From the Groupe d'Ingénierie des Protéines (CNRS URA
1129), Unité de Biochimie Cellulaire, Institut Pasteur, 28 rue du
Docteur Roux, 75724 Paris Cedex 15, France
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INTRODUCTION |
The unfolding of proteins by denaturing agents under thermodynamic
equilibrium conditions is useful to characterize their unfolding
mechanism and to quantify their conformational stability. Most
quantitative studies of unfolding have been performed on soluble
monomeric proteins or on dimeric proteins that unfold according to a
two-state mechanism, i.e. without an intermediate state
between the native protein and the unfolded polypeptide (1). Very few
quantitative studies have dealt with dimeric proteins that unfold
through an intermediate, whether monomeric (2, 3) or dimeric (4, 5),
yet most cellular proteins contain several domains or subunits.
Moreover, many proteins of therapeutic interest are dimeric, and it is
important to be able to compare their stability with those of
engineered mutants.
The aminoacyl-tRNA synthetases are divided into two classes. The 10 enzymes of class I are characterized by the common fold of their
catalytic domain and the existence of two conserved sequence motifs,
involved in the binding of ATP. All of the aminoacyl-tRNA synthetases
of class I are monomeric, except for the tyrosyl- and tryptophanyl-tRNA
synthetases, which are dimeric and structurally very homologous (6-8).
Tyrosyl-tRNA synthetase
(TyrRS)1 catalyzes the
aminoacylation of tRNATyr in a two-step reaction. Tyrosine
is first activated with ATP to form tyrosyladenylate, and then this
intermediate is attacked by tRNATyr to form
tyrosyl-tRNATyr and AMP. TyrRS shows half-of-the-sites
reactivity since it binds only one molecule of tyrosine and one
molecule of tRNATyr per molecule of dimer (9, 10).
The structure of TyrRS from Bacillus stearothermophilus has
been determined at high resolution (11). Each subunit of TyrRS comprises two structural domains in the crystal structure, an N-terminal domain (residues 1-319) and a C-terminal domain (residues 320-419). The N-terminal domain contains the interface of dimerization and the binding sites for tyrosine, tyrosyladenylate, and the acceptor
arm of tRNATyr. Its isolated form (i.e. unlinked
to the other domain) is dimeric, forms tyrosyladenylate normally, but
does not bind tRNATyr (12). The integrated form and the
isolated form of the N-terminal domain have the same crystal structure
(11, 13). The C-terminal domain is disordered in the crystals of
full-length TyrRS. It is essential for the binding of
tRNATyr to TyrRS (12, 14).
We have chosen to study the N- and C-terminal domains of TyrRS
separately to characterize its unfolding mechanism and its stability.
In a previous study, we have shown that the isolated form of the
C-terminal domain, TyrRS(
3), has secondary structure, is compact,
and unfolds through a cooperative transition (15). In the present work,
we studied the unfolding of the isolated N-terminal domain,
TyrRS(
1), by urea under equilibrium conditions. We detected a
monomeric intermediate between the native dimeric state of the
N-terminal domain and its unfolded state and quantified the variation
of free energy and its dependence on the concentration of urea for each
of the corresponding conformational transitions. This work opens new
prospects for the study of TyrRS by a mutational approach. For example,
it will be possible to analyze the recognition between the subunits,
the transmission of information between the active sites across the
subunit interface, or the molecular bases for the hyperstability of
TyrRS from B. stearothermophilus. Moreover, the quantitative
thermodynamic analysis developed in this work should be applicable to
other dimeric or oligomeric proteins.
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MATERIALS AND METHODS |
Proteins and General Conditions--
TyrRS(
1) was
overexpressed from phage M13-BY(
1) and purified as described (15,
16). The purified TyrRS(
1) was >95% pure as judged by gel
electrophoresis. It was stored at
70 °C in 20 mM
Tris-HCl (pH 7.78) and 5 mM 2-mercaptoethanol. The
concentration of protein in the purified samples of TyrRS(
1) was
measured with the Bio-Rad protein assay kit using bovine serum albumin
as a standard. The molecular mass of TyrRS(
1) was taken as 36,324 Da/subunit (12). Ultrapure urea was purchased from ICN and used directly as provided. All the experiments were performed at 25 °C. The reactions of unfolding and refolding of TyrRS(
1) in the presence of urea were performed and brought to equilibrium as described (15). In
particular, all the measurements were done after a prolonged incubation
of the reaction mixtures at 25 °C, between 12 and 16 h, a time
after which the equilibrium between the different conformational states
of TyrRS(
1) is reached (15). The concentration of urea in the
reaction mixtures was measured with a refractometer and a precision of
0.01 M after the completion of each experiment.
Intrinsic Fluorescence Experiments--
The samples were excited
at 278 nm; the slit width was equal to 2.5 nm for the excitation light
and 5 nm for the emission. The spectra of fluorescence intensity were
recorded between 310 and 380 nm with a Quanta Master spectrofluorometer
(Photon Technology International). The signal was acquired for 1 s
at each wavelength, and the increment of wavelength was equal to 1 nm.
