ACCELERATED PUBLICATION
Folding of Prion Protein to Its Native
-Helical Conformation
Is under Kinetic Control*
Ilia V.
Baskakov
§,
Giuseppe
Legname
§,
Stanley B.
Prusiner
§¶, and
Fred E.
Cohen
¶
**
From the
Institute for Neurodegenerative Diseases
and Departments of § Neurology, ¶ Biochemistry and
Biophysics, and
Cellular and Molecular Pharmacology,
Pharmaceutical Chemistry, and Medicine, University of California,
San Francisco, California 94143
Received for publication, April 10, 2001
 |
ABSTRACT |
The recombinant mouse prion protein
(MoPrP) can be folded either to a monomeric
-helical or oligomeric
-sheet-rich isoform. By using circular dichroism spectroscopy and
size-exclusion chromatography, we show that the
-rich isoform of
MoPrP is thermodynamically more stable than the native
-helical
isoform. The conformational transition from the
-helical to
-rich
isoform is separated by a large energetic barrier that is associated
with unfolding and with a higher order kinetic process related to
oligomerization. Under partially denaturing acidic conditions, MoPrP
avoids the kinetic trap posed by the
-helical isoform and folds
directly to the thermodynamically more stable
-rich isoform. Our
data demonstrate that the folding of the prion protein to its native
-helical monomeric conformation is under kinetic control.
 |
INTRODUCTION |
Although protein folding is commonly thought to be controlled by
thermodynamic preferences, it has been understood by many, including
Anfinsen and others (1,2), that kinetic issues can alter the folding
landscape. Whereas most small globular proteins will refold
spontaneously in vitro to a native conformation, in
vivo folding often exploits auxiliary molecules and defined
subcellular compartments to avoid the deposit of misfolded forms (3).
Increasingly, a role for protein misfolding in a variety of
neurodegenerative diseases has emerged. A common thread joining
prion-based diseases and Alzheimer's disease, and possibly
Parkinson's disease and frontotemporal dementia, is the conversion of
a normal, cellular, monomeric isoform of a protein into a
-sheet-rich, polymeric form (4-6). When the deposited polymeric
form is sufficiently ordered to bind Congo red and exhibit
birefringence to polarized light, the pathologic term amyloid is used
to cluster these and other maladies (7).
Recent studies by Dobson and others (8-12) have demonstrated that a
broad variety of proteins that rapidly fold into monomeric or
oligomeric cellular forms under native-like conditions can also be
refolded into
-rich, amyloid forms under conditions that destabilize
the native state. So far, these proteins have not been associated with
human deposition diseases. This finding has led to the suggestion that
the ability to adopt alternative
-rich folds capable of forming
amyloid is not a unique property of specific proteins associated with
conformational diseases but reflects a general property of polypeptide
chains (13). The interplay between protein concentration and the
conformational preferences of the monomeric chain in driving the
transition to a
-rich multimeric isoform remains to be more fully explored.
Glockshuber and colleagues (14) have shown that a fragment of the mouse
prion protein folds very rapidly into the
-helix-rich conformation
with a half-life of 170 µs as measured at 4 °C. Here, we report
that a
-sheet-rich conformation of the mouse prion protein
(MoPrP)1 is thermodynamically
more stable than its native
-helix-rich conformation. The
conformational transition from the
-helical to a
-sheet-rich
isoform is controlled by a large energetic barrier that is associated
with partial unfolding and oligomerization of an intermediate state.
Under partially denaturing conditions, it is possible to avoid the
kinetic trap that leads to the normal cellular isoform,
PrPC, and fold the prion protein directly to a
thermodynamically more stable, non-native
-isoform. Our data
demonstrate that folding the prion protein to its native
-conformation is under kinetic, not thermodynamic, control.
 |
EXPERIMENTAL PROCEDURES |
Protein Preparation--
The expression and purification of
recombinant MoPrP 89-231 was performed as described by Mehlhorn
et al. (15).
Circular Dichroism--
CD spectra were recorded with a J-720 CD
spectrometer (Jasco, Easton, MD) scanning at 20 nm/min, with a
bandwidth of 1 nm and data spacing of 0.5 nm using a 0.1-cm cuvette.
