From the Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106
Received for publication, August 28, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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It is believed that the critical step in the
pathogenesis of transmissible spongiform encephalopathies is a
transition of prion protein (PrP) from an Prion diseases, or transmissible spongiform
encephalopathies, comprise a group of fatal neurodegenerative
disorders that can arise sporadically or can have an infectious or
genetic etiology (1-3). The best known animal forms of the disease are
scrapie in sheep and bovine spongiform encephalopathy in cattle. The
human versions include kuru, Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker syndrome. These disorders are characterized by vacuolation of neurons, astroglyosis, and cerebral
accumulation of an abnormal (scrapie-like) form of prion protein,
PrPSc.1 Although
the molecular mechanism of transmissible spongiform encephalopathies is
controversial (4), numerous observations point to the central role of
PrPSc in the pathogenesis of these disorders (1-3).
According to the "protein only" hypothesis (1, 5, 6),
PrPSc constitutes the sole component of the infectious
prion pathogen.
PrPSc is derived from a normal (cellular) prion protein,
PrPC. Human PrPC is a 209-residue glycoprotein
that has two N-glycosylation sites and a disulfide bridge
linking Cys residues at positions 179 and 214. PrPC is
transported through the secretory pathway and ultimately anchored to
the cell surface by a glycosylphosphatidylinositol anchor (1, 7, 8). It
is localized in cholesterol-rich membrane microdomains called rafts or
caveo-like domains (9). The transition between PrPC and
PrPSc occurs post-translationally on the cell surface
and/or in an endocytic pathway (10, 11). No differences in the covalent structure have been observed between PrPC and
PrPSc (12). However, the two protein isoforms have
profoundly different biochemical and biophysical properties.
PrPC is soluble in mild detergents and easily degradable by
proteinase K, whereas PrPSc is insoluble in mild detergents
and highly resistant to proteinase K digestion (7, 13, 14).
Furthermore, spectroscopic studies have revealed that the two isoforms
have markedly different secondary structures; PrPC consists
largely of Because the critical step in the pathogenesis of spongiform
encephalopathies appears to be a conformational transition of PrP,
there is currently great interest in understanding the biophysical properties of the prion protein. Recent studies have provided a wealth
of data on the three-dimensional structure, folding pathway, and
thermodynamic stability of the recombinant model of PrPC
(18-28). However, the molecular mechanism of conformational
transition(s) underlying the conversion of PrPC to
PrPSc still remains unknown. In a recent study (29), it was
reported that, upon reduction of a single disulfide bridge, the
recombinant prion protein could reversibly switch between The Construction of Plasmids--
The plasmids encoding
huPrP23-231 and huPrP90-231 with an N-terminal linker containing a
His6 tail and a thrombin cleavage site were described
previously (25). Proteins obtained using these plasmids contain the
N-terminal extension Gly-Ser-Asp-Pro (25). To remove the last two
residues of this extension, the plasmids were amplified by polymerase
chain reaction using the primers 5'-CCGCGTGGTTCGAAGAAGCGCCCG and
5'-CGGGCGCTTCTTCGAACCACGC GG for huPrP23-231 or
5'-GCGTGGTTCGGGTCAAGGAG and 5'-CTCCTTGACCCGAACCA CGC for
huPrP90-231.
