From the Departamento de Bioquímica
Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ
21941-590, Brazil and the ¶ Department of Anesthesia and the
Johnson Research Foundation, University of Pennsylvania, Philadelphia,
Pennsylvania 19104
Received for publication, October 20, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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The stability and equilibrium unfolding of
a model three-helix bundle protein, Understanding the mechanisms by which a polypeptide adopts a
stable and functional three-dimensional structure still represents a
challenging problem (1). The folding of small proteins usually takes
place on timescales close to a millisecond or less, and is believed to
occur in a highly cooperative fashion without the presence of populated
folding intermediates (2-8). However, recent simulation studies have
suggested the existence of intermediate states during the folding of a
small model three-helix bundle protein (9-12). Three-helix bundles
represent a simple folding motif found in a variety of soluble and
membrane proteins, including spectrin (13) and the extramembranous
portion of Staphylococcus aureus protein A (14). Using
sequence patterns discovered in coiled coils, the synthesis of
amphiphilic Recently, Johansson and co-workers (18) reported the synthesis and
initial characterization of a native-like three-helix bundle protein,
designated In the last two decades, hydrostatic pressure has been extensively used
as a reversible thermodynamic variable to characterize subunit
association in oligomeric proteins (21, 22). In general, unfolding of
monomeric proteins requires significantly higher pressures
(i.e., 5-7 kilobars (kbar)) than those required for subunit
dissociation of oligomers (typically up to 3.5 kbar) (21). Few examples
to date demonstrate denaturation of monomeric proteins at pressures
below 3 kbar. In the present study, we have used a combination of
hydrostatic pressure (up to 3.5 kbar) and low temperatures to
investigate the folding stability of Chemicals--
All reagents were of the highest analytical grade
available. Distilled water was filtered and deionized through a
Millipore water purification system. Bis-ANS was from Molecular Probes
(Eugene, OR).
Peptide--
The design, synthesis, and purification of
Fluorescence Measurements--
Unless otherwise indicated,
fluorescence emission spectra were measured at 25 °C on a
spectrofluorometer (PC 1, ISS Inc., Champaign, IL). For intrinsic
fluorescence measurements, excitation was at 280 nm and emission
spectra were recorded from 300 to 420 nm. Bis-ANS fluorescence was
measured with excitation at 375 nm and emission from 420 to 600 nm.
Fluorescence measurements under pressure were performed using a
pressure cell similar to that originally described by Paladini and
Weber (23), equipped with sapphire optical windows. The temperature of
the pressure cell was controlled by means of a jacket connected to a
circulating bath and was monitored by a telethermometer. All
experiments were carried out in 20 mM Tris-HCl, pH 7.4, containing 130 mM NaCl. Protein concentration in all
experiments was 2 µM (determined with a UV-visible
Ultraspec 2000 spectrometer (Amersham Pharmacia Biotech) using
Fluorescence spectral centers of mass (intensity-weighted average
emission wavelengths, Unfolding of
Fig. 2 shows the effect of pressure on
the fluorescence spectral center of mass of
The pressure unfolding data for Cold Denaturation of
At constant pressure the temperature dependence of the equilibrium
constant for a two-state unfolding transition is described by the
van't Hoff equation,
Bis-ANS Binding Studies--
The environment-sensitive fluorescent
dye bis-ANS was used to characterize different partially folded
conformations of Structural transitions of a single-chain 65-amino acid three-helix
bundle polypeptide, 3-1, by
guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature
have been investigated. The combined use of these denaturing agents
allowed detection of two partially folded states of
3-1,
as monitored by circular dichroism, intrinsic fluorescence emission,
and fluorescence of the hydrophobic probe bis-ANS
(4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid). The overall
free-energy change for complete unfolding of
3-1,
determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native
state is stabilized by
1.4 kcal/mol relative to a partially folded
pressure-denatured intermediate (I1). Cold
denaturation at high pressure gives rise to a second partially
(un)folded conformation (I2), suggesting a
significant contribution of hydrophobic interactions to the stability
of
3-1. The free energy of stabilization of the
native-like state relative to I2 is evaluated
to be
2.5 kcal/mol. Bis-ANS binding to the pressure- and
cold-denatured states indicates the existence of significant residual
hydrophobic structure in the partially (un)folded states of
3-1. The demonstration of folding intermediates of
3-1 lends experimental support to a number of recent
protein folding simulation studies of other three-helix bundle proteins
that predicted the existence of such intermediates. The results are
discussed in terms of the significance of de novo designed
proteins for protein folding studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices that self-assemble into three- or four-helix
bundles stabilized by a hydrophobic core has been successfully achieved
(15-17). The de novo design of proteins represents a
versatile tool to gain insight into the interplay of forces resulting
in conformational stability. Artificial proteins are generally less
complex than their native counterparts but at the same time retain the
features responsible for the folding process.