Each spectrum represents the accumulation of three individual scans
after subtracting the background spectra. To monitor the refolding
curves, MoPrP was diluted from 10 M to various
concentrations of urea in 20 mM sodium acetate in the
absence or in the presence of 0.2 M NaCl, pH 3.6, and then
incubated at room temperature for different periods of time. No change
in pH value was detected during the time course of incubation. To
monitor the kinetic trace of the conformational transition,
-MoPrP
was rapidly mixed with 10 M urea in a 1:1 volume ratio,
whereas to monitor the kinetics of refolding to the
-MoPrP, MoPrP
unfolded in 10 M urea was mixed with buffer, again at a 1:1
volume ratio. All kinetic experiments were carried out in 20 mM sodium acetate and 0.2 M NaCl, pH 3.6.
Analysis of the Kinetic Data--
The rate constant and apparent
rate order of refolding were calculated from Equation 1,
|
(Eq. 1)
|
in which C0 is concentration of the
monomer MoPrP at zero time, C is concentration of monomer
MoPrP at time t, and n is apparent order of the
process.
E
was calculated from the
Arrhenius relation (1) with kobs measured experimentally and k0 determined from the
equation for diffusion-controlled reaction, assuming that the reaction
follows fifth-order kinetics.
Size-exclusion Chromatography--
All separations were
performed at 23 °C with a flow rate of 1 ml/min using TSK-3000 high
pressure liquid chromatography gel filtration column (300 mm × 7.80 mm) equilibrated in 20 mM sodium acetate, 0.2 M NaCl, pH 3.6, and the corresponding concentration of urea.
Thioflavin T Assay--
To follow the kinetics of amyloid
formation, 0.64 mg/ml
-MoPrP was incubated in 20 mM
sodium acetate and 0.2 M NaCl, pH 5.5, constantly shaken at
36 °C. In the time course of incubation, aliquots of MoPrP were
diluted 8 times by phosphate-buffered saline, pH 7.0, and the
fluorescence was measured using a LS50B fluorimeter (PerkinElmer Life
Sciences) at 482 nm (excitation at 450 nm, excitation slit is 5 nm,
emission slit is 10 nm, 0.4-cm rectangular cuvettes) with 5 µM thioflavin T.
Congo Red Binding--
Congo red (Sigma) was dissolved in 5 mM potassium phosphate, 150 mM NaCl, filtered 5 times with a 0.22-mm filter (Millipore, Bedford, MA), and adjusted to
0.2 mM. The difference spectra were obtained by subtracting
the Congo red spectra in the absence of MoPrP from the Congo red
spectra in the presence of 1.5 µM MoPrP amyloid,
corrected for MoPrP scattering.
Electron Microscopy--
Samples were absorbed on carbon-coated,
600-mesh copper grids for 30 s, stained with freshly filtered 2%
ammonium molybdate or 2% uranyl acetate, and were viewed in a JEOL JEM
100CX II electron microscope at 80 kV at standard magnifications of
40,000.
 |
RESULTS AND DISCUSSION |
To estimate the thermodynamic stability of
-MoPrP, its
urea-induced unfolding and refolding was measured using the far-UV CD as a probe of its secondary structure. In a low salt buffer, pH 3.6, the urea-induced unfolding profile of the molar ellipticity at
222 nm shows a single cooperative transition between the
-isoform and the unfolded state (Fig.
1a). When
-MoPrP is
unfolded in 10 M urea and then refolded by diluting the
urea concentration, its refolding curve expresses hysteresis, a
phenomenon indicative of a non-two-state process (Fig. 1a).
Both the unfolding and refolding limbs of the curve remain stable for
at least 5 weeks when MoPrP is kept in a low salt buffer (20 mM sodium acetate). However, when refolding of MoPrP at 10 µM concentration is performed in a high salt buffer (0.2 M NaCl, 20 mM sodium acetate), the refolding curve undergoes a gradual time-dependent transformation
from a single cooperative transition to a transition with local
intermediates (Fig. 1b). If a similar experiment is
performed at 30 µM MoPrP, the migration of refolding
curve occurs more rapidly (Fig. 1c).

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Fig. 1.
