Tài liệu Báo cáo khoa học: The elusive intermediate on the folding pathway of the prion protein pptx

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
Prion diseases, which include Creutzfeldt–Jakob dis-
ease in humans, bovine spongiform encephalopathy in
cattle and scrapie in sheep, are associated with conver-
sion of the normal cellular form of the prion protein
(PrP
C
) to an altered pathological form, generally desig-
nated as the scrapie isoform (PrP
Sc
). 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 associ-
ated 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
PrP
C
and PrP
Sc
contrast greatly. Whereas PrP
C
is a
globular protein, composed primarily of an unstruc-
tured N-terminal region and a C-terminal domain
comprising three a-helices and two short b-strands

(Fig. 1) [5–8], PrP
Sc
has a much higher proportion of
b-sheet structure [9]. Physicochemical studies have
shown that PrP
C
is monomeric, soluble in aqueous
buffer and sensitive to protease digestion, while, in
comparison, PrP
Sc
has a high propensity to aggregate,
is water- and detergent-insoluble, and partially resis-
tant to proteinases [2,10,11].
The details of prion conversion are not fully under-
stood, but it is generally accepted that the key mole-
cular event in sporadic and familial cases of prion
diseases involves a conformational transition of the
prion protein from its cellular state to the altered dis-
ease-associated form [12]. Thus, an understanding of
the folding and refolding mechanisms of the prion pro-
tein 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 uncharacter-
ized conditions, be recruited to form PrP
Sc
. Results

from equilibrium folding studies have been ambiguous
on defining the presence of an intermediate in the fold-
ing 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
Keywords
denaturant unfolding; molten globule; phase-
diagram; 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:
(Received 14 August 2007, revised 20
December 2007, accepted 15 January 2008)
doi:10.1111/j.1742-4658.2008.06293.x
A key molecular event in prion diseases is the conversion of the cellular
conformation of the prion protein (PrP
C
) to an altered disease-associated
form, generally denoted as scrapie isoform (PrP
Sc
). The molecular details
of this conformational transition are not fully understood, but it has been
suggested that an intermediate on the folding pathway of PrP
C
may be
recruited to form PrP

Sc
. In order to investigate the folding pathway of PrP
we designed and expressed two mutants, each possessing a single strategi-
cally 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.
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
[17]. Recent evidence from NMR [18–20] and fluores-
cence 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 PrP
Sc
[4,22]. Each
mutant possesses a single tryptophan residue, strategi-
cally located to produce a significant change in fluores-
cence 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 1 shows the location of the tryptophan residues
in the single Trp mutants of PrP employed in this
study.
Fluorescence and CD were used to establish the fold-
ing 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 PrP
C
and propose that this intermediate may
serve as a precursor for prion conversion.
Results
Denaturant unfolding of PrP
In its normal cellular form, PrP adopts a conformation
rich in a-helices with a small amount of b-sheet struc-
ture 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 wild-
type 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, PrP
W175
and
PrP
W198
, 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 do not intro-
duce 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 spec-
trum with the maximum intensity at k
max
$ 345 nm
for the folded protein at either pH 5.5 or 7.0. By
contrast, PrP
W175

and PrP
W198
have k
max
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 k
max
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).
Fig. 1. 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 Bio-
computing, Visualization and Informatics at the University of Califor-
nia, San Francisco (supported by NIH P41 RR-01081) [64].
Prion folding D. C. Jenkins et al.
1324 FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS
The equilibrium unfolding of PrP in urea was moni-

tored 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, PrP
W175
and PrP
W198
, a view of the unfolding of the tertiary
structure of PrP can be built up. Comparison of the
folding transition curves determined using these com-
plementary spectroscopic techniques gives a more com-
prehensive view of the folding of PrP.
To determine transition curves by CD, molar elliptic-
ity 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 unfold-
ing 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 1. The mid-
points 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 [DG
u
(H
2

O)],
which was calculated to be 18 ± 2 kJÆmol
)1
at pH 7.0
and 15 ± 2 kJÆmol
)1
at pH 5.5.
Unfolding transition curves of the two single-trypto-
phan mutants at pH 5.5 and 7.0 were also determined
from fluorescence data as a function of denaturant
concentration. Both changes in fluorescence k
max
(Fig. 4) and changes in intensity (Fig. 5) were exam-
ined. 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 behav-
iour for both mutants and are illustrated for PrP
W175
in Fig. 5. Intensity-derived transition curves reflect the
changes in emission spectra observed with increasing
concentrations of urea, where an initial decrease in flu-
orescence at lower urea concentrations was observed
followed by an increase in fluorescence at higher urea
concentrations (Fig. 5). In contrast to the transition
curves derived from k
max
shifts and CD (Fig. 4), which
have well-defined baselines in the native and denatured
A

