The elusive intermediate on the folding pathway of the prion protein David C Jenkins, Ian D Sylvester and Teresa J T Pinheiro Department of Biological Sciences, University of Warwick, UK Keywords denaturant unfolding; molten globule; phasediagram; prion conversion; prion diseases Correspondence T J T Pinheiro, Department of Biological Sciences, Gibbet Hill Road, University of Warwick, Coventry CV4 7AL, UK Fax: +44 2476 523 701 Tel: +44 2476 528 364 E-mail: t.pinheiro@warwick.ac.uk (Received 14 August 2007, revised 20 December 2007, accepted 15 January 2008) A key molecular event in prion diseases is the conversion of the cellular conformation of the prion protein (PrPC) to an altered disease-associated form, generally denoted as scrapie isoform (PrPSc) The molecular details of this conformational transition are not fully understood, but it has been suggested that an intermediate on the folding pathway of PrPC may be recruited to form PrPSc In order to investigate the folding pathway of PrP we designed and expressed two mutants, each possessing a single strategically located tryptophan residue The secondary structure and folding properties of the mutants were examined Using conventional analyses of folding transition data determined by fluorescence and CD, and novel phase-diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP The potential role of this intermediate in prion conversion is discussed doi:10.1111/j.1742-4658.2008.06293.x Prion diseases, which include Creutzfeldt–Jakob disease in humans, bovine spongiform encephalopathy in cattle and scrapie in sheep, are associated with conversion of the normal cellular form of the prion protein (PrPC) to an altered pathological form, generally designated as the scrapie isoform (PrPSc) Such diseases can be sporadic, inherited or acquired by transmission Sporadic Creutzfeldt–Jakob disease accounts for 85% of all cases of the disease; around 10–15% are associated with the familial cases and fewer than 5% are transmitted [1] Although coded for by the same gene [2,3] and covalently identical [4], the structure and properties of PrPC and PrPSc contrast greatly Whereas PrPC is a globular protein, composed primarily of an unstructured N-terminal region and a C-terminal domain comprising three a-helices and two short b-strands (Fig 1) [5–8], PrPSc has a much higher proportion of b-sheet structure [9] Physicochemical studies have shown that PrPC is monomeric, soluble in aqueous buffer and sensitive to protease digestion, while, in comparison, PrPSc has a high propensity to aggregate, is water- and detergent-insoluble, and partially resistant to proteinases [2,10,11] The details of prion conversion are not fully understood, but it is generally accepted that the key molecular event in sporadic and familial cases of prion diseases involves a conformational transition of the prion protein from its cellular state to the altered disease-associated form [12] Thus, an understanding of the folding and refolding mechanisms of the prion protein should provide insight into the process of prion conversion, and represent a major step forward in our understanding of the misfolding and aggregation of PrP during disease Studies of the folding kinetics of PrP have indicated that it may fold via an intermediate state [13–15], and that this intermediate may, under as yet uncharacterized conditions, be recruited to form PrPSc Results from equilibrium folding studies have been ambiguous on defining the presence of an intermediate in the folding of PrP Initial studies apparently revealed a folding intermediate rich in b-structure [16], later shown to be off-pathway aggregated species in the folding of PrP Abbreviations PrP, prion protein; SHaPrP, Syrian hamster prion protein FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1323 Prion folding D C Jenkins et al Fluorescence and CD were used to establish the folding transitions of PrP and derive their thermodynamic folding parameters at two different pH values, which represent two distinct cellular environments of PrP The study is complemented by phase-diagram analyses of the fluorescence data, which unequivocally revealed the presence of an intermediate state in the folding of PrP Furthermore, using CD, we present compelling evidence in support of a molten-globule intermediate state in the folding of PrPC and propose that this intermediate may serve as a precursor for prion conversion Results Denaturant unfolding of PrP Fig Structure of the prion protein Ribbon representation of the folded C-terminal domain of SHaPrP(90–231) based on the NMR structure in aqueous solution [6] The N- and C-termini are labelled N and C, respectively; the three main helices I, II and III running from the N- to the C-terminus are shown in red, and the strands S1 and S2 of the short antiparallel b-sheet are drawn in yellow Using ball-and-stick