The C179A/C214A variant of huPrP23-231 was obtained by site-directed
mutagenesis using the primers 5'-C TTT GTG CAC GAC GCC GTC AAT ATC AC
and 5'-GT GAT ATT GAC GGC GTC GTG CAC AAA G for Cys179 Protein Expression and Purification--
The wild type
huPrP23-231 and huPrP90-231 were expressed and purified as described
previously (25). The C179A/C214A variant was expressed by the same
procedure. However, the tendency of the mutant protein to precipitate
at neutral pH required different purification strategy. After
expression in BL21(DE3) cells, the latter protein was extracted by
sonication in a buffer (10 mM Tris, 100 mM
K2HPO4, pH 8.0) containing 6 M
GdnHCl. The protein was then applied onto a nickel-nitriloacetic
acid-agarose column (Amersham Pharmacia Biotech). Weakly bound proteins
were removed by washing the column with 10 mM Tris, 100 mM K2HPO4, 50 mM
imidazole, pH 8.0. The mutant huPrP23-231 was then eluted in 500 mM imidazole, 6 M GdnHCl, pH 5.8. Protein was
then dialyzed against a 10 mM sodium acetate buffer, pH
5.6, and the His tail was cleaved by 15 h of treatment at room
temperature with thrombin (10 units/mg of protein). The free peptide
and thrombin were removed by selective precipitation of C179A/C214A
huPrP23-231 in sodium phosphate buffer, pH 7.0. The pellet was
dissolved in 10 mM sodium acetate containing 8 M urea, pH 4.0. Finally, the purified protein was refolded
by 10-fold dilution in 10 mM sodium acetate, pH 4.0, followed by extensive dialysis against the same buffer. To remove the
residual aggregated protein, the dialyzed sample was filtered and
ultracentrifuged for 3 h at 100,000 × g. Protein
concentration was determined using a molar coefficient at 276 nm of
56,650 M Preparation of the Reduced huPrP--
To reduce the disulfide
bond, the wild type huPrP23-231 or huPrP90-231 was unfolded with 8 M urea or 6 M GdnHCl in 10 mM Tris, pH 8.0, and treated for 15 h at room temperature with 100 mM DTT. Upon reduction, the protein was refolded by
dialysis against 10 mM sodium acetate, 1 mM
DTT, pH 4.0. The dialyzed sample was filtered and ultracentrifuged at
100,000 × g for 3 h.
Circular Dichroism Spectroscopy and Equilibrium Unfolding in
Urea--
Far-UV CD spectra were recorded in a 1-mm quartz cell at a
protein concentration of 0.25 mg/ml. Near-UV CD spectra were obtained using a 1-cm cell at a protein concentration of 1 mg/ml. To obtain equilibrium unfolding curves, huPrP23-231, Cys-free huPrP23-231 and
reduced huPrP23-231 were diluted (to a final concentration of 0.024 mg/ml) in 10 mM sodium acetate, pH 4.0, containing
different concentration of urea. Samples were incubated for 24 h
at room temperature, and the ellipticity at 222 nm was measured in a
1-cm cell by averaging the signal over 1 min. The concentration of urea
was determined by refractive index measurements. All CD measurements were carried out at room temperature on a Jasco J-810 spectropolarimeter.
ANS Binding--
Protein samples were diluted to a concentration
of 0.05 mg/ml in 50 mM sodium acetate buffer, pH 4.0, containing 10 µM ANS. After 1 h of incubation in the
dark, fluorescence spectra were measured on an SLM 8100 spectrofluorimeter using the excitation wavelength of 375 nm.
Dynamic Light Scattering--
The hydrodynamic radius of
monomeric forms of PrP was measured by quasi-elastic light scattering
on a DynaPro-801 molecular size detector (Protein Solutions Inc.). Data
were analyzed with the software provided by the manufacturer using
appropriate viscosity and refractive index corrections.
Size-exclusion Chromatography--
Size-exclusion chromatography
was performed using Bio-Sil SEC-250 column (Bio-Rad) attached to a
RAININ HPLC system. The column was pre-equilibrated with 10 mM sodium acetate, 50 mM NaCl, pH 4.0, and the
protein was eluted using the same buffer at the flow rate of 1 ml/min.
The elution of the protein was monitored by absorbance at 280 nm.
Solubility and Oligomerization State of the Disulfide Bridge-free
huPrP23-231--
The disulfide bridge in the folded huPrP is buried
in a hydrophobic environment (18-21) and is not accessible to reducing
agents such as DTT. However, the bridge could be readily reduced by DTT upon unfolding of the protein in 8 M urea or 6 M GdnHCl. At neutral pH, attempts to refold the reduced
huPrP23-231 by rapid dilution or dialysis against the denaturant-free
buffer (10 mM sodium phosphate, pH 7.0 and 8.0 or Tris HCl,
pH 8.0) consistently resulted in a massive precipitation of the
protein, leaving no material in a soluble form. Very similar behavior
was observed for prion protein variants in which the disulfide bridge
was removed by a replacement of Cys residues with alanine (C179A/C214A
huPrP23-231).