3-1. The three different helices of this
65-amino acid polypeptide are joined by (glycine)4 linkers. NMR solution studies revealed a well structured conformation with
-helical secondary structure.
GdnHCl1-induced unfolding of
3-1 followed by CD revealed a Gibbs free-energy of
unfolding of +4.6 kcal/mol (18), comparable to that observed for small
monomeric natural proteins of similar size, such as myoglobin (7.6 kcal/mol) (19) or the 43-amino acid residue peripheral subunit-binding
domain of the pyruvate dehydrogenase complex (3.1 kcal/mol) (20).
3-1. Interestingly, our results revealed the existence of partially (un)folded intermediate states of
3-1, giving support to the predictions from
the above mentioned simulation studies. Bis-ANS binding studies
revealed the existence of significant residual hydrophobic structure in the pressure-denatured and especially in the cold-denatured state of
3-1, suggesting molten globule-like conformations for
these intermediates.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-1 have been previously described (18). The amino acid
sequence of
3-1 is given below in single-letter codes.
The sequence is arranged in heptads, with the coiled-coil heptad
positions labeled a through g (Table
I). The N terminus of the peptide is acetylated, and the C terminus is amidated.
Heptad sequences
280 = 5700 M
1
cm
1), and bis-ANS concentration, when used, was 1.0 µM. All samples were deoxygenated by bubbling with a
stream of nitrogen for 5 min prior to the experiments. In low
temperature experiments, the windows of the pressure bomb were flushed
with nitrogen to prevent condensation.
av) were calculated with software provided by ISS Inc. as follows,
where
(Eq. 1)
is the emission wavelength and I(
)
represents the fluorescence intensity at wavelength
. Shifts in the
spectral center of mass were converted into extent of denaturation
(
p) at each pressure according to the following
phenomenological relationship (24):
where
(Eq. 2)
N and
U are the spectral
centers of mass of native-like and fully unfolded protein obtained in
the absence of denaturant and in the presence of a high concentration
of GdnHCl, respectively,
p is the spectral center of
mass at pressure p, and Q is the ratio of
fluorescence quantum yields of unfolded and native-like
3-1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-1 by GdnHCl and Hydrostatic
Pressure--
The equilibrium unfolding of
3-1 by
GdnHCl was initially investigated. Control measurements showed that
unfolding was very rapid and was complete within a few minutes after
addition of GdnHCl. Samples were incubated at increasing GdnHCl
concentrations for 2 h at room temperature, and intrinsic
fluorescence emission spectra were recorded. Unfolding was accompanied
by a significant red shift of the fluorescence emission of
3-1 (Fig. 1), indicating increased exposure of the single tryptophan residue (Trp-32) to the aqueous medium. Fig. 1 (inset) shows the degree of
denaturation (
) of
3-1 as a function of GdnHCl
concentration. For comparison, data on the unfolding of
3-1 monitored by far-UV CD measurements (18) have also
been included. A single, cooperative transition from the native to the
unfolded state was observed in both fluorescence and CD measurements.