representation, phenylalanine residues Phe175 and Phe198, which were in turn mutated to a tryptophan residue, are highlighted in green, and the disulfide bond between Cys179 and Cys214 is shown in blue The picture was drawn from PDB file 1B10 using the UCSF Chimera package from the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081) [64] [17] Recent evidence from NMR [18–20] and fluorescence studies [21] are indicative of the presence of an intermediate state Further clarification is clearly required to ascertain the presence of an intermediate in the folding of PrP and the conditions under which it is observed To investigate the equilibrium folding of PrP, we designed and expressed two tryptophan mutants of the truncated Syrian hamster prion protein (SHaPrP), comprising residues 90–231 This fragment contains the folded C-terminal domain [6] and corresponds to the proteinase K-resistant core of PrPSc [4,22] Each mutant possesses a single tryptophan residue, strategically located to produce a significant change in fluorescence upon unfolding and refolding transitions of the protein The mutations were made in a conservative fashion, replacing the bulky hydrophobic side chain of phenylalanine with that of tryptophan, as pioneered and validated previously [23,24] Such an approach has been used to great effect in previous folding studies of related PrP constructs from other species [14,15,25] Figure shows the location of the tryptophan residues in the single Trp mutants of PrP employed in this study 1324 In its normal cellular form, PrP adopts a conformation rich in a-helices with a small amount of b-sheet structure and a long unstructured N-terminus [7,8,26] The truncated protein PrP(90–231) contains the folded C-terminal domain, but most of the unstructured N-terminus is not present To establish that the single tryptophan variants of PrP made here have the same structure and behave in a manner similar to the wildtype protein, the secondary structure content of the Trp variant proteins was compared with that of the wild-type protein, using CD The results showed that under native conditions both mutants, PrPW175 and PrPW198, adopt the same a-helical conformation as the wild-type protein, displaying the characteristic minima at 209 and 220 nm and a maximum at 192 nm (Fig 2) Both variants were seen to be fully unfolded at high concentration of urea and to refold to the native conformation upon dilution of the denaturant Equivalent results were observed for the corresponding measurements at pH 7.0 (data not shown) Both Trp mutants displayed reversible folding transitions as observed with the wild-type protein (Fig 2) These results indicate that the point mutations not introduce major changes in the global structure and folding properties of PrP, as also observed for similar Trp constructs of mouse and human PrP [15,25] The wild-type protein has a broad fluorescence spectrum with the maximum intensity at kmax $ 345 nm for the folded protein at either pH 5.5 or 7.0 By contrast, PrPW175 and PrPW198 have kmax at 337 nm (pH 5.5) and 338 nm (pH 7.0), which reflect the more buried position of the single Trp residues in the mutants (Fig 1) Upon unfolding in 7.5 m urea, mutants and wild-type protein exhibit a kmax at around 351 nm This gives a peak shift upon unfolding in excess of 13 nm for the Trp mutants compared with only 5–6 nm for the wild-type PrP (Fig 3) FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS D C Jenkins et al Fig Structure of single tryptophan variants of PrP Far-UV CD spectra of (A) PrPW175 (green lines) and (B) PrPW198 (red lines) under native conditions (solid line), unfolded by 7.5 M urea (shortdashed line), and refolded from 7.5 M urea to 0.6 M urea (longdashed line) compared with the CD spectrum of PrPwt under native conditions (black line) (C) Near-UV CD spectra of PrPW175 (green line) and PrPW198 (red line) compared with that of PrPwt (black line) in their folded oxidized state Spectra were collected at 20 °C on samples containing lM protein in 1-mm cuvette for far-UV CD or 50–60 lM protein in 5-mm cuvette for near-UV CD, all at pH 5.5 (see Experimental procedures) The equilibrium unfolding of PrP in urea was monitored by far-UV CD and tryptophan fluorescence Whereas far-UV CD is sensitive to the secondary structure of a protein, fluorescence is used to monitor changes in the environment of the tryptophan residues as a protein unfolds Using the two mutants, PrPW175 and PrPW198, a view of the unfolding of the tertiary structure of PrP can be built up Comparison of the folding transition curves determined using these complementary spectroscopic techniques gives a more comprehensive view of the folding of PrP To determine transition curves by CD, molar ellipticity at 222 nm at increasing concentrations of denaturant was plotted as a function of urea concentration (Fig 4) Similar curves