In contrast to the results obtained at neutral pH, dialysis refolding
of the disulfide bridge-free huPrP23-231 under acidic conditions (10 mM sodium acetate, pH 4.0) did not lead to visible precipitation of the protein. However, upon complete removal of urea
the samples were slightly turbid, indicating that at least part of the
protein was self-associated. The aggregated material could be removed
by ultracentrifugation for 3 h at 100,000 × g. An
analysis of the remaining fraction by quasi-elastic light scattering revealed the presence of a monomeric protein with an apparent Stokes
radius of ~2.8 nm. A comparison of the latter number with the Stokes
radius of 2.4 nm for the oxidized wild-type huPrP23-231 suggests that
the loss of the disulfide bridge in prion protein is associated with a
transition to a less compact conformation. The recovery of the protein
in a monomeric form was ~20% for the reduced huPrP23-231 and less
than 10% for the Cys-free mutant. It should be noted that under
present experimental conditions, freshly prepared monomeric protein was
stable for at least 10 h. However, upon longer incubation at room
temperature the protein had a tendency to self-associate. The behavior
of the reduced and Cys-free protein contrasts with that of the
disulfide bridge-containing huPrP23-231. The latter protein could be
unfolded reversibly and refolded both at neutral and acidic pH. The
refolded species retained native Conformational Properties of the Monomeric Form of the Disulfide
Bridge-free huPrP23-231--
The effect of disulfide bridge on the
secondary structure of huPrP23-231 was assessed by far-UV circular
dichroism spectroscopy. As shown in Fig.
1, the spectrum at pH 4 for the wild-type
protein with an intact disulfide bridge exhibits a double minimum at
208 and 222 nm (with a mean residue ellipticity at 222 nm,
The perturbation of structural integrity upon removal of the disulfide
bridge in huPrP is also indicated by near-UV circular dichroism
spectroscopy. Near-UV CD spectra provide a very sensitive probe of a
global tertiary structure of proteins (31). As shown in Fig.
2, upon removal of the disulfide bridge
in huPrP23-231, there is a major reduction of the CD signal in the
near UV region. This spectral change most likely reflects at least a
partial collapse of native tertiary interactions in the protein (31).
However, a small part of the signal reduction could also be due to the loss of spectral contribution from the disulfide bond.
The conformational properties of the reduced and Cys-free variants of
huPrP23-231 were further probed using the hydrophobic dye ANS. This
dye, the fluorescence of which greatly increases upon binding to
hydrophobic sites, has been used widely to assess the surface
hydrophobicity of proteins (32). Fig. 3
shows that there is very little fluorescence of ANS in the presence of
huPrP23-231 with an intact disulfide bridge. However, incubation of
the dye in the presence of the reduced PrP or the Cys-free variant
results in a dramatic increase in the fluorescence intensity. This
indicates binding of the probe to disulfide bridge-free proteins, most
likely because of an increased exposure of hydrophobic patches (32, 33).
The effect of the disulfide bridge on the thermodynamic stability of
prion protein was studied by equilibrium unfolding in urea. As shown in
Fig. 4, the unfolding curve for the
oxidized wild-type huPrP23-231 at pH 4 (10 mM sodium
acetate) is highly cooperative. Analysis of this curve using a
two-state transition model (34) yields a midpoint unfolding urea
concentration of 3.7 M and a free energy difference between
the native and unfolded state of ~12 kJ/M. In
contrast to the disulfide bridge-containing protein, under similar
experimental conditions the reduced huPrP23-231 and the Cys-free
variant unfold over a very broad range of urea concentration, showing
no well defined cooperative transition.
Salt-induced Conformational Transition--
The reduced (or
Cys-free) PrP monomer with the properties described above was observed
only at acidic pH in a low ionic strength buffer (10 mM
sodium acetate) and in the absence of NaCl. Upon addition of NaCl,
there was a rapid change in the CD spectrum to one with a minimum at
~215 nm (Fig. 5), indicating a
conformational transition to a form rich in
The oligomerization state of different forms of huPrP23-231 was
assessed by size-exclusion chromatography (Fig.
6). Under the conditions corresponding to
Experiments similar to those described above for huPrP23-231 were also
performed with the reduced human prion protein fragment 90-231.
In close resemblance to the full-length protein, formation of
the The central event in the pathogenesis of spongiform
encephalopathies is a profound conformational change of the prion
protein from an Within the context of the "protein folding problem," the
fundamental question is whether the In this study, we have examined the biophysical properties of the
disulfide bridge-free recombinant full-length human prion protein. This
protein provides a valuable model for probing the role of protein
folding intermediates in the PrPC The present study demonstrates that, upon addition of NaCl, the
molten-globule-like (disulfide-free) form of huPrP23-231 undergoes a
transition to a The finding that the reduced PrP at acidic pH adopts an oligomeric
-helical conformation,
PrPC, to a
-sheet-rich form, PrPSc.