The intensities of fluorescence at 330 nm were measured with a
Perkin-Elmer LS-5B spectrofluorometer. The signal was averaged during
17 s. The fluorescence signal for the protein was corrected by
substraction of the signal for the solvent alone.
Circular Dichroism Experiments--
Far-UV CD experiments were
performed with a Jobin-Yvon CD6 apparatus. The spectra were recorded
between 210 and 250 nm with a 0.2-cm path length cell when the
concentration of TyrRS(
1) was equal to 100 µg/ml and between 225 and 250 nm with a 1.0-cm path length cell when its concentration was 20 µg/ml. The signal was acquired for 1 s at each wavelength, and
the increment of wavelength was equal to 1 nm. The signal for the
protein was corrected by substraction of the signal for the solvent
alone.
Size-exclusion Chromatography--
The hydrodynamic properties
of TyrRS(
1) and their variations with the concentration of urea were
measured by size-exclusion chromatography through a Superdex 200 HR
10/30 column connected to a fast protein liquid chromatography system
(Amersham Pharmacia Biotech). The effluent was continuously monitored
at A280 nm. Before each chromatographic run,
the column was equilibrated with more than 4 column volumes of elution
buffer. An aliquot (100 µl) of either an unfolding reaction or a
refolding reaction, containing 100 µg/ml TyrRS(
1), was injected at
the top of the column. The column was eluted with the same buffer that
was used in the unfolding or refolding reaction. The measures were
expressed using the partition coefficient Kav = (Ve
VO)/(Vt
V0), where Ve is the elution
volume corresponding to the maximum of the protein elution peak and
Vt and V0 are the total volume and the void volume of the column, respectively. The relation between the Stokes radius (RS) and the rate of
elution (1000/Ve) was established with the proteins
in the calibration kits from Amersham Pharmacia Biotech. Blue dextran
200 and acetone were used to measure V0 and
Vt, respectively. All the chromatographic runs were
performed at a flow rate of 0.3 ml/min and at room temperature
(22-26 °C).
Pyrophosphate Exchange--
The active-site titration and the
pyrophosphate exchange assay were performed essentially as described
(16, 17) with the following modifications. TyrRS(
1) (0.40 µM active sites) was first unfolded by different
concentrations of urea as described above. The pyrophosphate exchange
reaction was then started by diluting four times the unfolded enzyme in
a reaction mixture containing the various substrates
([32P]pyrophosphate, ATP, and tyrosine) and the same
concentration of urea as in the unfolding reaction.
ANS Binding--
A stock solution of ANS (either 28 or 280 mM in methanol) was prepared, and its concentration was
determined using a molar extinction coefficient equal to 6.8 × 103 M
1·cm
1 at 370 nm in methanol (18). ANS (0.5 µl, either 14 or 140 µM final concentration) was added to a pre-equilibrated unfolding reaction
of TyrRS(
1) (1 ml, 100 µg/ml = 0.1 µM protein)
or to a control reaction without protein. The mixture was incubated for
1 h, and then the fluorescence spectrum of ANS was recorded between 400 and 600 nm, with excitation at 380 nm.
Analysis of the Unfolding Profiles--
The profiles of
unfolding of TyrRS(
1) by urea, monitored by its fluorescence
intensity or its CD ellipticity, were analyzed with the thermodynamic
models described in Table I. Equation 13 was fitted to the unfolding
profiles with the software pro Fit 5.0 for Macintosh (Cherwell
Scientific Publishing Ltd., Oxford, United Kingdom) and 106
iterations of the Monte Carlo algorithm, followed by the
Levenberg-Marquardt algorithm. The error on the concentration of urea
(x) was set up to zero, and the error on the signal
(Y) was set up to unknown for both algorithms. The fitting
range for the Monte Carlo algorithm was set up to the default one,
i.e. ±10% of the starting values of the parameters.
However, the autosearch option was activated so that the limits of the
parameters were adapted during the fit. Care was taken to reset these
limits to ±10% of the starting values before each new fit. The values
of the molar fractions fn, fi,
and fu (Equations 10-12) and of their roots were
calculated with the same software.