were obtained by plotting ellipticity at 217 nm, resulting in curves that overlaid with those from the data at 222 nm (data not shown) The unfolding transition data measured by CD were reliably fitted to a two-state transition model using the combined data sets for the three proteins (Fig 4) The thermodynamic parameters determined are shown in Table The midpoints of unfolding indicate that PrP is more stable at pH 7.0 than at pH 5.5, showing values of 5.0 and 4.4 m urea, respectively This is reflected in the free energy of unfolding in the absence of denaturant [DGu(H2O)], which was calculated to be 18 ± kJỈmol)1 at pH 7.0 and 15 ± kJỈmol)1 at pH 5.5 Unfolding transition curves of the two single-tryptophan mutants at pH 5.5 and 7.0 were also determined from fluorescence data as a function of denaturant concentration Both changes in fluorescence kmax (Fig 4) and changes in intensity (Fig 5) were examined A comparative analysis of the total spectral intensity and intensities at single wavelengths across the spectrum was conducted for each mutant The resulting transition curves exhibited a similar behaviour for both mutants and are illustrated for PrPW175 in Fig Intensity-derived transition curves reflect the changes in emission spectra observed with increasing concentrations of urea, where an initial decrease in fluorescence at lower urea concentrations was observed Prion folding A B C followed by an increase in fluorescence at higher urea concentrations (Fig 5) In contrast to the transition curves derived from kmax shifts and CD (Fig 4), which have well-defined baselines in the native and denatured FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1325 Prion folding D C Jenkins et al A A B B Fig Fluorescence of single tryptophan variants of PrP Fluorescence spectra of PrPW175 (green lines) and PrPW198 (red lines) compared with spectra of PrPWT (black lines) in their native (folded) state (solid lines) and unfolded in 7.5 M urea (dashed lines) at pH 5.5 (A) and pH 7.0 (B) Spectra were acquired at 20 °C on samples containing lM protein and using an excitation at 295 nm regions, the intensity curves have slopping native baselines and indistinct baselines at high urea concentrations (Fig 5) As apparent from the unfolding parameters calculated from CD (Table 1), the fluorescence-derived results confirmed that the two mutants have very similar thermodynamic stability, with DGu(H2O) values at pH 5.5 of 13 ± and 12 ± kJỈ mol)1 for PrPW175 and PrPW198, respectively, and at pH 7.0 of 11 ± and 12 ± kJỈmol)1 for PrPW175 and PrPW198, respectively (Table 1) This is consistent with the small size of the folded domain of PrP 1326 Fig Equilibrium unfolding of PrP Unfolding transitions of PrP monitored by fluorescence (circles) and CD (triangles) at pH 5.5 (A) and pH 7.0 (B) Data points were collected from PrPW175 (red), PrPW198 (green) and PrPwt (black) Fluorescence-derived transition curves were measured through shifts in kmax of tryptophan spectra and those derived from CD were calculated from signal intensity changes at 222 nm (see Experimental procedures) Lines represent the best fit, assuming a two-state model, to the fluorescence data set from PrPW175 (red line) and PrPW198 (green line), and to the collective CD data sets from all three proteins (black line) Fluorescence and CD measurements were carried at 20 °C on samples containing lM protein Table shows the thermodynamic parameters derived from the analysis of intensities at 350 nm as at this wavelength the greatest difference in fluorescence intensity was observed between the folded and FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS D C Jenkins et al Prion folding Table Thermodynamic parameters for the equilibrium unfolding of the prion protein Thermodynamic parameters for the equilibrium unfolding of PrP were determined from CD (top two rows of values) and fluorescence transition curves (four lower rows) CD-derived parameters were generated from global fits to the data for all three proteins and fluorescence-derived parameters were from fits to individual data sets for each protein, monitoring changes in fluorescence intensity at 350 nm (Fig 5) compared with values derived from fluorescence kmax shifts (Fig 4) shown in parenthesis DGu(H2O) is the free energy of unfolding extrapolated to zero concentration of urea, the parameter m represents the co-operativity of the transition, and [D]50% is the concentration of urea at the midpoint of unfolding, i.e the concentration of urea required to denature 50% of the protein Intersection points were determined from phase diagram plots (Fig 6) Protein pH DGu(H2O) ⁄ kJỈmol)1 m ⁄ kJỈmol)1ỈM)1 PrPwt, PrPW175, PrPW198 5.5 7.0 5.5 7.0 5.5 7.0 15 18 13 11 12 12 3.4 3.6 3.3 1.9 3.5 2.6 PrPW175 PrPW198 ± ± ± ± ± ± 2 1 (8.3 ± 0.9) (10 ± 1) (7.9 ± 0.3) (9.4 ± 0.8) unfolded states The calculated values from this