Urea-induced unfolding and refolding
transitions of MoPrP monitored by CD at pH 3.6. a,
unfolding (squares) and refolding (circles)
curves monitored upon incubation of 10 µM MoPrP for 10 min (filled symbols) and for 5 weeks (open
symbols) at various concentrations of urea. Data were analyzed
according to a two-state transition model; the result of the fitting is
represented by solid curves (18). b, refolding
curves measured upon incubation of 10 µM MoPrP for 10 min
(filled circles), 72 h (open circles), 1 week (filled triangles), and 5 weeks (open
triangles) after dilution at a particular concentration of urea
and 0.2 M NaCl. The solid line represents the
unfolding curve from panel a. c, refolding curves
measured upon incubation of 30 µM MoPrP for 10 min
(filled circles), 72 h (open circles), 1 week (filled triangles), and 5 weeks (open
triangles) after dilution at a particular concentration of urea
and 0.2 M NaCl. The solid line represents the
unfolding curve from panel a. d, far-UV CD
spectra of original -MoPrP (solid line), recorded
immediately after dilution from 10 M urea
(long-dashed line) and after a 5-week incubation following
dilution (short-dashed line). e, SEC profile of
original -MoPrP (1) and profiles obtained upon incubation for 10 min
(2), 72 h (3), 1 week (4), and 5 weeks (5) after dilution from 10 to 1 M urea.
|
|
Unfolded MoPrP folds first to the
-helical form upon dilution
from 10 M urea (Fig. 1d). During incubation for
5 weeks in the high salt buffer, it undergoes a slow conformational
transition to the
-rich form as illustrated by the change in the
overall CD spectra, as well as by reduction of the CD signal at 222 nm (Fig. 1, b and d). The conformational transition
from the
-helical to a
-sheet-rich isoform is accompanied by
oligomerization as judged by size-exclusion chromatography (SEC) (Fig.
1e). Immediately after dilution from 10 M urea,
a new peak corresponding to an oligomer appears, in addition to the
peak that represents a monomer. During the conformational transition,
the population of monomer decreases whereas the fraction of oligomer
grows. Although the square variance analysis of the oligomer peak
indicates that there is certain heterogeneity of the oligomer species,
electrospray mass spectrometry suggests that an octamer is the dominant
multimeric assembly (data not shown).
The unfolding and refolding behavior of MoPrP demonstrates hysteresis,
a time-dependent transformation of the single
transition curve into a double transition curve, and a
concentration-dependence for this process. These observations challenge
the application of either of the two possible classical three-state
models used previously to estimate the thermodynamic parameters
for PrP unfolding (16, 17). In contrast, a
model with two independent transitions, one between the
-isoform and
unfolded and the other between the
-isoform and unfolded, can be
used to fit the data. We have observed that the refolding to the
-isoform is much faster than the refolding to a
-isoform, whereas
the time-dependent accumulation of a
-isoform indicates
that it is thermodynamically more stable than the
-isoform. Thus,
MoPrP diluted out of urea folds predominantly to the
-isoform with
little
-isoform present. The presence of a
-isoform would account
for the hysteresis between the unfolding and the refolding curves (Fig.
1b). With time, the refolding curve transforms from an
apparent single transition to the double transition, demonstrating equilibration of the
- and the
-isoforms.
Direct comparison of the thermodynamic stability of the
-and the
-isoforms illustrate that the
-isoform is not the lowest energy
state. First, we estimated the thermodynamic parameters for the
-isoform using the urea-induced unfolding curve and applying a
classical two-state model (see Fig. 1a and Table
I) (18). To evaluate the thermodynamic
stability of the
-isoform, two parameters, the molar
ellipticity at 222 nm and the fraction of the oligomer, were monitored
in parallel as a function of urea concentration after re-equilibration
of MoPrP for 5 weeks. Despite the fact that a small fraction of MoPrP
remains trapped in the
-helical conformation even after 5 weeks, we
have exploited the fraction of the
-oligomer as directly measured by
SEC to analyze the "unfolded
-isoform" equilibrium
using the two-state model (Fig.
2a). The unfolding curve
measured by CD requires deconvolution, because it is composed of
signals from the
-isoform, the unfolded state, and the
-isoform.
Using the population of the monomer measured by SEC as a function of
urea concentration and the thermodynamic parameters estimated
previously for the "
-isoform
unfolded" equilibrium,
we calculated the contribution of the
-isoform to the CD curve (Fig.
2b). When this contribution is subtracted from the original
curve, a curve reflecting the unfolded
-isoform transition results. As shown in Fig. 2c, the transition
curves for the
-isoform are superimposible, with
G, m, and
C1/2 determined from the two techniques equal within the
uncertainty of the experiment (Table I). Both
G and C1/2
demonstrate that the
-isoform is thermodynamically more stable than
the
-isoform (see Fig. 2c and Table I). Because both
isoforms can be refolded directly from the unfolded state, we have used
the unfolded state as a reference in the free energy diagram (Fig.
