direct ¹H dimension over a spectral width of 12 p.p.m. Sixty-four complex points and 160 complex points were collected for the indirect ¹⁵N and ¹H dimensions respectively over a ¹⁵N spectral width of 25 p.p.m. and a ¹H spectral width of 12 p.p.m. in both experiments. Both spectra were zero filled to 2048 data points in the direct ¹H dimension, 128 data points in the indirect ¹⁵N dimension and 512 data points in the indirect ¹H dimension. All NMR data were processed in XWINNMR (Bruker) and analysed in XEASY running on a Silicon Graphics O2 computer (SGI). Resonance assignments were made manually using sequential ¹H NOE (nuclear Overhauser effect) connectivities.

¹⁵N spin spin T₂ relaxation times were obtained from running phase-sensitive two-dimensional ¹⁵N-HSQC spectra acquired with Echo/antiecho gradient selection as described above. The variable relaxation delays used were 0.016, 0.032, 0.048, 0.064, 0.096, 0.128, 0.196 and 0.256 s [35]. ¹⁵N transverse relaxation times were obtained by fitting two-dimensional signal volumes or peak heights [36] to the following exponential decay curve: I_t=I₀ e^-t/T₂, where I_t is the two-dimensional signal intensity at time t, I₀ is the initial two-dimensional signal intensity and T₂ is the transverse relaxation time (note, T₂=1/R₂).

Translational diffusion experiments were carried out using the STE (stimulated echo) method incorporating bipolar gradients to reduce eddy current effects [37,38]. The gradient strength was calibrated using the value of 2.299×10^-5 cm²/s as the translation diffusion constant for water at 25 °C. Water in the protein samples was suppressed with a 3-9-19 WATERGATE sequence. A spoiler gradient of 17.13% was applied at the start of the diffusion time D. For protein and water diffusion measurements, the total duration of the gradient pulse d was 0.006 and 0.002 s respectively. For both measurements the time between gradients (D) was 0.1 s. An exponential line-broadening function (1 Hz) was applied prior to Fourier transformation. The integral of the baseline-corrected amide/aromatic region of the proton spectra was measured at 16 different gradient strengths (G) ranging from 0.741 to 35.21 G/cm and fitted to the following equation:

I₀ is the initial integral, I is the measured integral, D_t is the translational diffusion coefficient (cm²/s), and g_H is the ¹H gyromagnetic ratio (2.675197×10⁴ G^-1·s^-1) [37,38]. Plotting ln(I) against G² produces a straight line whose slope divided by g_H²d²(D-d/3) yields D_t. The D_t values for water in the native, b-intermediate and acid denatured samples of mPrP-(23–231) were 2.30±0.01×10^-5, 1.87±0.01×10^-5 and 1.89±0.01×10^-5 cm²/s respectively. The small decrease in the latter two D_t values for water was presumably due to the increased viscosity of these solutions arising from the presence of 3.5 M urea. In order to correct for this effect, the D_t value of the b-intermediate form of mPrP-(23–231) was multiplied by 1.23 (2.30/1.87), while the D_t value of the acid denatured form of mPrP-(23–231) was multiplied by 1.22 (2.30/1.89). Similarly there was also a small correction made for D_t measurements recorded at 37 °C. The D_t values for water in 20 mM sodium acetate at 30 and 37 °C were 2.35±0.01×10^-5 and 2.71±0.01×10^-5 cm²/s respectively. The D_t value at 37 °C was therefore corrected by a factor of 1.15 (2.71/2.35) for a direct comparison of diffusion coefficient at the two temperatures.