wavelength are very similar to the average values over all wavelengths and match the values calculated from the total intensity analysis It is notable that the DGu(H2O) values calculated from CD are consistently higher than those originating from fluorescence data, particularly when compared with values derived from kmax shifts (Table 1) These observations are consistent with different folding events being probed by CD and Trp fluorescence Refolding was also measured by fluorescence and the resulting equilibrium transition curves were found to overlay with the unfolding curves at pH 5.5 and 7.0 (data not shown for simplicity) This was also reflected in the thermodynamic parameters calculated from the curve-fitting process, which showed very similar values for the unfolding and refolding of PrP for individual mutants at each pH value These results support the reversibility of the folding transition, also measured by CD (Fig 2) The transitions determined using both CD and fluorescence occur over a broad concentration range of urea from to m (Figs and 5) Although none of the transition curves exhibits an obvious plateaux characteristic of stable partially folded intermediates, the broadness of the transitions indicate that the folding of PrP may not be via a single all-or-none transition, as suggested by the two-state transitions fit to the data Also, comparison of the transition curves determined by following the change in the a-helix signal with those determined by the change in the fluorescence kmax of tryptophan residues reveals some striking differences (Fig 4) The native baseline region of the CD transition curves extends further than the baseline of the fluorescence transition curves, implying that at urea concentrations at which the secondary structure of the ± ± ± ± ± ± 0.3 0.4 0.3 0.1 0.7 0.3 (3.0 (3.2 (2.7 (2.7 ± ± ± ± [D]50% ⁄ M 0.3) 0.3) 0.1) 0.2) 4.4 5.0 4.8 5.5 5.1 5.5 Intersection point ⁄ M N⁄A 3.1 3.7 3.1 3.3 protein remains intact, the tertiary structure, as monitored by the tryptophan probes, begins to break down Hence, at the midpoints of denaturation reported by fluorescence only a small reduction in the fraction of folded protein as determined by CD is observed Conversely, at the midpoint of denaturation reported by CD, the unfolding transition reported by fluorescence is nearly complete This lack of coincidence is an observation commonly made when partially folded intermediate states accumulate on the folding pathway of a protein [27–30] These results have prompted us to further analyse the fluorescence data in more detail in an attempt to reveal this elusive intermediate on the folding pathway of PrP Phase diagram analysis of the unfolding of PrP The low co-operativity of the unfolding transitions shown in this study, the non-coincidence of transition curves determined by two complementary spectroscopic techniques and the observation of equilibrium folding intermediates in other studies [18,31,32] lead to our further investigation into the capture of this elusive intermediate on the folding pathway of PrP The fluorescence spectra used to generate the transition curves were also analysed in terms of ‘phase diagrams’ for the unfolding of the two mutant proteins, PrPW175 and PrPW198, at pH 5.5 and 7.0 (Fig 6) The method of phase diagrams applied to protein folding was first developed by Uversky’s group [29,30] The essence of this analysis, which is based on a generic approach to the analysis of fluorescence data, is to construct a phase diagram by plotting fluorescence intensity at a wavelength k1, I(k1), against the intensity at second wavelength k2, I(k2), for the different experimental conditions inducing the structural FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1327 Prion folding D C Jenkins et al A B C D E F Fig Unfolding transitions of PrP Unfolding transitions curves for PrPW175 monitored by total fluorescence intensity and at various wavelengths, as noted in the individual legend for each panel, across the series of fluorescence spectra at various denaturant concentrations for unfolding at pH 5.5 (left) and pH 7.0 (right) Experiments were performed at 20 °C with protein at lM concentration Solid lines represent the best fit assuming a two-state model change of the protein In this study, these conditions were various concentrations of denaturant, but the analysis can also be applied to any extensive parameter generated by other methods of folding ⁄ unfolding proteins As an extensive parameter, fluorescence intensity for any two-component system will result from the sum of the component intensities associated with each species in proportion to their individual 1328 concentrations at a particular experimental condition A linear correlation for the plot of I(k1) = f(I(k2)) reflects an all-or-non transition between two conformations, whereas nonlinear correlations