The almost exact superimposition of the unfolding profiles revealed by
fluorescence and CD suggests that, in the presence of GdnHCl, the
transition between native and unfolded
3-1, at
atmospheric pressure and room temperature, is essentially a two-state
transition with no evidence for the existence of populated folding
intermediates. Fig. 1 also shows that unfolding of
3-1 takes place between 1 and 3 M GdnHCl, with a transition
mid-point at 2.4 M GdnHCl.
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Fig. 1.
GdnHCl unfolding of
3-1 followed by the red-shift in
intrinsic fluorescence emission (
) and CD spectroscopy (
).
CD data were taken from Ref. 18. The data shown represent means of four
independent experiments. The inset shows an overlay of the
degree of unfolding (
)for intrinsic fluorescence (
) and CD
measurements (
). Standard deviations in the measurements are smaller
than the symbols used.
3-1 in the
absence or in the presence of GdnHCl. In the absence of GdnHCl
(triangles) the center of mass did not reach a plateau even
at the highest pressure used (3.5 kbar), indicating that complete
unfolding was not achieved by the pressurization of
3-1.
From the equilibrium GdnHCl unfolding experiments (Fig. 1) it is
apparent that GdnHCl concentrations up to 1 M are
subdenaturing for
3-1. Pressure unfolding experiments were then repeated in the presence of different subdenaturing concentrations of GdnHCl (0.4 M and 1 M, Fig.
2) to poise the system toward unfolding. Although addition of 0.4 M GdnHCl had little effect on the pressure sensitivity of
3-1, pressure denaturation in the presence of 1 M GdnHCl exhibited a clearly defined plateau of the
spectral centers of mass at about 342 nm (Fig. 2). It is important to
note that fully unfolded
3-1 (i.e. in the
presence of 6 M GdnHCl) exhibited a very red-shifted
fluorescence emission, with a spectral center of mass of 355 nm. Thus,
the plateau observed for the spectral center of mass of the
pressure-denatured state at 342 nm seems to correspond to a stable
partially unfolded intermediate. Upon stepwise release of pressure, the
fluorescence spectra underwent a blue shift and reached complete
recovery of the spectral center of mass at atmospheric pressure (data
not shown), indicating reversible refolding of
3-1 to a
state qualitatively similar to the native-like protein. The
fluorescence changes thus indicate that application of pressure in the
presence of a subdenaturing concentration of GdnHCl (1 M)
induced a transition to a stable conformation different from the fully
denatured state induced by 6 M GdnHCl.
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Fig. 2.
Upper panel, pressure-induced spectral
shift of the intrinsic fluorescence emission of 3-1 in
the absence of GdnHCl (
), or in the presence of 0.4 M
GdnHCl (x) or 1.0 M GdnHCl (
). Lower panel,
plot of ln[
p/(1
p)] as a function of
pressure. The errors are smaller than the symbols used.
3-1 were analyzed using
a two-state model for monomer unfolding. The dimensionless equilibrium denaturation constant at atmospheric pressure
(K0) and the molar volume change of folding
(
V) can be calculated from the following thermodynamic relation,
where Kp is the denaturation constant at
pressure p, and R and T have their
usual meanings. The equation can be rewritten by introducing the degree
of unfolding,
(Eq. 3)
p, at pressure p:
where ln[
(Eq. 4)
p/(1
p)] equals
lnKp for the denaturation of a monomer. Thus, a plot
of ln[
p/(1
p)] versus
pressure (Fig. 2, lower panel) yields the molar volume
change of folding (
V) from the slope and
lnK0 from the intercept on the ordinate. The
parameters obtained for pressure unfolding of
3-1 are
shown in Table II.