Native prion protein contains a single disulfide bond linking Cys
residues at positions 179 and 214. To elucidate the role of this bridge
in the stability and folding of the protein, we studied the reduced
form of the recombinant human PrP as well as the variant of PrP in
which cysteines were replaced with alanine residues. At neutral pH, the
reduced prion protein and the Cys-free mutant were insoluble and formed
amorphous aggregates. However, the proteins could be refolded in a
monomeric form under the conditions of mildly acidic pH. Spectroscopic
experiments indicate that the monomeric Cys-free and reduced PrP have
molten globule-like properties, i.e. they are characterized
by compromised tertiary interactions, an increased exposure of
hydrophobic surfaces, lack of cooperative unfolding transition in urea,
and partial loss of native (
-helical) secondary structure. In the
presence of sodium chloride, these partially unfolded proteins undergo
a transition to a
-sheet-rich structure. However, this transition is
invariably associated with protein oligomerization. The present data
argue against the notion that reduced prion protein can exist in a
stable monomeric form that is rich in
-sheet structure.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-helices, whereas PrPSc is rich in
-sheet
structure (15-17).
-helical
conformation and a monomeric form rich in
-sheet structure. It was
also postulated that the latter form represents a key monomeric
precursor of PrPSc. The notion that a monomeric
protein could exist in two profoundly different conformations is
highly intriguing and has potentially far reaching implications. To
obtain further insight into the molecular basis of conformational
transitions in prion protein, we have undertaken detailed studies on
the effect of disulfide bridge on the folding and conformational
stability of the recombinant human PrP. Our data show that the removal
of a disulfide bridge greatly destabilizes the native structure of the
protein. In the presence of salt, the reduced or Cys-free protein is
highly prone to oligomerization. However, under no experimental
conditions could we observe a monomeric
-sheet-rich form of the
protein. Both in the presence (28) and absence of a disulfide bridge,
-structure was formed only upon oligomerization of the recombinant PrP.
EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Ala replacement and 5'-GTT GAG CAG ATG GCG ATC ACC CAG TAC and 5'-GTA
CTG GGT GAT CGC CAT CTG CTC AAC for Cys214
Ala
replacement. All DNA manipulations were carried out according to
standard protocols (30). DNA sequences of the final constructs were
verified by automated sequencing at the Case Western Reserve University
Molecular Biology Core Facility.
1
cm
1.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-helical conformation and was
stable as a monomer for many days or even weeks.
222, of
8000° cm2
dmol
1) typical for
-helix-rich proteins
(31). This spectrum is consistent with the structure of PrP containing
-helical C-terminal domain and largely unstructured N-terminal
region (19, 21). The CD spectrum of the monomeric huPrP23-231 with
reduced disulfide bridge is profoundly different; it shows a minimum at
~204 nm, whereas the mean residue ellipticity at 222 nm is reduced to
3000° cm2 dmol
1 (Fig. 1). A
very similar spectrum was obtained for the Cys-free variant of
huPrP23-231, although in this case
222 is somewhat higher (approximately
4000° cm2
dmol
1). The spectral data presented above
indicate that in the absence of the disulfide bridge, prion protein
refolds (as a monomer) into a conformation that is characterized by a
major loss of
-helix and increased proportion of an unordered
structure.
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Fig. 1.
Far-UV circular dichroism spectra for the
wild-type huPrP23-231 with native disulfide bridge ( ),
reduced huPrP23-231 (- - -), and the Cys-free (C179A/C214A)
variant of huPrP23-231 (
· ·
). The spectra were
obtained in 10 mM sodium acetate, pH 4.0.
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Fig. 2.
Near-UV circular dichroism spectra for the
wild-type huPrP23-231 with native disulfide bridge ( ) and the
Cys-free (C179A/C214A) variant of huPrP23-231 (
· ·
).
As a reference, the spectrum of huPrP23-231 unfolded in 8 M urea is also included (····).
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Fig. 3.
Fluorescence spectra of ANS alone
(····) and in the presence of the wild-type huPrP23-231 with
native disulfide bridge ( ), reduced huPrP23-231 (- - -), and
the Cys-free (C179A/C214A) variant of huPrP23-231
(
· ·
). Spectra were obtained in 10 mM
acetate buffer, pH 4.0, containing 10 µM ANS and 0.05 mg/ml protein. The excitation wavelength was 375 nm.
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Fig. 4.