indicate a sequential structural transition The number of such linear portions on a phase diagram reflects the number of intermediate species involved in the folding pathway of the protein FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS D C Jenkins et al Prion folding A Fig Phase-diagram analysis of unfolding of PrP Phase diagrams plotted using fluorescence intensities measured at 320 and 365 nm at individual denaturant concentrations for the unfolding of PrPW175 (A, B) and PrPW198 (C, D) (see Experimental procedures for the rationale behind the choice of these wavelengths) Experiments were conducted at pH 5.5 (A, C) and pH 7.0 (B, D) Linear regions representing all-or-nontransitions were determined by eye and straight lines fit by linear regression N denotes the native state, I the intermediate state, and U the unfolded state B C D Each phase diagram clearly shows two linear portions with a single intersection, indicating that two all-or-non transitions are involved, and therefore the presence of three distinct conformational species on the folding pathway of PrP, comprising the native state (N), the unfolded state (U) and a partially folded intermediate state (I) Each linear portion of the phase diagram represent the sequential transitions ‘N to I’ and ‘I to U’ The urea concentration at which the intersection occurs is similar between constructs and pH values, with intersection points at the average urea concentrations of 3.1 m at pH 5.5 and 3.5 m at pH 7.0 (Table 1) The higher urea concentration at which the intermediate forms at pH 7.0 reflects the higher thermodynamic stability of the protein at higher pH Examination of the transition curves determined by CD at the urea concentrations at which the intersection points occur, indicates that the intermediate is rich in secondary structure However, the transition curves determined by tryptophan suggest that the tertiary structure is more open than in the native conformation The persistent secondary structure is illustrated in Fig by the CD spectra collected at urea concentrations across a range close to the intersection points determined by the phase diagrams These spectra indicate that the secondary structure content of the intermediate state strongly resembles that of PrP under native conditions This is consistent with the folding intermediate of PrP being a ‘molten-globule’ state, which is characterized by a native-like expanded conformation possessing native levels of secondary structure and disrupted tertiary structure [33,34] Discussion According to the prion hypothesis, a key molecular event in the pathogenesis of prion diseases is the conversion of the normal cellular form of the prion protein, PrPC, to the disease-associated PrPSc conformation [12,22] Although this event has yet to be characterized, it is possible that PrPSc is formed by recruitment of a partially folded intermediate on the folding pathway of PrPC [35] The presence of equilibrium folding intermediates in the folding of PrP has been a controversial issue [16,17,36], but NMR [18–20] and fluorescence experiments [21] are now providing evidence that an intermediate may indeed be present Folding kinetic studies also support this view [13–15] To further investigate the folding of PrP and clarify the existence of a folding intermediate we produced two mutants of the truncated form of the prion protein, each with a single tryptophan residue (Fig 1) Comparison of the unfolding transition curves determined by fluorescence for the two mutants revealed that there is little difference between their unfolding, as would be expected for a protein possessing only a FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1329 Prion folding D C Jenkins et al A B Fig Structure of the folding intermediate of PrP CD spectra of PrP at (A) pH 5.5 and (B) pH 7.0 in the presence of 3.0 M (red line), 3.3 M (green line) and 3.9 M (blue line) urea compared with the spectra of PrP under native conditions (solid black line) and unfolded in 7.5 M urea (dashed black line) Spectra were collected at 20 °C on samples containing lM protein Fig Folding of PrP in health and disease The folding mechanism of PrP incorporating the normal folding pathway (blue) predominant in healthy conditions, and the off-pathway aggregation of PrP (brown) occurring in disease U represents the unfolded state; I symbolizes a normal folding intermediate state, which can feed into the off-pathway aggregation either directly or via the unfolded state U; N, is the native folded state; PrPn indicates an oligomeric state; and PrPSc denotes the highly aggregated state of PrP, which comprises amyloid plaques, ordered fibrillar structures and amorphous aggregates 1330 single folded domain A small destabilization of PrP at pH 5.5 relative to pH 7.0 was detected through the thermodynamic analysis (Table 1) and is consistent with previous