Thermodynamic parameters of different (partially) denatured states of
3-1
3-1--
To further
characterize the existence of folding intermediates of
3-1, we carried out low temperature unfolding
experiments under pressure. The freezing point of water is
significantly decreased under pressure (25), allowing aqueous samples
to be analyzed at sub-zero temperatures without the need for addition
of cryosolvent additives. Fig. 3 shows
the fluorescence spectral centers of mass of
3-1 as a
function of decreasing temperature at 3.5 kbar in the absence and in
the presence of 1.0 M GdnHCl. The starting points of the
two curves (at 25 °C) are similar to the spectral centers of mass
obtained in the pressure denaturation experiments at the corresponding
GdnHCl concentrations. In the absence of GdnHCl (circles)
the spectra became progressively red-shifted but did not reach a
plateau at low temperatures (down to
12 °C), indicating that a
stable partially (un)folded intermediate had not been reached. By
contrast, in the presence of 1.0 M GdnHCl, a further red
shift of the fluorescence emission occurred, with a low temperature
plateau observed at about
10 °C. Interestingly, the fluorescence
spectra of the cold-denatured state and the completely unfolded protein
(i.e., in 6 M GdnHCl) differ by about 8 nm in spectral center of mass (Fig. 4). After
return of the sample to room temperature the fluorescence spectral
center of mass returned to the original value, reflecting the
reversibility of the process (Fig. 3, open symbols).
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Fig. 3.
Upper panel, cold denaturation of
3-1 under pressure (3.5 kbar) in the absence (
) or in
the presence of 1 M GdnHCl (
). During cooling, samples
were allowed to equilibrate for 20 min at each temperature prior to the
acquisition of the emission spectra. Open symbols correspond
to data acquired with increasing temperature, and show the
reversibility of the process. Lower panel, van't Hoff plot
for the cold denaturation of
3-1 at 3.5 kbar and 1 M GdnHCl. The degree of unfolding (
) at each temperature
was calculated using the values for spectral centers of mass of the
native and the fully unfolded states of
3-1. Gibbs
free-energy changes of unfolding were calculated from the
relationship:
Gunf =
RT ln[
/(1-
)], where R is the gas
constant and T is the absolute temperature. The errors are
smaller than the symbols used.
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Fig. 4.
Fluorescence emission spectra of native
(1), pressure-denatured (2),
cold-denatured (3), and fully unfolded
3-1 in the presence 7 M
GdnHCl (4). Pressure unfolding was obtained by
incubation with 1 M GdnHCl and increasing the pressure to
3.5 kbar and 25 °C (see Fig. 1). Cold denaturation was obtained by
applying pressure up to 3.5 kbar in the presence of 1 M
GdnHCl and decreasing the temperature to
10 °C (see Fig. 2).
Spectra are normalized for maximal emission intensity.
where KT is the equilibrium constant for
denaturation at temperature T and
(Eq. 5)
G
is the corresponding Gibbs free-energy change. From a plot of
G/T versus the inverse temperature,
the changes in enthalpy (
H) and entropy
(
S) of unfolding can be extracted (Fig. 3,
lower panel). The thermodynamic parameters obtained from
such analysis are summarized in Table II.
3-1. Bis-ANS tends to bind exposed
hydrophobic surfaces in partially folded intermediates more tightly
than both the native and random coil states of proteins (26). Bis-ANS
binding is accompanied by an increase in its fluorescence quantum
yield, as well as by a blue shift of the fluorescence emission. On the
basis of the increase in bis-ANS fluorescence, the pressure-denatured
state of
3-1 bound more bis-ANS than the native-like
state, and the cold-denatured state exhibited significantly stronger
binding (Fig. 5). In addition, bis-ANS
binding to the cold-denatured state was also accompanied by a 14-nm
blue shift of the fluorescence emission.
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Fig. 5.
Bis-ANS fluorescence emission spectra in the
presence of native (b), pressure-denatured
(c), cold-denatured (d), or
completely denatured (a) (in the presence of 6 M GdnHCl) 3-1.