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RESULTS |
Unfolding Monitored by Fluorescence Intensity--
The sequence of
TyrRS(
1) contains six tryptophan residues (at positions 9, 97, 126, 196, 240, and 255) and 11 tyrosine residues. Several tryptophan
residues are buried inside the protein, according to its crystal
structure (13). We recorded the fluorescence emission spectra of
TyrRS(
1) in 0 and 8 M urea at an excitation wavelength
of 278 nm. The spectrum of unfolded TyrRS(
1) had a
max at 349 nm, a value similar to that of tryptophan as
a free amino acid. The
max of native TyrRS(
1) was
blue-shifted to 341 nm, and this shift was accompanied by an increase
in intensity. The blue shift and the increase in intensity confirmed
that some of the tryptophan side chains were buried in the hydrophobic
interior of the native protein. The difference in fluorescence
intensity between the native and unfolded states of TyrRS(
1) was
maximal at an emission wavelength of 330 nm. This difference was larger when the excitation wavelength was equal to 278 nm than when it was 295 nm, as expected since both tyrosine and tryptophan are excited at 278 nm, whereas only tryptophan is excited at 295 nm. We therefore used the
intensity of fluorescence emission at 330 nm, upon excitation at 278 nm, to monitor the unfolding of TyrRS(
1) by urea under equilibrium
conditions. Fig. 1 shows the unfolding profile of TyrRS(
1) at a concentration of 10 µg/ml. The
fluorescence intensity remained roughly constant at low concentrations
of urea, decreased non-linearly between 2 and 6 M urea, and
then increased linearly at high concentrations of urea.

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Fig. 1.
Unfolding of TyrRS( 1) by urea, as
monitored by its fluorescence intensity (excitation at 278 nm and
emission at 330 nm). The concentration of TyrRS( 1) was 10 µg/ml. The corrected signal (Equation 6 in Table I) is given along
the y axis. The solid line
was obtained by fitting Equation 13 (mechanism with a monomeric
intermediate) in Table I to the experimental data.
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The molar fractions of the different conformational states depend on
the total concentration of protein for a dimer (Table I). In contrast, the equilibrium
constants K, K1, and
K2 between these states and the associated free
energies
G,
G1, and
G2 do not depend on this concentration and
should remain constant when it varies. We used this invariability of
the thermodynamic parameters as a criterion to characterize the
mechanism through which TyrRS(
1) unfolds. We recorded its profile of
unfolding by urea, monitored by its intensity of fluorescence, at
different concentrations of protein, 10 µg/ml (138 nM), 5 µg/ml (69 nM), and 2.5 µg/ml (34 nM). Each
profile was recorded three times in independent experiments. We fitted
Equation 13, which links the global intensity of fluorescence and the
concentration of urea, to the unfolding profiles for each of the three
mechanisms in Table I, i.e. without an intermediate state
between the native and unfolded states, with a monomeric intermediate,
or with a dimeric intermediate. The thermodynamic parameters
corresponding to these fittings varied with the total concentration of
protein for the unfolding mechanism without an intermediate state or
with a dimeric intermediate. In contrast, they remained constant,
within experimental error, for the mechanism with a monomeric
intermediate (Table II). Thus, the values
of the thermodynamic parameters and their comparison at different
protein concentrations showed that TyrRS(
1) unfolded through a
monomeric intermediate.
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Table I
Equations for the equilibrium unfolding of dimeric proteins, as
monitored by spectrometry
The abbreviations used are: N2, native dimeric
state; J2, dimeric intermediate state; I,
monomeric intermediate state; U, unfolded state;
C, total concentration of protein (M), expressed
as dimer equivalent; x, concentration of denaturant
(M); Y', measured global signal; Y,
corrected signal; Yn, Yi,
Yj, and Yu, molar signals of the
various protein states; Yd, molar signal of the
denaturant; T, temperature (K); R, gas constant.
The other parameters are defined below. The final fitting equation was
obtained by replacing Yn, Yi,
Yj, Yu, fn,
fi, fj, and fu
with their expressions as a function of x in Equation 13. Equations 7 and 8 have already been discussed (31, 34).
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Table II
Comparison of different mechanisms for the unfolding of TyrRS( 1)
The notations are the same as described for Table I. The unfolding of
TyrRS( 1) was monitored by its fluorescence intensity, as described
in the legend to Fig. 1. The thermodynamic parameters listed in the
first column were obtained by fitting equation 13 in Table I to the
experimental data. Each entry corresponds to the mean ± S.E. of
three independent experiments.