reports [16,36] Examination of the folding transitions determined by the change in the environment of tryptophan residues measured by fluorescence, and the transitions monitored by the change in the a-helical content reported by CD, reveals striking differences At $ m urea, when 50% of the protein molecules are seen to be unfolded in terms of their tertiary structure (as measured by tryptophan fluorescence), close to 100% have their full a-helical content (Fig 4) This indicates that at least some of the tertiary structure forms simultaneously with the secondary structure This is consistent with a nucleation–condensation type folding model, in which the secondary and tertiary structure form simultaneously from a diffuse nucleus [37], a mechanism not uncommon among globular proteins which show the formation of intermediates on their folding pathways [38,39] In addition, the non-coincidence of transition curves determined by different spectroscopic techniques, as seen in Fig 4, is indicative of the existence of stable, partially folded intermediates on the folding pathway of a protein [27–30] The presence of an intermediate on the folding pathway of PrP was further disclosed through phasediagram analyses of the folding transition data Each of the resulting phase-diagram plots clearly showed a single point of intersection (Fig 6), indicating that the folding of PrP at both pH 5.5 and 7.0 proceeds via a single folding intermediate In this way we show that the folding of PrP follows a three-state mechanism: U M I M N, where U is the protein in the unfolded state, I is the intermediate, and N the native state The examination of the CD spectra of PrP at the intermediate concentrations of urea disclosed through the phase diagram plots (Fig 6) revealed that the intermediate state I has a native-like secondary structure content (Fig 7), akin to a molten-globule state These CD spectra also revealed that the a-helical content of I is consistently higher at pH than at pH 5.5 The thermodynamic parameters for the equilibrium unfolding of PrP also showed D50% and DGu(H2O) values consistently higher at pH 7.0 than at pH 5.5 for all three proteins (Table 1) The non-coincidence of CD-derived unfolding transitions with those determined from fluorescence data (Fig 4) is a classical signature of the accumulation of an intermediate state with molten-globule properties The fluorescence curves reflect the breakdown of tertiary structure whilst no changes occur in the secondary structure, as reported by the CD curve Therefore, FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS D C Jenkins et al the fluorescence-derived curves have contributions from both N to I and I to U transitions, whereas the CD curves are dominated by the unfolding of I to U Comparison of the free energies of unfolding (DGu) derived from both methods (Table 1) reveals that most of DGu for the unfolding of PrP is associated with the unfolding of I to U, and conversely that I is easily accessible (low energy barrier) from the native state Interestingly, in vitro fibrillization conditions of prion protein generally employ partially denaturing conditions [40–43], including urea concentrations at which we identify the accumulation of a moltenglobule species This would suggest that the helical intermediate (I) identified here on the folding pathway of PrP could also serve as a precursor for the off-pathway aggregation leading to the formation of PrPSc, which not only refers to fibrillar material but also to less ordered and amorphous aggregated protein states The proposal of a molten-globule state, nearly native-like in secondary structure content, serving as a precursor in the formation of PrPSc, contrasts with other suggestions implying that extensive unfolding of PrPC is required for the generation of PrPSc [44,45], but is in line with other studies indicating that partial denaturation of native PrP is more conducive to the formation of fibrillar material than are fully unfolded or native protein [18,46] Therefore, a plausible scheme combining the normal folding pathway of PrP with the possible off-pathways to aggregation is presented in Fig In this scheme, I serves as a precursor to the formation of PrPSc either via an oligomeric state (PrPn) or via the unfolded state (U) In a recent NMR study, a native-like helical monomeric state of PrP with molten-globule characteristics was shown to convert to a b-sheet oligomer [47], which in Fig is represented by PrPn Recruitment of partially folded states into off-pathway aggregation has also been seen in the fibrillization pathway of other proteins [48,49], but whether the molten-globule-like state (I) identified here is on- or off-pathway to the formation of PrPSc remains to be unequivocally demonstrated In vivo conversion of PrPC into PrPSc is perceived to occur at the membrane surface [50,51] or via the