B
C
Fig. 2. Structure of single tryptophan variants of PrP. Far-UV CD
spectra of (A) PrP
W175
(green lines) and (B) PrP
W198
(red lines)
under native conditions (solid line), unfolded by 7.5
M urea (short-
dashed line), and refolded from 7.5
M urea to 0.6 M urea (long-
dashed line) compared with the CD spectrum of PrP
wt
under native
conditions (black line). (C) Near-UV CD spectra of PrP
W175
(green
line) and PrP
W198
(red line) compared with that of PrP
wt
(black line)
in their folded oxidized state. Spectra were collected at 20 °Con
samples containing 5 l
M protein in 1-mm cuvette for far-UV CD or
50–60 l
M protein in 5-mm cuvette for near-UV CD, all at pH 5.5
(see Experimental procedures).

D. C. Jenkins et al. Prion folding
FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1325
regions, the intensity curves have slopping native
baselines and indistinct baselines at high urea con-
centrations (Fig. 5). As apparent from the unfolding
parameters calculated from CD (Table 1), the fluores-
cence-derived results confirmed that the two mutants
have very similar thermodynamic stability, with
DG
u
(H
2
O) values at pH 5.5 of 13 ± 1 and 12 ± 2 kJÆ
mol
)1
for PrP
W175
and PrP
W198
, respectively, and at
pH 7.0 of 11 ± 1 and 12 ± 1 kJÆmol
)1
for PrP
W175
and PrP
W198
, respectively (Table 1). This is consistent
with the small size of the folded domain of PrP.
Table 1 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
A

B
Fig. 4. 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 PrP
W175
(red),
PrP
W198
(green) and PrP
wt
(black). Fluorescence-derived transition
curves were measured through shifts in k
max
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 PrP
W175
(red line) and PrP
W198
(green line), and to the col-
lective CD data sets from all three proteins (black line). Fluores-
cence and CD measurements were carried at 20 °C on samples
containing 5 l
M protein.

A

B
Fig. 3. Fluorescence of single tryptophan variants of PrP. Fluores-
cence spectra of PrP
W175
(green lines) and PrP
W198
(red lines) com-
pared with spectra of PrP
WT
(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 sam-
ples containing 5 l
M protein and using an excitation at 295 nm.
Prion folding D. C. Jenkins et al.
1326 FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS
unfolded states. The calculated values from this wave-
length 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 DG
u
(H
2
O)
values calculated from CD are consistently higher than
those originating from fluorescence data, particularly
when compared with values derived from k

max
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 fluo-
rescence occur over a broad concentration range of
urea from 3 to 5 m (Figs 4 and 5) Although none of
the transition curves exhibits an obvious plateaux char-
acteristic 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 k
max
of
tryptophan residues reveals some striking differences
(Fig. 4). The native baseline region of the CD transi-

tion curves extends further than the baseline of the
fluorescence transition curves, implying that at urea
concentrations at which the secondary structure of the
protein remains intact, the tertiary structure, as moni-
tored 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. Con-
versely, 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 spectro-
scopic 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 transi-
tion curves were also analysed in terms of ‘phase dia-
grams’ for the unfolding of the two mutant proteins,
PrP
W175

and PrP
W198
, 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 k
1
, I(k
1
), against the inten-
sity at second wavelength k
2
, I(k
2
), for the different
experimental conditions inducing the structural
Table 1. 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 indi-
vidual data sets for each protein, monitoring changes in fluorescence intensity at 350 nm (Fig. 5) compared with values derived from fluo-
rescence k
max
shifts (Fig. 4) shown in parenthesis. DG
u
(H
2
O) 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 DG
u
(H
2
O) ⁄ kJÆmol
)1
m ⁄ kJÆmol
)1
ÆM
)1
[D]
50%
⁄ M Intersection point ⁄ M
PrP
wt
, PrP
W175
, PrP
W198
5.5 15 ± 2 3.4 ± 0.3 4.4 N ⁄ A
7.0 18 ± 2 3.6 ± 0.4 5.0
PrP
W175
5.5 13 ± 1 (8.3 ± 0.9) 3.3 ± 0.3 (3.0 ± 0.3) 4.8 3.1
7.0 11 ± 1 (10 ± 1) 1.9 ± 0.1 (3.2 ± 0.3) 5.5 3.7