CD spectroscopy

CD spectra were recorded in the far-UV region (185–260 nm) at 25 °C in a 0.02 cm path length cuvette (for samples containing urea) on an AVIV 202 CD spectrometer. CD spectra of tag-free mPrP-(23–231) and His-tagged mPrP-(112–231) (urea-free) were recorded in the far-UV region at 37 °C in a 0.1 cm path length cuvette on an Applied Photophysics Chirascan CD spectrometer. Protein samples were prepared by diluting an aliquot from each of the NMR samples described previously with the appropriate buffer system. Spectra were recorded using three to four scans, a bandwidth of 1 nm and a wavelength step of 0.5 nm. CD spectra were background corrected and smoothed using AVIV software; smoothing for the urea-free samples was not required. The protein concentration of CD samples was measured using the absorbance at 280 nm. Estimates of percentage secondary structure were obtained using the K2D and CDSSTR analysis programs available on the Dichroweb website [39]. CD spectra are presented as mean residue molar CD De_MRE units (M^-1·cm^-1).

RESULTS

Two-dimensional ¹⁵N-HSQC and CD spectroscopy of mPrP-(23–231)

Chemical shift dispersion in NMR is a powerful method of determining the extent of structure in proteins on a per-residue basis [40–42]. For this reason two-dimensional ¹⁵N-HSQC was used to characterize the folding of PrP under various conditions. The two-dimensional ¹⁵N-HSQC spectrum of native mPrP-(23–231) (Figure 1A) possesses amide ¹H signals in the ¹H dimension from 6.5 to 9.5 p.p.m., characteristic of a folded protein [40–42]. However, for conditions promoting formation of the b-intermediate i.e. 3.5 M urea and 150 mM NaCl at pH 4.1 (Figure 1B), ¹H signals lie between 7.8 and 8.7 p.p.m. This contraction in the chemical shift dispersion is typical of a completely unfolded protein [40–42]. The 0.9 p.p.m. ¹H dispersion range for amides in the two-dimensional ¹⁵N-HSQC of mPrP-(23–231) was very similar to that of the acid denatured species (Figure 1C), although the chemical shifts of the amide resonances are not the same. An aliquot from each of the three NMR samples was prepared for UV-CD spectroscopy. The CD spectrum of native mPrP-(23–231), Figure 2 (spectrum A), is typical of a protein possessing a-helices. However, the CD spectrum taken from the NMR sample of mPrP-(23–231) under b-intermediate-forming conditions, is characteristic of a b-sheet species with a strong negative CD band at 217 nm (Figure 2, spectrum B). The acid denatured spectrum, Figure 2 (spectrum C), shows less ellipticity, suggesting a more random conformation.

pH 4 intermediate created at 37 °C

A recent study has indicated that the pH 4 intermediate can also be produced in the absence of chemical denaturant by simply raising the temperature to 37 °C [24,25]. Figure 3(A) shows the two-dimensional ¹⁵N-HSQC of mPrP-(23–231) at pH 3.6 incubated for approx. 2 h at 37 °C. Incubation of the NMR sample for a further 30 h caused no further change in the two-dimensional ¹⁵N-HSQC spectra (results not shown). A CD spectrum of this NMR sample was then obtained at 37 °C. The incubation at 37 °C causes a loss of helical content and the appearance of a strong band at 217 nm (Figure 3B). After incubation at 37 °C, both the two-dimensional ¹⁵N-HSQC and CD spectra (Figures 3A and 3B) have a strong resemblance to the corresponding spectra of the urea-induced pH 4 intermediate shown in Figures 1 and 2; although there is not a direct correspondence between amide resonances obtained at 30 °C in urea and those obtained at 37 °C in water. It was possible to obtain estimates of the amount of secondary structure from the CD spectra of the pH 4 intermediate at 37 °C using deconvolution algorithms. Analysis with the CDSSTR algorithms suggested 31% b-sheet present and only 3% a-helix. However, using deconvolution analysis with the K2D secondary structure estimation program, 31% b-sheet was indicated but a higher estimate of the a-helical content at 16%.