Urea-induced unfolding of the wild-type
huPrP23-231 with native disulfide bridge ( ), reduced huPrP23-231
(
), and the Cys-free (C179A/C214A) variant of huPrP23-231
(
). The experiments were performed in 10 mM sodium
acetate, pH 4.0, and the degree of unfolding was monitored by
ellipticity at 222 nm.
-sheet structure (31).
Under the present experimental conditions, this conformational
transition was very fast, precluding detailed kinetic studies. For
example, at a protein concentration of 0.25 mg/ml and a NaCl
concentration of 50 mM, the changes in the spectrum were
complete within less than 3 min. In contrast to the reduced and
Cys-free proteins, the oxidized huPrP23-231 remained
-helical even
after 24 h of incubation in the presence of 50 or 100 mM NaCl.
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Fig. 5.
Far-UV circular dichroism spectra of the
reduced huPrP23-231 (A) and the Cys-free
(C179A/C214A) variant of huPrP23-231 (B) in the
absence ( ) and presence (
) of 50 mM
NaCl. Spectra were obtained in 10 mM sodium acetate
buffer, pH 4.0, at a protein concentration of 0.25 mg/ml.
-sheet formation (10 mM sodium acetate, 50 mM NaCl), the reduced and Cys-free proteins consistently eluted as high molecular aggregates larger than the 600 kDa
fractionation limit of the Bio-Sil SEC-250 column. The elution profile
presented above was highly reproducible, and repeated experiments
provided no indication for the presence of a monomeric
-sheet-rich
species. This is in contrast with the behavior of the oxidized
(
-helical) huPrP23-231 that, under the same buffer conditions,
eluted at a time corresponding to a monomeric prion protein. Attempts
to obtain size exclusion data in the absence of NaCl were unsuccessful because, under these conditions, huPrP23-231 adsorbs to Bio-Sil column
and other chromatographic media tested (Superdex, Sephacryl, Superose).
However, the monomeric state of the oxidized, reduced, and
Cys-free forms of huPrP23-231 in the NaCl-free buffer is clearly indicated by quasi-elastic light scattering experiments as described above.
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Fig. 6.
Size-exclusion chromatography profiles for of
the wild-type huPrP23-231 with native disulfide bridge
(top), reduced huPrP23-231 (middle)
and the Cys-free (C179A/C214A) variant of huPrP23-231
(bottom). The chromatograms were obtained
following a 1-min incubation of the proteins (0.5 mg/ml) in 10 mM sodium acetate, 50 mM NaCl, pH 4.0. The
wild-type huPrP23-231 with native disulfide bond eluted as a monomeric
protein (elution volume of 10.2 ml), whereas the reduced huPrP23-231
and the Cys-free variant eluted in the void volume of the column (5.6 ml). The size exclusion limit of the Bio-Sil SEC-250 column is 600 kDa
for globular proteins.
-sheet structure by the reduced huPrP90-231 was invariably associated with rapid oligomerization of the protein, with no indication of the presence of a stable monomeric
-sheet rich form
(data not shown for brevity).
DISCUSSION
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ABSTRACT
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DISCUSSION
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-helical form, PrPC, to a
-sheet-rich
form, PrPSc (1-3, 15-17). Recent NMR studies have
provided high-resolution structural data for the recombinant model of
PrPC (18-21), whereas a detailed structure of the
PrPSc isoform is still unknown. It is believed that the
propagation of the disease can be described according to the
heterodimer (template-assistance type) model (35) or the
nucleation-dependent polymerization model (36). However,
the molecular mechanism of the conformational transition and potential
folding intermediates underlying the PrPC
PrPSc conversion remain unknown.
-sheet-rich form of PrP can exist in a stable monomeric state or, as implied by the
nucleation-dependent polymerization model, the above form
is stable only as a high molecular weight oligomer. Recently, we
demonstrated that, under appropriate solvent conditions, the
recombinant human PrP undergoes a transition from
-helix to a
-sheet-rich structure (28). The key requirements for this conversion
include acidic pH, mildly denaturing conditions, and the presence of
certain ions.2 The formation
of
-sheet structure was associated invariably with oligomerization
of prion protein into high molecular weight aggregates. A transition to
an oligomeric
-sheet-rich structure was also reported in a related
study with a redacted chimeric mouse-hamster PrP consisting of 106 amino acids (37). Importantly, the above two studies provide no
evidence for the presence of a monomeric
-sheet-rich conformer of
the prion protein. A markedly different behavior was reported for prion
protein with a reduced disulfide bond. According to Jackson et
al. (29), at neutral pH the reduced huPrP90-231 exists as a
native
-helical conformer, whereas at acidic pH it adopts a
monomeric form rich in a
-sheet structure. These authors also
proposed that the reduction of prion protein in intracellular
compartments could play a crucial role in the PrPC
PrPSc conversion. However, this hypothesis is controversial
(3), and certain observations argue against the involvement of
disulfide bond reduction in the pathogenesis of prion disease (38,
39).