PrP
W198
5.5 12 ± 2 (7.9 ± 0.3) 3.5 ± 0.7 (2.7 ± 0.1) 5.1 3.1
7.0 12 ± 1 (9.4 ± 0.8) 2.6 ± 0.3 (2.7 ± 0.2) 5.5 3.3
D. C. Jenkins et al. Prion folding
FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1327
change of the protein. In this study, these conditions
were various concentrations of denaturant, but the
analysis can also be applied to any extensive parame-
ter 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
concentrations at a particular experimental condition.
A linear correlation for the plot of I(k
1
)=f(I(k
2
))
reflects an all-or-non transition between two confor-
mations, whereas nonlinear correlations indicate a
sequential structural transition. The number of such
linear portions on a phase diagram reflects the num-
ber of intermediate species involved in the folding
pathway of the protein.
A B
C D
E F
Fig. 5. Unfolding transitions of PrP. Unfolding transitions curves for PrP

W175
monitored by total fluorescence intensity and at various wave-
lengths, 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 5 l
M concentration. Solid lines represent
the best fit assuming a two-state model.
Prion folding D. C. Jenkins et al.
1328 FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 inter-
mediate state (I). Each linear portion of the phase dia-
gram represent the sequential transitions ‘N to I’ and
‘I to U’. The urea concentration at which the inter-
section 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 ther-
modynamic stability of the protein at higher pH.
Examination of the transition curves determined by
CD at the urea concentrations at which the intersec-
tion 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 conforma-
tion. The persistent secondary structure is illustrated in

Fig. 7 by the CD spectra collected at urea concentra-
tions across a range close to the intersection points
determined by the phase diagrams. These spectra indi-
cate that the secondary structure content of the inter-
mediate 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, PrP
C
, to the disease-associated PrP
Sc
conformation [12,22]. Although this event has yet to
be characterized, it is possible that PrP
Sc
is formed by
recruitment of a partially folded intermediate on the
folding pathway of PrP
C
[35]. The presence of equilib-
rium 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 pro-
tein, each with a single tryptophan residue (Fig. 1).
Comparison of the unfolding transition curves deter-
mined 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
B
D
A
C
Fig. 6. Phase-diagram analysis of unfolding
of PrP. Phase diagrams plotted using fluo-
rescence intensities measured at 320 and
365 nm at individual denaturant concentra-
tions for the unfolding of PrP
W175
(A, B) and
PrP
W198
(C, D) (see Experimental proce-
dures for the rationale behind the choice of
these wavelengths). Experiments were con-
ducted at pH 5.5 (A, C) and pH 7.0 (B, D).
Linear regions representing all-or-nontransi-
tions 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.
D. C. Jenkins et al. Prion folding
FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1329
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 resi-
dues measured by fluorescence, and the transitions
monitored by the change in the a-helical content
reported by CD, reveals striking differences. At $ 3 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-coinci-
dence 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 path-
way of PrP was further disclosed through phase-

diagram 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 interme-
diate concentrations of urea disclosed through the
phase diagram plots (Fig. 6) revealed that the interme-
diate state I has a native-like secondary structure con-
tent (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 7 than at pH 5.5. The ther-
modynamic parameters for the equilibrium unfolding
of PrP also showed D
50%
and DG
u
(H
2
O) values consis-
tently higher at pH 7.0 than at pH 5.5 for all three
proteins (Table 1).
The non-coincidence of CD-derived unfolding transi-
tions 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 ter-

tiary structure whilst no changes occur in the second-
ary structure, as reported by the CD curve. Therefore,
A
B
Fig. 7. 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 5 l
M protein.
Fig. 8. Folding of PrP in health and disease. The folding mecha-
nism of PrP incorporating the normal folding pathway (blue) pre-
dominant 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; PrP
n
indicates an oligomeric state;
and PrP
Sc
denotes the highly aggregated state of PrP, which com-
prises amyloid plaques, ordered fibrillar structures and amorphous
aggregates.
Prion folding D. C. Jenkins et al.
1330 FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS

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 (DG
u
)
derived from both methods (Table 1) reveals that most
of DG
u
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 molten-
globule 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-path-
way aggregation leading to the formation of PrP
Sc
,
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 PrP
Sc
, contrasts with
other suggestions implying that extensive unfolding of

PrP
C
is required for the generation of PrP
Sc
[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. 8. In this scheme, I serves as a precursor to the
formation of PrP
Sc
either via an oligomeric state
(PrP
n
) 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 con-
vert to a b-sheet oligomer [47], which in Fig. 8 is repre-
sented by PrP
n
. 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 PrP
Sc
remains to be unequivocally demonstrated.

In vivo conversion of PrP
C
into PrP
Sc
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-glob-
ule 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 inter-
mediate state (I) closely related to the native state (N)
of PrP. The I state could be accessed from the N state
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 PrP
Sc
that is a common intermediate in
the normal folding of PrP
C
and with structural proper-
ties 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 trypto-
phan residues at positions 99 and 145 mutated to phenyl-
alanine (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 posi-
tion 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 plas-
mids 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 trun-
cated 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 mgÆmL
)1
in an oxidizing buffer consisting of 6 m
guanidine hydrochloride solution, 50 mm Tris ⁄ HCl (pH 8),
and 30 lm copper sulfate. This was agitated at room tem-
perature and the progress of the oxidation reaction fol-
lowed 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

2mm MES buffer, pH 5.5. The purity of the final product
was determined by SDS ⁄ PAGE and electrospray ionization
MS. PrP concentration was determined spectrophotometri-
cally, using e
280
=24420m
)1
Æcm
)1
for the wild-type pro-
tein, and e
280
=18730m
)1
Æcm
)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: wild-
type protein (PrP
wt
) 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
(PrP
W175
or PrP
W198

, respectively).
D. C. Jenkins et al. Prion folding
FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS 1331
CD and denaturant unfolding
Far-UV CD spectra were collected on a JASCO J-715 spe-
ctropolarimeter using 1-mm pathlength quartz cuvette on
samples containing 5–7 lm protein in 2 mm MES buffer
pH 5.5. Near-UV CD spectra employed high protein con-
centration 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 nmÆmin
)1
, a time constant of 1 s, a
bandwidth of 2 nm and a resolution of 0.5 nm. Both far-
and 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 pro-
tein 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 concen-
tration.
For each CD spectrum obtained at an individual dena-
turant concentration, the molar ellipticity at 222 nm ([h]
222
)
was determined. These values were normalized to a fraction
of folded protein (f
N

) using f
N
=(y
D
) y) ⁄ (y
D
) y
N
) [62],
where y
D
is the [h]
222
of the CD spectrum measured for
protein in the denatured state, y is [h]
222
measured at a par-
ticular denaturant concentration, and y
N
is the [h]
222
of
protein in the native state. The f
N
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(H
2
O)) was calculated by assuming that the observed
free energy of folding (DG
obs
) is linearly dependent on urea
concentration, following the relationship DG
obs
= DG(H
2
O)
) 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 ¼ e
ÀððDG
obs
Àm½ureaÞ=RTÞ
, where R is the universal gas con-
stant 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 exci-
tation wavelength of 295 nm (4 nm bandwidth) and col-
lected 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-
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 path-
length cuvette. Fluorescence spectra were recorded immedi-
ately after the two solutions were mixed. A longer
incubation was not necessary as the system reaches equilib-
rium in < 1 s, because of the very fast unfolding ⁄ refolding
of the prion protein [14,25].
Unfolding curves were plotted using the total fluores-
cence intensity (integrated area under fluorescence spec-
trum) or intensities at single wavelengths spanning the
fluorescence spectra at each denaturant concentration. For
transition curves based on fluorescence peak shifts the k
max
were determined and normalised to fraction of folded
protein (f
N
) as described in the previous section, but using
fluorescence k
max

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 concen-
trations ranging across the denaturation curve. The result-
ing 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 inter-
sect. 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),
Prion folding D. C. Jenkins et al.
1332 FEBS Journal 275 (2008) 1323–1335 ª 2008 The Authors Journal compilation ª 2008 FEBS

the Biotechnology and Biological Science Research
Council (BB ⁄ D524516 ⁄ 1) and the Royal Society.
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