A ‘head’ count of the number of observable ¹⁵N amide resonances in the pH 4 intermediate suggests a loss of some signals; from approx. 150 native full-length PrP (207 amino acids minus proline residues and octa-repeat degeneracy of signals) to half that in the pH 4 intermediate. Both the urea- and temperature-induced pH 4 intermediate shows a loss of half the amide cross-peaks but no loss of amide signal intensity. Using the two-dimensional ¹⁵N-HSQC alone it was not clear if this loss of signals was due to signal overlap in the less dispersed spectra or the possibility that only a portion of the PrP was detected by the two-dimensional ¹⁵N-HSQC in the pH 4 intermediate. The formation of a high-molecular-mass soluble polymeric species would become largely undetectable in the two-dimensional ¹⁵N-HSQC experiment due to their increased line widths. Alternatively, the formation of a molten-globule conformation could cause exchange broadening of many signals. The retention of signals attributable to the tryptophan side chain and glycine residues (for both the urea unfolded and the temperature induced pH intermediate) hints strongly at the possibility that only the N-terminal half of the protein is being detected. Most of the tryptophan and glycine residues occur within the N-terminal half of the protein; residues 23–118 contain 27 out of 35 glycine and seven out the eight tryptophan residues of mPrP.

Truncated PrP, residues 112–231

To test this hypothesis, PrP-(112–231), a truncated version of full-length PrP, was studied. This fragment lacks nearly all the unstructured N-terminal residues of native PrP^C. Similar truncated fragments have been shown to form a pH 4 intermediate [18,19]. Figure 3 presents a comparison of the pH 4 intermediate for full-length PrP-(23–231) with that of PrP-(112–213). There is a dramatic change in the appearance of the two-dimensional ¹⁵N-HSCQ spectrum for the truncated PrP-(112–231) pH 4 intermediate, with only a few residues (~16 amide signals) being detected. This observation strongly suggests that the two-dimensional ¹⁵N-HSQC of full-length PrP for the pH 4 intermediate only detects N-terminal residues (23–121). This is confirmed in the following section using three-dimensional ¹⁵N-HSQC-TOCSY and ¹⁵N-HSQC-NOESY assignment methods.

Estimates of the secondary structure for the PrP-(112–231) pH 4 intermediate from the CD spectra using CDSSTR fitting suggest that it is composed of approx. 46% b-sheet, 27% turns and 23% random and almost no a-helix (5%). However, the K2D secondary structure estimates suggest that the pH 4 intermediate contains as much as 21% a-helix and 31% b-sheet.

Main chain NMR assignments for the pH 4 intermediate

In order to understand the ¹⁵N-HSQC spectra further, three-dimensional ¹⁵N-edited HSQC-TOCSY and NOESY experiments were obtained for the pH 4 intermediate for mPrP-(23–231) in water at 37 °C. Almost all of the observed amide resonances from the ¹⁵N-HSQC have been assigned. All assigned resonances come from the N-terminus, between residues Gly³⁰ and Gly¹¹⁸, and are indicated on the ¹⁵N-HSQC (Figure 3A). Taken from the three-dimensional ¹⁵N-HSQC-NOESY spectra, a section of a strip-plot for residues Thr⁹⁴–Asn¹⁰⁷ is shown in Figure 4; sequential NOEs are indicated. There are a limited number of missing assignments between Gly²⁹ and Gly¹¹⁸, attributed to glycine residues. The N-terminus contains a high percentage of GG sequences, which contributes to the overlap and makes a number of glycine amide peaks ambiguous to assign. Amide resonances within the four octa-repeats are degenerate and have four times the signal intensity of other amide signals. In addition, there are a further four amide peaks with observable TOCSY side-chain resonances; these amides lack appreciable NOE connectivities but can tentatively be attributed to lysine and arginine residues at the N-terminus, residues Lys²³ to Lys²⁷.

Chemical shift values have minimal deviations from ‘random-coil’. For example, there are no Ha deviations that exceed 0.15 p.p.m. and the overwhelming majority have deviation less than 0.1 p.p.m. CSI (chemical shift indexing) criteria [43,44] indicate that all the residues, 23–118, are indeed unstructured in the pH 4 intermediate (37 °C). Ha chemical shift assignments and deviations from random coil are available as supplementary material (see http://www.BiochemJ.org/bj/401/bj4010533add.htm). We note that signals up to residue 115 are quite intense, comparable with intense signals from native PrP^C-(23–231). Resonances for Ala¹¹⁷ and Gly¹¹⁸ have strongly attenuated signals while residues beyond 118 are not observed, due to the increased line widths of these signals.