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We recorded the statistical parameter
2, which measures
the agreement between the function and the experimental values, for each experimental unfolding profile and each mechanism in Table I. We
found that the relative values of
2 were equal to
2.4 ± 0.3, 1.3 ± 0.1, and 1.0 (mean ± S.E. in nine independent experiments) for the mechanisms without an intermediate, with a monomeric intermediate, and with a dimeric intermediate, respectively. Thus, the equation corresponding to the mechanism with a
dimeric intermediate gave the best formal fit to the experimental data,
even though it did not satisfy the thermodynamic criteria described
above. To reconcile the conclusions obtained with the thermodynamic and
fitting criteria, we established the equation describing the mechanism
with both a dimeric intermediate and a monomeric intermediate. This
equation (not shown) comprised a total of 11 fitting parameters. The
S.D. values that were associated with these parameters during the
fitting process were very large, which indicated that the parameters
were too numerous to be accurately determined by the fitting
process.
Characteristic Parameters of Unfolding Monitored by Fluorescence
Intensity--
We calculated the molar fractions
fn, fi, and fu
of the different conformational states of TyrRS(
1) from the
thermodynamic parameters for an unfolding mechanism with a monomeric
intermediate. We deduced several characteristic concentrations of urea
from these molar fractions: fn
1(0.5),
at which half of the TyrRS(
1) molecules were dissociated; fi
1(max fi), at
which the concentration of monomeric intermediate was maximal; and
fu
1(0.5), at which half of the
molecules were unfolded. Table III gives
the average values of these characteristic concentrations for the
experiments performed at 10, 5, and 2.5 µg/ml. These values showed an
effect of the total concentration of protein on the molar fractions of
the different conformational states, as expected for the unfolding of a
dimeric protein.
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Table III
Characteristic concentrations of urea for the unfolding of
TyrRS( 1), as monitored by its fluorescence intensity
The notations are the same as described for Table I. The abbreviations
used are: max fi, maximal value of
fi; fi 1(max
fi), concentration of urea at which
fi is maximal; fn 1(0.5)
and fu 1(0.5), concentrations of urea
(x) at which fn(x) = 0.5 and
fu(x) = 0.5, respectively. Thermodynamic
parameters were obtained by fitting Equation 13 (mechanism with a
monomeric intermediate) in Table I to the experimental data. The values
of the molar fractions fn, fi,
and fu and of their roots were calculated from these
thermodynamic parameters using Equations 10-12. Each entry corresponds
to the mean ± S.E. of three independent experiments.
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Table IV gives the global average values
of the thermodynamic parameters, calculated from the results of nine
experiments performed at three different protein concentrations. The
unfolding reaction is characterized by four thermodynamic parameters:
the free energies for the dissociation of the native dimer and for the
unfolding of the monomeric intermediate in the absence of urea
(
G1(H2O) and
G2(H2O), respectively) and their
coefficients of dependence on the concentration of urea
(m1 and m2,
respectively). The values of the equilibrium constants
K1(H2O) and
K2(H2O), calculated from them by
Equation 8 in Table I, were 84 pM and 68 × 10
12, respectively. The values of the total parameters
G(H2O) and m, calculated by
Equation 14 in Table I, were 41 ± 1 kcal·mol
1 and
5.9 ± 0.2 kcal·mol
1·M
1,
respectively.
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Table IV
Mean parameters for the unfolding of TyrRS( 1), as monitored by
its fluorescence intensity
The notations are the same as described for Table I. The parameters
were obtained by fitting Equation 13 (mechanism with a monomeric
intermediate) in Table I to the experimental data. The molar signals of
the different conformational states, Yn = yn + mnx and
Yi and Yu = yu + mux, where x = [urea], were
expressed as fractions of the molar signal of the native protein in the
absence of urea, yn. Each entry corresponds to the
mean ± S.E. of nine independent experiments at three different
concentrations of TyrRS( 1): 2.5, 5.0, and 10.0 µg/ml.
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Whereas the thermodynamic parameters
G1,
G2, m1, and
m2 are characteristic of the studied molecule
and can be directly compared between experiments, the molar signals of
the different conformational states (Yn,
Yi, and Yu) depend on the experimental setting. However, they can be compared between experiments if they are expressed as fractions of the molar signal of the native
protein in the absence of urea (yn) provided that the native protein is the only conformational state under these conditions. Table IV gives the average values of these molar signals, calculated from the results of the same nine independent experiments. The molar fluorescence of the monomeric intermediate in 0 M
urea was equal to 9.8% of its value for the dimeric protein and
therefore to 19.6% of its value for one subunit of the native protein.
The molar fluorescence of the unfolded monomer in 0 M urea
was equal to 15.2% of its value for one subunit of the native protein
and to 78% of its value for the monomeric intermediate.