2d).
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Table I
Thermodynamic parameters for the urea-induced unfolding of the -
and -isoforms of recombinant MoPrP 89-231
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Fig. 2.
Estimated thermodynamic stability of
-MoPrP. MoPrP unfolded at 10 M
urea was diluted to various concentrations of urea at pH 3.6 in the
presence of 0.2 M NaCl and incubated for 5 weeks at
23 °C before SEC and CD measurements. a, examples of SEC
profiles monitored at different concentrations of urea, from
bottom to top: 1, 2, 3, 3.5, 4, 5, 6, 7, 8, and 9 M. To apply a two-state model, the population of the
-oligomer versus urea concentration was normalized
relative to the amount of the -oligomer observed with 1 M urea taken as 100%. b, CD measurements of the
original refolding curve (filled circles) and the transition
curve after subtracting the contribution of the -isoform (open
circles). The solid line represents the result of the
fitting to the two-state model. c, normalized transition of
the -isoform (filled circles) and the -isoform
(open circles) as determined by SEC (solid line)
and by CD (dashed line) against varying concentrations of
urea. d, free energy diagram for the - and the
-isoform of MoPrP determined at pH 3.6. U represents the
unfolded state.
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|
Although the
-isoform is thermodynamically more stable than the
-isoform, it might be not a true global energy minimum state, because the
-isoform can undergo an additional
time-dependent transition to a polymeric amyloid form.
Incubation of
-MoPrP at 37 °C and constant shaking lead to the
formation of higher molecular weight aggregates that possess amyloid
properties. The process of amyloid formation monitored by thioflavin T
binding displays an apparent latent period and then an exponential
accumulation of the aggregate (Fig.
3a). In addition to thioflavin
T, the amyloid of MoPrP binds Congo red in a specific manner as judged
by birefringence of polarized light and typical red shift of absorbance
spectra (Fig. 3b). Aggregated MoPrP forms numerous twisted
fibrilar filaments as seen by electron microscopy (Fig.
3c).

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Fig. 3.
-MoPrP assembles into amyloid
fibrils. a, the kinetics of assembly of 40 µM MoPrP monitored by thioflavin T binding. b,
difference spectra obtained at 1.7 (solid line), 3.4 (dotted line), 5.1 (dashed line), and 6.8 µM (dotted-dashed line) Congo red in the
presence of 1.5 µM MoPrP taken after 160 h.
c, electron micrograph of fibrils negatively stained with
ammonium molybdate.
|
|
Why is the thermodynamically more stable
-isoform not
accessible during folding under native conditions? Previously, it has been shown that the folding of PrP to the
-isoform is an extremely fast, first-order process (14). Folding to the
-isoform is slower by
several orders of magnitude and is concentration-dependent. To prevent the conformational conversion, the
-isoform has to be
separated by a large energetic barrier from the
-isoform. Although
the free energy diagram does not provide a view of the actual kinetic
pathway for the conformational transition, several important
observations can be made concerning the origin of the energetic
barrier. First, the
-isoform has to unfold substantially on route to
the
-isoform. As we have seen before, the
-isoform converts very
slowly to the
-isoform at pH 3.6 in the absence of urea (Fig.
1b). This process can be accelerated by shifting the
-isoform
unfolded equilibrium toward the unfolded state. After
jumping the urea concentration from 0 to 5 M, we observed a
very fast loss of secondary structure by the
-monomer within the
dead time of manual mixing, followed by an accumulation of a
-sheet-rich conformation (Fig.
4a). This result illustrates that a substantial portion of the energetic barrier requires partial unfolding of the
-isoform. The connection between the structural complexity of the pretransition state and the energetic barrier is
demonstrated by previous observations that conversion of PrP-derived peptides with low structural complexity into
-rich isoforms occurs spontaneously and does not require partially denaturing conditions (19-21). Whether the transition state on the way from
the
- to the
-isoform is predominantly unfolded under native
conditions or whether it has residual
-sheet or
-helical
structure remains to be established.