The ¹⁵N-HSQC spectrum for mPrP-(112–231), pH 4, 37 °C (Figure 3C), shows a large reduction in the number of observed amide resonances; only 16 amide signals are observed. A number of these signals directly overlay residues 114–118 from the pH 4 b-intermediate of mPrP-(23–231).

Signal intensities of the b-intermediate in the two-dimensional ¹⁵N-HSQC spectra are very similar to that of the native two-dimensional ¹⁵N-HSQC, indicating that the majority of the protein is detected in the pH 4 two-dimensional ¹⁵N-HSQC. A single set of resonances is observed for each spin-system, between residues 23 and 118. Based on peak intensity more than 90% of the protein is detected by the two-dimensional ¹⁵N-HSQC spectra of the pH 4 intermediate. This is true even when the more intense signals in the unstructured tail of native PrP^C are compared with resolved resonances of the b-intermediate.

Formation of the pH 4 intermediate is reversible and not subject to proteolytic cleavage

There remained the possibility, albeit unlikely given the presence of a bacterial inhibitor reagent sodium azide, that the detection of the N-terminal residues in the mPrP-(23–231) pH 4 intermediate is the result of proteolytic cleavage of PrP under these conditions. However, the SDS/PAGE of the native pH 4 intermediate and the refolded native PrP-(23–231) samples all showed intact PrP with no appreciable cleavage products (see supplementary data at http://www.BiochemJ.org/bj/401/bj4010533add.htm). Studies indicate that the formation of the pH 4, 37 °C, b-intermediate is reversible. Taking the pH 4 intermediate of mPrP-(112–231) back to pH 5.5 causes the reappearance of appreciable amounts of natively folded mPrP-(112–231), as indicated by the two-dimensional ¹⁵N-HSQC spectra and UV-CD spectra shown in Figure 5.

¹⁵N transverse relaxation times of mPrP-(23–231)

We then went on to characterize the molecular dynamics of the soluble PrP pH 4 intermediate using ¹⁵N transverse (T₂) relaxation measurements. R₂ relaxation is sensitive to the overall rotational correlational time (t_c) of a molecule. The presence of additional main-chain flexibility on a nanosecond-to-picosecond timescale can reduce R₂ values considerably.

R₂ relaxation values for amide resonances of the native mPrP-(23–231) are comparable with values reported for full-length Syrian hamster PrP [7,45] as would be expected. For example, typical ¹⁵N R₂ relaxation values for amide resonances in helices A, B and C were 12–19 s^-1 for full-length Syrian hamster PrP at 30 °C [7]. In the present study, amides in the same region of mPrP-(23–231) gave comparable R₂ values, e.g. 13.2, 16.7 and 18.7 s^-1 for Tyr¹⁵⁶, Lys²⁰³ and Arg²⁰⁷ respectively. In addition, amides from the unstructured tail (residues 29–120) gave R₂ values of approx. 4 s^-1 [7]. Similar values are obtained here for mPrP-(23–231), for example, Ala¹¹² has an R₂ of 3.0 s^-1.

R₂ values for individual resonances for the pH 4 intermediate (3.5 M urea) were nearly all below 4 s^-1, typically 3.6 s^-1. These R₂ values are considerably smaller than those of a folded protein 200 amino acids in size. It is clear that R₂ values are dominated by local picosecond timescale motions indicating a high degree of flexibility in the N-terminal half of the protein, residues 23–114.