Pressure denaturation was carried out in the presence of 1 M GdnHCl at 25 °C (see Fig. 1). Cold denaturation was
carried out at 3.5 kbar in the presence of 1 M GdnHCl and
decreasing the temperature to
10 °C (see Fig. 2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-1, induced by hydrostatic pressure and by a combination of low temperature and high pressure revealed the
existence of partially folded intermediate states, which are not
observable in GdnHCl unfolding experiments of this model protein. Bis-ANS binding studies support the idea that organized hydrophobic surfaces persist, or can form, at both high pressures and low temperatures. Taken into account the "new view" of protein
(un)folding, which models the chain collapse of a polypeptide by a
multiple pathways "funnel," our results suggest that one possible
unfolding transition of
3-1 can be summarized by the
following scheme,
where N is the native-like and U is the unfolded state, and
I1 and I2 represent the
two partially (un)folded intermediates revealed in high pressure and
low temperature experiments, respectively (Fig.
6).
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Fig. 6.
Free-energy diagram for the (un) folding
transitions of 3-1.
Pressure-induced changes in the intrinsic fluorescence emission
spectrum of 3-1 took place between atmospheric pressure
and 2.5 kbar in the presence of a subdenaturing concentration (1 M) of GdnHCl. The shift in spectral center of mass from 335 to 342 nm indicates partial exposure of the previously solvent-shielded tryptophan at the central heptad a position of helix II to
the aqueous environment. The single-chain polypeptide nature of
3-1 renders this native-like three-helix bundle of
particular interest for pressure unfolding studies. It is generally
assumed that pressures below 5 kbar do not significantly disturb the
secondary or tertiary structures of proteins (27). Hydrogen bonds, the stabilizing elements of helices and
-sheets, are permanent dipoles and relatively insensitive to pressure changes. Moreover, the volume
change attendant on replacement of protein-protein hydrogen bonds by
protein-water hydrogen bonds is rather small. Therefore, pressure-induced unfolding of small monomeric proteins is generally only observed at high temperature (28), at low pH (29, 30), or with
mutant proteins (31, 32). The finding that
3-1 can be
unfolded by pressure in the presence of a subdenaturing concentration of GdnHCl opens interesting possibilities for further studies of the
stability of helical bundles and, in particular, of the structure of
the pressure-stabilized partially folded state.
It is interesting to compare the volume change measured for the
unfolding of 3-1 with the volume changes reported for
pressure denaturation of other proteins. The specific volume changes
observed upon dissociation of dimeric proteins are dependent on the
molecular weight. Arc repressor (Mr 13,000), for
example, shows a specific volume change of
7.7 µl/g (33), whereas
Enolase (Mr 80,000) is reported to have a change
in volume of
0.7 µl/g (23). This can be explained by a larger
proportion of buried amino acid residues, which becomes exposed to the
solvent upon dissociation in smaller dimers, because in these cases the
subunit interfaces involve a larger fraction of the entire structure.
Alternatively, volume changes can also be interpreted in terms of the
balance of forces responsible for protein stability. Disruption of
electrostatic interactions leads to a large decrease in volume caused
by electrostriction of water around the unpaired charged residues
(34). By contrast, breaking of hydrophobic interactions is accompanied
by much smaller volume changes. The denaturation of monomeric proteins
is accompanied by similar effects, resulting in stronger hydration and
the replacement of longer dispersion bonds by shorter dipolar
interactions. Therefore, the relatively large specific volume change of
2.3 µl/g observed for the folding transition of the intermediate
I1 to the native-like state N of
3-1 (Table II) occurs most likely with the burial of
polar side-chain groups.
Cold denaturation experiments at high pressures take advantage of the
depression of the freezing point of water (25). Such an experimental
setup and the presence of a subdenaturing concentration of GdnHCl
allowed characterization of another folding intermediate, which showed
strong bis-ANS binding. Destabilization of proteins at low temperatures
indicates a significant contribution of hydrophobic interactions to the
folding process. Studies of the small dimeric protein Arc repressor
showed that folding and association are accompanied by the displacement
of solvent molecules, suggesting the burial of previously
solvent-exposed nonpolar side chains (35). According to Privalov (36),
hydration of polar residues decreases the entropy of the folding
process. On the other hand, Weber (37) described the entropy-driven
condensation of proteins as a consequence of the conversion of stronger
solvent-protein interactions into weaker (entropy-rich) protein-protein
interactions (London dispersion forces). In several cases,
protein-protein interactions involved in folding and subunit
association have indeed been found to be predominantly entropy-driven.