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Fig. 4.
a, the kinetic trace of transition from
the -isoform to the -isoform (10 µM MoPrP) induced
by jumping the urea concentration from 0 to 5 M at pH 3.6, as monitored simultaneously by SEC (open squares) and CD
(filled circles). b, the kinetic trace of folding
the -isoform induced by jumping the urea concentration from 10 to 5 M at pH 3.6 monitored by CD. In the inset, the
linearity of the fifth-order (1/ normalized
n 1 versus time) plot suggests that the
process of folding may follow an apparent fifth-order kinetics.
c, free energy diagram of the conformational transition,
representing the activation energy (kcal/mol) estimated at pH 3.6 and
10 µM of MoPrP. TS represents the transitional state. The
-MoPrP undergoes an additional transition to the amyloid form,
represented by the dotted line. d, the activation
energy versus the concentration of MoPrP shown at different
pH values, as calculated from the Arrhenius relation applying the
diffusion-controlled reaction rates. The physiological concentration
range of MoPrP is shown in the shaded bar.
|
|
A significant contribution to the energetic barrier seems to be
associated with the process of oligomerization. As shown on Fig.
4b, the accumulation of a
-rich conformation is
accompanied by oligomerization. The fact that both kinetic curves are
superimposible illustrates that the two processes are coupled (Fig.
4a). MoPrP can be refolded directly to the
-isoform if
the unfolded protein is diluted first to 5 M urea (Fig.
4b). When dialyzed out of urea and salt,
-MoPrP is stable
for months at room temperature with no detectable conversion to the
-isoform. Analysis of the kinetic traces indicates that the process
of folding to the
-isoform represents a single transition with
apparent reaction order of 5, regardless of whether the refolding
is initiated by dilution of urea from 10 to 5 M, a jump of
the urea concentration from 0 to 5 M, or if the
conformational transition occurs in the absence of urea. Such a high
order of reaction suggests that the conformational transition will
depend upon the concentration of the transition state.
To estimate the energy of activation
(
E
) of the conformational
transition, the Arrhenius relation,
|
(Eq. 2)
|
can be used, in which kobs is the constant
rate of the conformational transition measured experimentally, and
k0 is the rate of the process under diffusion
control. Under experimental conditions employed (pH 3.6 and 10 µM MoPrP), we found that the
-isoform is separated
from the
-isoform by an energy barrier of 20 kcal/mol (Fig.
4c). The energetic barrier is predicted to be much higher under physiological conditions because of the lower concentration of
PrP and the higher thermodynamic stability of the
-isoform at pH
5-7 (Fig. 4d). For wild-type MoPrP, the calculated energy barrier of 35-45 kcal/mol is sufficient to prevent the process of
conformational transition over the functional lifetime of the protein.
Hence, a large energetic barrier prevents the conversion of the
-isoform to the thermodynamically more stable
-isoform. From the
kinetic perspective, the process of conformational transition can be
facilitated by the reduction of the energetic barrier (22). Thus,
single point mutations associated with inherited forms of prion
diseases might reduce the energetic barrier by stabilizing the
transition state. Additionally, if PrPSc provides a
template for the conversion of PrPC to PrPSc by
binding and stabilizing the transition state, this would also speed up
the conformational conversion.
Our results clearly indicate that the folding of native
PrPC is under kinetic control. The observations that many
proteins are able to adopt alternative amyloid-like folds require us to revisit the role of kinetic traps in protein folding (8-12). If a
-rich amyloid competent structure is an intrinsic preference especially at a high protein concentration, then compartmentalization of partially folded intermediates and proteins that mediate unfolding and clearance of misfolded proteins play critical roles in cellular health. In addition, side-chain patterns that favor the formation of
amyloid, such as alternating polar and non-polar amino acid residues,
will be avoided (23). Despite these strategies, some proteins,
including PrP, A
,
-synuclein, parkin and tau, find a route
to a
-rich, multimeric structure with unfortunate consequences.
 |
FOOTNOTES |
*
This work was supported in part by grants from the National
Institutes of Health (AG0Z132, AG10770, and NS14069), as well as by a
gift from the G. Harold and Leila Y. Mathers Foundation. I.B. was
supported by the John Douglas French Foundation for Alzheimer's Disease Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Cellular and
Molecular Pharmacology, University of California at San Francisco, Box
0450, San Francisco, CA 94143. Tel.: 415-476-8519; Fax: 415-476-6515;
E-mail: cohen@cmpharm.ucsf.edu.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.C100180200
 |
ABBREVIATIONS |
The abbreviations used are:
MoPrP, mouse prion
protein;
PrP, prion protein;
PrPC, cellular isoform;
PrpSc, scrapie isoform;
SEC, size-exclusion
chromatography.
 |
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