Translational diffusion measurements of mPrP-(23–231)

Translational diffusion measurements can indicate the hydrodynamic radius or molecular size of a protein. The techniques measure the bulk property of the protein rather than local internal motions. Using translational diffusion to indicate the molecular size of the b-intermediate has some advantages over previous methods, such as size-exclusion chromatography, as the studies are performed in solution under conditions similar to that used for the CD measurements. The intensities of the ¹⁵N-HSQC signals indicate that for the N-terminal residues all the protein is being detected and not a small monomeric fraction. Figure 6 shows the ¹H spectra for native PrP acquired at different gradient strengths used to calculate the translational diffusion for PrP-(23–231) in the three states: native, b-intermediate and acid denatured. The translational diffusion coefficient (D_t) of native PrP-(23–231) in 20 mM sodium acetate at pH 5.23 and 30 °C was 0.78±0.01×10^-6 cm²/s. This value is identical with the D_t value reported for Syrian hamster PrP-(29–231) and is typical for a monomeric protein of this size [7].

The D_t for mPrP-(23–231) in 3.5 M urea and 150 mM NaCl at pH 4.4 (the b-intermediate form) was 0.81±0.02×10^-6 cm²/s. This is a value corrected for the increased viscosity due to the presence of 3.5 M urea. The close similarity of the b-intermediate D_t value to that of native mPrP-(23–231) indicated that the protein in its b-intermediate form is also predominantly monomeric. Uncorrected, the D_t (0.66±0.02×10^-6 cm²/s) of the b-intermediate is comparable with that for dimeric PrP.

The D_t for mPrP-(23–231) in 3.5 M urea and 150 mM NaCl at pH 1.65 (the acid denatured form) was 0.50±0.01×10^-6 cm²/s. The D_t for the acid denatured form, after correcting for the increased viscosity of the solution due to the presence of urea, was 0.61±0.01×10^-6 cm²/s. The decreased D_t of the acid denatured mPrP-(23–231) compared with the other two forms may indicate that it has aggregated into an oligomer. It is possible to calculate the increase in frictional coefficient, f, upon going from a monomer to an oligomer of n subunits [46]. The frictional coefficient of a molecule is related to the reciprocal of its translational diffusion by the Stokes Einstein equation, D_t=k_BT/f, in which k_B is the Boltzmann constant and T is the absolute temperature [37,46]. The increase in f upon going from a monomer to an oligomer of n subunits for different values of n have been calculated using the equation: F_n=n^1/3(f_m/f_n); F_n is a geometric factor, n is the number of subunits in the oligomer, while f_m and f_n are the frictional coefficients of the monomer and oligomer respectively [46]. The experimental D_t ratio of the acid denatured form of mPrP-(23–231) to that of native mPrP-(23–231) in the present study is 0.78. This ratio is very similar to a D_t ratio of 0.75 expected for a theoretical monomer/dimer model; note that the analysis assumes a globular (spherical) oligomer [46]. It is also similar to an experimental D_t ratio of 0.72 for the monomer/dimer system of ubiquitin and of the dimeric cytokine MCP-1 (monocyte chemoattractant protein-1) proteins [37]. Thus the translational diffusion data indicate that the native and b-intermediate forms of mPrP-(23–231) are predominantly monomeric, while the D_t of the acid denatured form implies a dimer.

The translational diffusion coefficient for the PrP sample obtained for the pH 4 intermediate at 37 °C but without urea present, exhibits a D_t of 0.62±0.02×10^-6 cm²/s. This value did not need to be corrected for the viscosity of urea as the denaturant was not present. The pH 4 intermediate, produced under these conditions, gives a translational diffusion coefficient close to that expected for a dimer of PrP. However, correction for the increased temperature (37 °C) gave a value of 0.54×10^-6 cm²/s. This value might suggest a larger molecule close to a trimer in size. These diffusion coefficients represent an average value for the PrP oligomer in equilibrium between monomer and higher molecular mass species. Small amounts of monomer would have the effect of increasing the average rate of translational diffusion, while large oligomers would have the opposite effect.