Fig. 2 gives the molar fractions
fn, fi, and fu
of the different conformational states of TyrRS(
1) as functions of
the concentration of urea, at the three protein concentrations used
experimentally. These fractions were calculated from the global average
values of the thermodynamic parameters given in Table IV.

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Fig. 2.
Fraction of each TyrRS( 1) state as a
function of urea concentration in the unfolding process. The
fractions fn, fi, and
fu of TyrRS( 1) in the native, monomeric, and
unfolded states were calculated using Equations 10-12 in Table I, the
mean thermodynamic parameters in Table IV, and concentrations of
TyrRS( 1) equal to 2.5, 5.0, and 10 µg/ml. See also Table
III.
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Unfolding Monitored by Circular Dichroism--
We used the
circular dichroism of TyrRS(
1) in the absorption band of the peptide
bond (from 200 to 230 nm) to monitor the variation of its content in
secondary structure during its unfolding by urea. We first recorded the
CD spectra of TyrRS(
1) in 0 and 8 M urea. Unfolded
TyrRS(
1) showed no ellipticity for wavelengths >212 nm. In
contrast, native TyrRS(
1) showed a broad peak of negative
ellipticity, around 222 nm, consistent with its high helical content in
the crystal structure (13). We used higher total concentrations of
TyrRS(
1) in the CD experiments (20 µg/ml (0.28 µM)
and 100 µg/ml (1.4 µM)) than in the fluorescence
experiments for sensitivity reasons. We chose a wavelength equal to 229 nm to monitor quantitatively the unfolding of TyrRS(
1) for the same reason. Fig. 3 shows the unfolding
profile of TyrRS(
1) at 100 µg/ml, monitored by CD at 229 nm under
equilibrium conditions. The ellipticity of TyrRS(
1) decreased slowly
and linearly between 0 and 4.5 M urea, increased sharply
and non-linearly between 4.5 and 6 M urea, and then
increased slowly and linearly between 6 and 8.5 M urea.

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Fig. 3.
Unfolding of TyrRS( 1) by urea, as
monitored by its CD ellipticity at 229 nm. The concentration of
TyrRS( 1) was equal to 100 µg/ml. The corrected signal (Equation 6 in Table I) is given along the y axis. The
solid line was obtained by fitting Equation 13 (mechanism with a monomeric intermediate) in Table I to the
experimental data. mdeg, millidegrees.
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Because the ellipticity of molecules can be considered as an additive
property, we could fit the equations in Table I to the unfolding
profiles of TyrRS(
1) monitored by CD. Table
V gives the corresponding thermodynamic
parameters and characteristic concentrations of urea and compares them
with the results of the fluorescence experiments. We first tried the
mechanism with a monomeric intermediate. When the concentration of
TyrRS(
1) was equal to 20 µg/ml, the thermodynamic parameters
deduced from the CD experiments were close to those predicted from the
fluorescence experiments. When TyrRS(
1) was at 100 µg/ml, the S.D.
values that were associated with the values of
m1 and
G1(H2O) during the fitting
process were high. Moreover, the characteristic concentrations of urea
deduced from the CD experiments, in particular
fn
1(0.5), varied less with the
concentration of TyrRS(
1) than predicted from the fluorescence
experiments. We also tried the mechanism without an intermediate. The
values of m and
G(H2O) deduced
from the CD experiments and the mechanism without an intermediate were close to the values of the total m and
G(H2O) parameters deduced from the
fluorescence experiments and the mechanism with a monomeric intermediate, 5.9 ± 0.2 kcal·mol
1·M
1 and 41 ± 1 kcal·mol
1, respectively, especially at higher
concentrations of TyrRS(
1). However, the urea concentration of
half-transition, fn
1(0.5) = 5.60 M, deduced from the CD experiments did not vary with the
concentration of TyrRS(
1). These results showed that the CD signal
was less sensitive to the dissociation of the native dimer than the
fluorescence signal, but was as sensitive to the unfolding of the
monomeric intermediate. They indicated that the dissociation of the
subunits did not lead to a large change of the content in secondary
structure.
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Table V
Comparison of the characteristic parameters for the unfolding of
TyrRS( 1) by urea, as monitored by its CD ellipticity and its
fluorescence intensity
The notations are as described for Tables I and III. The values of the
parameters in the second and third columns correspond to the fitting of
Equation 13 (mechanism without intermediate) to the CD data. Those in
the fourth and fifth columns correspond to the fitting of Equation 13 (mechanism with a monomeric intermediate). The values of the four
thermodynamic parameters in the sixth and seventh columns were taken
from Table IV. The values of the molar fractions fn,
fi, and fu and of their roots
were calculated from the thermodynamic parameters in the first four
rows using Equations 10-12 and TyrRS( 1) concentrations of 20 and
100 µg/ml.