For example, the subunit association of hexokinase is characterized by
a strong entropic contribution (TS = +38
kcal/mol), which outweighs the unfavorable enthalpy of +17 kcal/mol
(38). The folding of
3-1 reveals an entropy-driven
transition from I2 to I1,
with T
S = +8.2 kcal/mol at 25 °C and a
van't Hoff enthalpy of
H = +7.1 kcal/mol, resulting in
1.1 kcal/mol of conformational stability (
G). The entropy-driven nature of the
I2
I1 transition
suggests that a hydrophobic collapse may be involved at this stage of
folding of
3-1.
The design of 3-1 contains six distinct hydrophobic core
layers, each consisting of three amino acids of either two a and one d or one a and two d heptad
positions of the corresponding helices I, II, and III. Very
likely, these areas are involved in formation of the organized
hydrophobic domains revealed by bis-ANS binding at low temperatures. Of
note is that the changes in intrinsic fluorescence emission of
3-1 induced by pressure and low temperature were fully
reversible, indicating that the protein refolds to a state that is
qualitatively similar to the native-like state upon return to
atmospheric conditions.
The folding of small (<100 amino acid residues), single-domain
proteins is assumed to occur in a concerted fashion well accounted for
by a two-state transition without well populated intermediates (39,
40). On the other hand, there is strong evidence, especially from
hydrogen exchange experiments, that partially folded conformations can
be present (41, 42). For a number of proteins, residual structure has
been detected and linked to partially folded states, which are believed
to be important as nucleation sites for condensation (43, 44). For
example, small patterns of stable residual structure were found in
barnase during acid denaturation and were assumed to be formed during
the early stage of folding (45). In addition, urea denaturation of the
chaperonin GroEL also revealed persistent hydrophobic surfaces
at high urea concentrations as probed by bis-ANS binding (46). Low
temperature unfolding studies of -lactamase provided evidence for
the existence of two equilibrium intermediates between native and
unfolded states (47).
Small synthetic helical proteins that undergo metal-directed
transitions from molten globule-like to native-like states via folding
intermediates have also been reported (48, 49). Thermodynamic calculations of a model three-helix bundle using either a simple off-lattice or an all-atom approach revealed the existence of a
metastable minimum (9-11). In addition, Zhou and Karplus (12) have
very recently used the same model protein to calculate different folding trajectories (12). The phase diagram could be varied by
changing a single parameter related to the relative stability of native
and non-native contacts. The simulation revealed that the folding
mechanism for helical proteins changes from a cooperative (diffusion-collision) transition to one that involves on-pathway intermediates depending on the difference between the strength of
native and non-native interactions (12). Our results on
3-1 give direct support to the idea that even small
helical bundle proteins may indeed present metastable folding
intermediates, which can be detected under appropriate experimental
conditions designed to stabilize them.
In conclusion, the present results, together with the small size of
this three-helix bundle protein, make 3-1 an ideally suited system for detailed protein folding studies. In this regard, an
interesting possibility could be the use of high pressure NMR studies
to characterize the structure of the folding intermediates.
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ACKNOWLEDGEMENTS |
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A. C. thanks Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro for a previous fellowship.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM55876 (to J. S. J.) and by a Howard Hughes Medical Institute International Research Scholar Award (to S. T. F.).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.
§ A visiting professor at the Universidade Federal do Rio de Janeiro.
To whom correspondence should be addressed. Tel.:
5521-270-5988; Fax: 5521-270-8647; E-mail:
ferreira@bioqmed.ufrj.br.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M009622200
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ABBREVIATIONS |
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The abbreviations used are: GdnHCl, guanidine hydrochloride; bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid; CD, circular dichroism; kbar, kilobars.
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