acidic conditions in the endosomal pathway [52,53] Partial unfolding of native proteins, resulting in molten-globule states, can be driven by low pH [54,55] and upon binding to lipid membranes [56,57] In previous studies we have shown that the interaction of PrP with lipid membranes can partially unfold the compact native structure of PrP leading to the aggregation of PrP [58,59] Our findings highlight the existence of an intermediate state (I) closely related to the native state (N) of PrP The I state could be accessed from the N state Prion folding through changes in the cellular environment of PrP, such as the low endocytic pH or the interaction with other cellular components of the plasma membrane A precursor of PrPSc that is a common intermediate in the normal folding of PrPC and with structural properties so closely related to the native state would also explain the inherent difficulties in the detection of early precursor states associated with the development of the disease Experimental procedures Mutagenesis and protein purification A plasmid encoding the SHaPrP with the intrinsic tryptophan residues at positions 99 and 145 mutated to phenylalanine (pTrcSHaPrPMet23–231 F99, F145) was prepared On the background of this plasmid two further constructs were made, one possessing a tryptophan residue at position 175, and the other possessing a tryptophan residue at position 198 These were used as PCR templates for insertion of the truncated (SHaPrP(90–231)) genes into the pIngPrP plasmid, as described previously [60] The resulting plasmids were termed pIngPrPTrp175 and pIngPrPTrp198 These plasmids were used to transform E coli 27C7 cells The single tryptophan variants and wild-type protein were expressed and purified as described previously [58,60], with the yield of oxidised protein maximised by the inclusion of an active oxidation step, similar to that used for the truncated construct of the human prion protein [32] Briefly, following the size-exclusion chromatography step, protein was immediately purified using RP-HPLC Protein was freeze-dried and dissolved to a concentration of $ 0.2 mgỈmL)1 in an oxidizing buffer consisting of m guanidine hydrochloride solution, 50 mm Tris ⁄ HCl (pH 8), and 30 lm copper sulfate This was agitated at room temperature and the progress of the oxidation reaction followed by RP-HPLC Once the oxidation reaction was seen to be complete (typically within 1–2 h), PrP was purified by RP-HPLC to remove the oxidizing buffer Protein was refolded to the a-helical conformation by dialysis against mm MES buffer, pH 5.5 The purity of the final product was determined by SDS ⁄ PAGE and electrospray ionization MS PrP concentration was determined spectrophotometrically, using e280 = 24 420 m)1Ỉcm)1 for the wild-type protein, and e280 = 18 730 m)1Ỉcm)1 for the single tryptophan variants [61] The abbreviation PrP used throughout the text refers to the Syrian hamster protein truncated domain 90–231 Three constructs are employed in this study: wildtype protein (PrPwt) and two single tryptophan mutants with the intrinsic tryptophan residues at positions 99 and 145 mutated to phenylalanine and the phenylalanine residue either at position 175 or 198 mutated to tryptophan (PrPW175 or PrPW198, respectively) FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1331 Prion folding D C Jenkins et al CD and denaturant unfolding Far-UV CD spectra were collected on a JASCO J-715 spectropolarimeter using 1-mm pathlength quartz cuvette on samples containing 5–7 lm protein in mm MES buffer pH 5.5 Near-UV CD spectra employed high protein concentration between 50 and 60 lm in 20 mm sodium acetate buffer, pH 5.5, and a 5-mm pathlength quartz cuvette Spectra were collected in continuous scanning mode at a scanning rate of 100 nmỈmin)1, a time constant of s, a bandwidth of nm and a resolution of 0.5 nm Both farand near-UV spectra were measured at 20 °C and final spectra are an average of 16 scans and have the appropriate buffer background subtracted Individual samples of protein at desired urea concentrations were prepared using a high concentration stock of folded protein in buffer (20 mm sodium acetate, pH 5.5 or 20 mm MOPS at pH 7.0), diluted into a buffer containing urea at the desired concentration For each CD spectrum obtained at an individual denaturant concentration, the molar ellipticity at 222 nm ([h]222) was determined These values were normalized to a fraction of folded protein (fN) using fN = (yD ) y) ⁄ (yD ) yN) [62], where yD is the [h]222 of the CD spectrum measured for protein in the denatured state, y is [h]222 measured at a particular denaturant concentration, and yN is the [h]222 of protein in the native state The fN value was plotted as a function of denaturant concentration to give unfolding and refolding transition curves Data were analysed according to a two-state model (N M