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Unfolding Monitored by Fast Size-exclusion
Chromatography--
Thorough studies with monomeric proteins have
shown that fast size-exclusion chromatography (fast-SEC) can be used to
monitor the unfolding of proteins by denaturants and to measure the
molecular dimensions of their different conformational states. Fast-SEC is an inert technique, and it does not perturb the thermodynamic equilibrium between the different states of a protein (19, 20). In
particular, the V0 and Vt of
a Superose 12 column (Amersham Pharmacia Biotech) are practically
independent of the concentration of denaturant, and a unique linear
relation, valid for all the concentrations of denaturant, links the
RS of a polypeptide and its migration rate
(1000/Ve) through this type of column (20).
We used fast-SEC on a Superdex 200 HR 10/30 column to monitor the
unfolding of TyrRS(
1). We found that the values of
V0 (6.88 ml) and Vt (19.86 ml), measured with blue dextran 200 and acetone, did not vary with the
concentration of urea in the elution buffer and that a linear relation
linked RS and 1000/Ve in 0 M urea with a correlation coefficient of 0.99:
RS = (1.03 ± 0.8)·(1000/Ve)
(49.46 ± 7.24). We
injected 100 µl of either an unfolding reaction or a refolding
reaction, containing TyrRS(
1) at a concentration of 100 µg/ml, at
the top of the column and observed that the protein eluted from the
bottom of the column as a single chromatography peak, whatever the
concentration of urea. This peak was narrow at low (0-3 M)
and high (6.25-8 M) concentrations of urea. It was less
narrow and slightly trailed toward the high values of Ve at intermediate concentrations of urea (4-6
M).
We used the partition coefficient Kav = (Ve
V0)/(Vt
V0) of TyrRS(
1) to monitor its unfolding by
urea (Fig. 4). The linear decrease in
Kav between 0 and 4 M urea and then
between 6.5 and 8 M urea suggested that the dimeric and
unfolded forms of TyrRS(
1) swelled when the concentration of urea
was increased. These swellings corresponded to increases in the value of RS for the dimeric protein from 32.5 Å in 0 M urea to 36.7 Å in 4 M urea and for the
unfolded polypeptide from 43.7 Å in 6.5 M urea to 46.1 Å in 8 M urea. Such swellings have already been observed. One
assumes that they are due to a massive penetration of the folded
protein by molecules of urea, compatible with crystallographic data,
and to the destruction of residual structures in the unfolded polypeptides (19, 21-23). The RS value for the
native dimer (32.5 Å in 0 M urea) was compatible with the
value that can be predicted from its molecular mass (34.7 Å) and with
the dimensions of the crystal structure (34 × 40 × 117 Å3; (3V/4
)1/3 = 33.6 Å, where
V is the x-ray volume) (13, 20). The RS value for the unfolded polypeptide (46.1 Å in 8 M urea)
was significantly lower than the predicted value (54 Å) (20).

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Fig. 4.
Unfolding of TyrRS( 1) by urea, as
monitored by its partition coefficient Kav in
fast size-exclusion chromatography experiments. The injected
sample (100 µl) contained TyrRS( 1) at a concentration of 100 µg/ml. The solid line was obtained by fitting
Equation 13' (see below; corresponding to a mechanism of unfolding
through a monomeric intermediate) to the experimental data. The
measured signal Y = Kav was
equal to the weighted average of the specific signals for the different
conformational states of TyrRS( 1) (35) so that Equations 5 and 6 were replaced by Equation 5',
Y([N2] + [I] + [U]) = Yn[N2] + Yi[I] + Yu[U], and Equation 13 by Equation 13',
Y = (Yn + (2Yi Yn + K2(2Yu Yn))fi)/(1 + (1 + K2)fi). The closed
circles correspond to unfolding experiments and were the
only ones to be taken into account for the fitting. The open
circles overlap with the closed
circles and correspond to refolding experiments.
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The sharp increase in Kav between 4.5 and 5.5 M urea, up to a value equal to that of the native dimer in
0 M urea, showed that the dimer of TyrRS(
1) dissociated
cooperatively into a compact monomeric intermediate. Its sharp decrease
between 5.5 and 6.5 M urea showed that this monomeric
intermediate unfolded cooperatively.
Unfolding Monitored by the Average Exposure of the Tryptophan
Residues--
The tryptophan residues have a maximal emission of
fluorescence around 330 nm in an apolar solvent and 350 nm in water.