DISCUSSION

For the first time, the nature of the PrP pH 4 b-intermediate has been characterized on a per-residue basis. The NMR chemical shifts and assignments of the b-intermediate indicate that most of the PrP molecules within a pH 4 oligomer possess a long flexible tail incorporating residues 23–118. The exact nature of the C-terminal half of PrP remains elusive, as large line widths have rendered these residues undetectable by NMR. However, deconvolution of the CD spectra indicates a high proportion of b-sheet for this region: 31–46% b-sheet for PrP-(112–231). The variability in the secondary structure estimates by CD is perhaps due to the nature of the oligomer, which may contain extended conformations in a molten-globule state. Standard secondary structure elements used by the algorithms will therefore fit poorly to the observed spectra. Protein oligomers have a tendency to form extended conformations that produce CD spectra with a negative CD band at 217 nm similar to that observed for b-sheet proteins. It is therefore not clear whether either an extended b-like polypeptide chain or a more ordered hydrogen-bonded network, found in cross-b-sheets of amyloids, is present in the b-intermediate. It is clear that the formation of the soluble pH 4 oligomer is reversible; returning the pH to 5.5 results in the refolding of the monomeric native species.

The studies described here have been carried out with the native disulfide bond intact. It is generally believed that the PrP^Sc exists with the native disulfide bond present [47]. A model of amyloids of PrP^Sc based predominantly on EM data suggests that much of helices B and C remains intact in the fibrils, while residues 89–174 form a cross-b structure in a b-helix conformation [48]. Fourier-transform infrared spectroscopy suggests that some of the a-helical structure is retained in the pH 4 oligomeric species [24]. Estimates of a-helix content from the CD spectra (although variable between 3 and 21%) suggest that there is little a-helix content for the pH 4 intermediate. It remains to be established if helices remain in the pH 7 amyloid species.

The translational diffusion measurements suggest that the b-intermediate is a trimer in size. This is a surprise as a number of studies using guanidinium chloride, urea or elevated temperature as partial denaturants have indicated that the pH 4 b-intermediate is oligomeric. Investigations using size-exclusion chromatography and dynamic light scattering measurements suggest that this b-intermediate is approximately octameric [20,21,23]. Multi-angle laser light scattering experiments suggest that this oligomeric species contains 8–15 monomers [24]; small-angle X-ray scattering has also indicated a large oligomer [25]. Our own two-dimensional ¹⁵N-HSQC spectra indicate a complete loss of signal from the C-terminus of PrP, suggesting a molecule of high molecular mass that renders the signal too broad for detection. However, we do note that a loss of signal might also be due to exchange broadening due to slow millisecond–microsecond motions of the main chain of the C-terminal domain in a molten-globule state. The translational diffusion value represents a weighted average of various oligomeric species; however, the presence of, for example, 10% monomer would not reduce the D_t value by a significant amount.

Residues 90–121 have been shown to be essential for prion propagation, suggesting that a conformational change in this region is key to prion propagation [49,50]. Furthermore, a number of mutations associated with familial prion diseases are also found in this region. However, in native PrP, these residues are unstructured despite structure-prediction studies of native PrP that suggest that residues 109–122 might form an a-helix. Models of PrP^Sc suggest that residues 89–174 might form a b-helical structure [48]. However, interestingly as with native PrP these residues (23–118) remain unstructured with a high degree of flexibility in the soluble pH 4 b-intermediate.

Characterizing folding intermediates of the PrP is an important goal towards understanding the mechanism of prion replication. For example, it has been shown that the rate of formation of the pH 4 b-intermediate is influenced by polymorphism at residue 129. Met¹²⁹ has a higher propensity to form the pH 4 b-intermediate than Val¹²⁹ [28]. Interestingly, to date only individuals homozygous for methionine (Met/Met) have contracted variant CJD.

This work was funded by BBSRC (Biotechnology and Biological Sciences Research Council) project grants. C. E. J. is the recipient of a C. J. Martin Postdoctoral Fellowship from the National Health and Medical Research Council, Australia. We thank Marcus Fries for assistance with the SDS/PAGE.

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Received 5 May 2006/17 August 2006; accepted 8 September 2006

Published as BJ Immediate Publication 8 September 2006, doi:10.1042/BJ20060668

The Biochemical Society, London ©2007