PrPSc
conversion. The disulfide bond in huPrP23-231 was reduced by treatment
with 100 mM DTT. However, some spectroscopic experiments are not feasible in the presence of concentrated DTT. Dilution (or removal) of the reducing agent could potentially lead to a recreation of the disulfide bond. Therefore, to avoid any ambiguity, in
addition to using the reduced protein, the present experiments were
also performed with a huPrP23-231 variant in which the two Cys
residues were replaced with alanine. Our data show that at neutral pH
both the reduced PrP as well as the Cys
Ala variant form insoluble
aggregates. The proteins could be recovered in a monomeric form only
under the conditions of acidic pH in a NaCl-free buffer. Compared with
the PrP with an intact disulfide bond, the monomeric forms of the
disulfide-free variants are characterized by a major loss of secondary
structure, disruption of native tertiary interactions, a greatly
increased affinity for hydrophobic dyes, and a loss of cooperative
unfolding transition in urea. Some of these properties are reminiscent
of a flexible folding intermediate often referred to as a "molten
globule" state (40). However, typical molten globules are
characterized by a substantially more ordered (native-like) secondary
structure than observed for the disulfide bridge-free variants of the
prion protein. The finding that removal of the disulfide bridge results
in partial unfolding of PrP is not surprising, because disulfide bonds
are known to stabilize the folded conformation of many proteins. The
contribution of a single disulfide bond to the thermodynamic stability
of proteins is usually in the range of several kJ/M
(41), although in certain cases it may be as high as 20 kJ/M (42). The above described stabilizing effect is
believed to be due largely to the reduction of conformational entropy
of the unfolded state (41). Examples of proteins that upon reduction of
one or more disulfide bonds adopt molten globule-like or partially
unfolded conformation include
-lactalbumin (43) and papain (44). It
was reported previously that at acidic pH the reduced recombinant prion
protein refolds to a stable monomeric form that is rich in
-sheet
structure (29). However, our present results are at odds with
this report. We are also not aware of any other protein that would
switch from a monomeric
-helical conformation in the
oxidized state to a monomeric
-sheet structure in the
reduced state.
-sheet-rich structure. This transition was invariably associated with protein self-association. In contrast to the
previous report (29), no monomeric
-sheet-rich conformer could be
detected in our experiments with huPrP23-231 and huPrP90-231. The
protein self-association reaction and changes in CD spectra occurred
very rapidly (within the dead-time of the present experiments), suggesting that the helix
-sheet transition is an integral part
of the oligomerization process. However, the present data do not allow
us to exclude the possibility that the self-association step may be
preceded by the formation of a transient monomeric
-sheet rich
conformer. Such a conformer would be, however, very short-lived and
highly prone to aggregation.
-sheet structure is in line with previous observations for
huPrP90-231 with an intact disulfide bond (28). However, compared with
the latter protein, the reduced form is characterized by a
substantially increased propensity to form a polymeric
-sheet-rich form. In contrast to the disulfide bridge-containing PrP, the conversion of the reduced (molten globule-like) protein is triggered by
salt alone and does not require denaturing agents such as guanidine HCl
or urea. This suggests that destabilization or partial unfolding of the
native
-helical conformation is a key factor in the conversion of
PrP into a scrapie-like form. The view expressed above is consistent with recent findings that the helix
-sheet transition of the nonreduced PrP may be modulated by changes in the concentration of
urea.2
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant NS38604 (to W. K. S.).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 Pathology,
Case Western Reserve University, 2085 Adelbert Rd., Cleveland, OH 4106. E-mail: wks3@pop.cwru.edu.
Published, JBC Papers in Press, November 11, 2000, DOI 10.1074/jbc.M007862200
2 M. Morillas, D. Vanik, and W. K. Surewicz, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PrP, prion protein; PrPC, cellular PrP isoform; PrPSc, scrapie (proteinase-resistant) PrP isoform; hu, human; GdnHCl, guanidine hydrochloride; DTT, dithiothreitol; ANS, 8-anilino-1-naphthalene sulfonic acid.
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