Therefore, the ratio
Y330/Y350, between the
fluorescence intensities of a protein at 330 nm and 350 nm, measures
the average environment of its tryptophans, which can be buried in its
apolar interior or exposed to the solvent at its surface. We used the
ratio Y330/Y350 to
monitor the unfolding of TyrRS(
1) (at a total concentration of 10 µg/ml) by urea under equilibrium conditions. This ratio increased
slowly and linearly between 0 and 4.5 M urea, decreased sharply between 5 and 6 M urea, and then increased slowly
and linearly between 7 and 9 M urea (Fig.
5). Because the ratio
Y330/Y350 is not an
additive property of the molecules, we could not apply the equations in
Table I. We therefore fitted a sigmoidal function to the unfolding
profile. Remarkably, the urea concentration of half-transition
b = 5.67 ± 0.02 M and the
cooperativity coefficient a = (3.0 ± 0.2)/RT for this sigmoid (where R is the gas
constant in kcal·mol
1·K
1 and
T = 298.16 K) (Fig. 5) were close to the parameters
fu
1(0.5) and m2
found in the fluorescence experiments above, equal to 5.9 ± 0.2 M and 2.6 ± 0.2 kcal·mol
1·M
1, respectively
(Tables II and III). These results showed that the tryptophan residues
of TyrRS(
1) became exposed to the solvent mainly during the
unfolding of its monomeric intermediate. We found that
Y330/Y350 measured more
precisely the average environment of the tryptophan residues during the
unfolding of TyrRS(
1) than its
max of emission (data
not shown).

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Fig. 5.
Unfolding of TyrRS( 1) by urea, as
monitored by the ratio
Y330/Y350 of
its fluorescence intensities at 330 and 350 nm.
Y330 and Y350 are the
corrected signals of fluorescence intensity (Equation 6 in Table I)
upon excitation at 278 nm. The concentration of TyrRS( 1) was equal
to 10 µg/ml. The signal Y = Y330/Y350 was assumed to
vary linearly with the concentration of urea in the pre- and
post-transition regions (Equation 7 in Table I). The solid
line corresponds to a fit of a simple sigmoidal function to
the experimental data: Y = Yu + (Yn Yu)·(1 1/(1 + exp(a(b x)))).
|
|
Unfolding Monitored by Activity--
The truncated form of
tyrosyl-tRNA synthetase, TyrRS(
1), is fully active for the reactions
of tyrosyladenylate formation and pyrophosphate exchange (12). We used
the latter reaction to monitor the inactivation of TyrRS(
1) by urea.
The total concentration of TyrRS(
1) in the reaction was 0.1 µM. The initial rate of pyrophosphate exchange decreased
sharply when the concentration of urea increased, with
half-inactivation around 0.5 M urea (Fig.
6). We calculated from the results of the
fluorescence experiments that, at this concentration of TyrRS(
1),
98% of the TyrRS(
1) molecules were dimeric in 0.5 M
urea and that the urea concentration of half-dissociation should be
equal to 5.0 M. Therefore, the results showed that the dimer of TyrRS(
1) was inactivated much before it dissociated into
monomers when the concentration of urea increased. This inactivation could be due to a local conformational change of TyrRS(
1), to a
weakening of the noncovalent interactions between the enzyme and its
substrates by urea, or to a competition between urea and the substrates
for binding to the enzyme.

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Fig. 6.
Rate of pyrophosphate exchange by TyrRS( 1)
(0.1 µM) as a function of the urea concentration.
The solid line corresponds to a fit of an
exponential function to the data: Vmax = a + b·exp( c·x).
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ANS Binding--
Some proteins unfold through an intermediate
conformational state that binds ANS more than the native state (24).
ANS is a hydrophobic dye that fluoresces little in water, where its
quantum yield is equal to 0.0091 and its maximum of emission occurs at 516 nm, much more in apolar solvents like dioxane, where its quantum yield is 0.68 and its maximum of emission occurs at 432 nm (25). We
measured the binding of ANS by TyrRS(
1) in the presence of urea
under equilibrium conditions. We calculated that the concentration of
monomeric intermediate should be equal to 0.32 µM in 4.5 M urea, 0.59 µM in 5.5 M urea,
and negligible in 0 and 8 M urea at the total concentration
of TyrRS(
1) dimer used (1.4 µM) according to the
results of the fluorescence experiments. ANS was in either 10- or
100-fold molar excess over TyrRS(
1) and excited at 380 nm. Similar
conditions have been used to show the binding of ANS to the molten
globular state of other proteins (24). We compared the emission
spectrum of the mixture of ANS and TyrRS(
1) with the spectrum of ANS
alone by calculating the ratio of or the difference in the fluorescence
intensities under the two conditions. For ANS at 14 µM,
the ratio of the intensities was at most equal to 1.32 in 0 M urea, 1.08 in 4.5 M urea, 1.10 in 5.5 M urea, and 0.98 in 8 M urea. The difference in
the intensities was maximal at 502 nm in 0 M urea, 501 nm
in 4.5 M urea, and 484 nm in 5.5 M urea. For
ANS at 140 µM, the difference in the fluorescence intensities was within the background signal. We concluded that TyrRS(
1) did not bind ANS, whatever its conformational state, native
dimer, monomeric intermediate, or unfolded protein.