U, where N is protein in the native state and U is protein in the unfolded state) The free energy of folding in the absence of denaturant (DG(H2O)) was calculated by assuming that the observed free energy of folding (DGobs) is linearly dependent on urea concentration, following the relationship DGobs = DG(H2O) ) m[urea] where m is a constant reflecting the gradient of a plot of DG as a function of denaturant concentration [63] For each concentration of urea the equilibrium constant (K) of the native and unfolded states was calculated by K ẳ eDGobs mẵureaị=RTị , where R is the universal gas constant and T is the absolute temperature (293 K) Data were fit to two-state transition curves by non-linear least-squares regression using sigmaplot (Systat Software, Richmond, CA, USA) Fluorescence and denaturant unfolding Fluorescence emission spectra were recorded on a Photon Technology International spectrofluorimeter using an excitation wavelength of 295 nm (4 nm bandwidth) and collected from 305 to 405 nm (2 nm bandwidth) Typically, four scans were averaged per spectrum Corresponding appropriate backgrounds of buffer alone or buffer and denaturant were subtracted from final spectra In a typical unfolding experiment, two stock solutions of PrP at identi- 1332 cal protein concentrations (5 lm) were prepared: one in buffer only (native protein) and one in buffer containing a high concentration of urea (unfolded protein) Stock urea solutions were made fresh at a concentration of 10 m, and treated for 14–16 h with Amberlite deionising resin (Merck, Darmstadt, Germany) to minimize chemical modification of protein The buffers were 20 mm sodium acetate for pH 5.5 or 20 mm MOPS for pH 7.0 For an unfolding curve the sample of unfolded protein was titrated (in increments of $ 0.2 m urea) to a sample of native protein in a 1-cm pathlength cuvette Fluorescence spectra were recorded immediately after the two solutions were mixed A longer incubation was not necessary as the system reaches equilibrium in < s, because of the very fast unfolding ⁄ refolding of the prion protein [14,25] Unfolding curves were plotted using the total fluorescence intensity (integrated area under fluorescence spectrum) or intensities at single wavelengths spanning the fluorescence spectra at each denaturant concentration For transition curves based on fluorescence peak shifts the kmax were determined and normalised to fraction of folded protein (fN) as described in the previous section, but using fluorescence kmax data Transition curves were analyzed according to a two-state model, as described in the previous section Phase-diagram analysis of fluorescence data A novel, qualitative approach to the analysis of folding data, complementary to the conventional presentation and analysis of unfolding and refolding transition curves, is to plot ‘phase diagrams’ This technique has been described in detail elsewhere [29,30], but briefly, phase diagrams are drawn by plotting the measured fluorescence intensity at two wavelengths against one another at denaturant concentrations ranging across the denaturation curve The resulting diagrams show one or more linear portions Each linear portion describes an individual all-or-non transition, with partially folded intermediate species stabilized at denaturant concentrations at which linear portions of the plots intersect It has been observed that phase diagrams are more informative if the two wavelengths are on different slopes of the spectrum, hence the wavelengths selected in this study are 320 and 365 nm Linear portions and regions of intersection were determined by eye, and straight lines fit by linear regression analysis Acknowledgements We thank Andrew Gill (IAH, Compton) for mass spectrometry of mutant prion proteins, Matthew Hicks for technical advice This project was funded by the Wellcome Trust (053914 ⁄ Z ⁄ 98 ⁄ Z), the Engineering and Physical Sciences Research Council (DCJ studentship), FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS D C Jenkins et al the Biotechnology and Biological Science Research Council (BB ⁄ D524516 ⁄ 1) and the Royal Society References Collinge J (2005) Molecular neurology of prion disease J Neurol Neurosurg Psychiatry 76, 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Discussion According to the prion hypothesis, a key molecular event in the pathogenesis of prion diseases is the conversion of the normal cellular form of the prion protein, PrPC, to the disease-associated... indicative of the existence of stable, partially folded intermediates on the folding pathway of a protein [27–30] The presence of an intermediate on the folding pathway of PrP was further disclosed... a mechanism not uncommon among globular proteins which show the formation of intermediates on their folding pathways [38,39] In addition, the non-coincidence of transition curves determined by