 |
DISCUSSION |
Experimental Conditions of the Unfolding Reaction--
We used
various experimental signals to monitor the unfolding of the
TyrRS(
1) dimer by urea. We allowed the unfolding reactions to reach
equilibrium before performing any measurements (see "Materials and
Methods"). In the spectrofluorometry, circular dichroism, and ANS
binding experiments, this equilibrium was not perturbed by the
measurement. In the fast size-exclusion chromatography experiments, it
was perturbed by a non-uniform and progressive dilution of the protein
during the run. However, the elution of the protein from the column as
a single chromatography peak showed that the exchange between the
different conformational states of TyrRS(
1) was fast when compared
with the length of the run. Therefore, the different states of
TyrRS(
1) were in quasi-equilibrium.
The unfolding of TyrRS(
1) by urea is reversible, according to
several criteria. The unfolding and refolding profiles of TyrRS(
1) in spectrofluorometry, urea gradient gel electrophoresis (15), and
fast-SEC (Fig. 4) experiments were identical. We did not observe soluble aggregates when we analyzed the content of the refolding reactions by fast-SEC (see "Results"). The unfolding of TyrRS(
1) in 8 M urea and then its refolding do not cause a
significant change in the kinetic parameters Km for
tyrosine, Km for ATP, and
kcat in the pyrophosphate exchange reaction.
This lack of change indicates that the reversible unfolding of
TyrRS(
1) by urea has no apparent effect on its functioning (26).
Stability and Energetics of Unfolding--
TyrRS(
1) unfolded
according to a three-state mechanism at low concentrations in protein
and to a two-state mechanism at high concentrations. We found close
values for the total free energy of unfolding under the different
experimental conditions, as expected (Table V). The high value of
G(H2O) (41 ± 1 kcal·mol
1) is compatible with the thermophilic origin
of TyrRS(
1). It makes TyrRS(
1) one of the two most stable dimeric
enzymes, with organophosphorus hydrolase as the other one (5). Neet and
Timm have shown the existence of a rough linear correlation between the
number of amino acid residues (N) in the monomer and the
value of
G(H2O) for a collection of dimeric
proteins that unfold according to a two-state mechanism (1). The
measured value of
G(H2O) for TyrRS(
1)
(41 ± 1 kcal·mol
1) was in reasonable agreement
with the predicted value (34.4 kcal·mol
1). The measured
value of
G2(H2O) for the
unfolding of the monomeric intermediate (13.9 ± 0.6 kcal·mol
1) was compatible with the free energy of
unfolding for soluble globular monomeric proteins (27). The values of
G1(H2O) and
G2(H2O), and thus those of
K1(H2O) and
K2(H2O), were close (Table IV).
Thus, about one-third of the global energy of stabilization for
TyrRS(
1) came from the association between the two subunits, and
one-third came from the secondary and tertiary interactions stabilizing
each of the two molecules of the monomeric intermediate. The closeness
of the
G1(H2O) and
G2(H2O) values made possible the
observation of a monomeric intermediate of TyrRS(
1) in the presence
of urea, contrary to other dimeric proteins for which
G2
G1
(i.e. K2
K1). Up to 21% of the subunits were in this intermediate state for a total concentration of dimer equal to 100 µg/ml and up to 72% for a total concentration equal to 2.5 µg/ml
(Tables III and V).
The amount of tyrosyl-tRNA synthetase in an E. coli cell is
~1400-2000 molecules (28, 29). One molecule/E. coli cell, which has a mean volume of 1.15 fl, corresponds to a molarity equal to
1.4 nM. Therefore, the calculation of
fn(0) with the thermodynamic parameters in Table IV
shows that 99.7% of the TyrRS molecules should be dimeric in the cell
at 25 °C. The pyrophosphate exchange and tRNATyr
charging reactions are usually performed in vitro at
25 °C at concentrations of enzyme above 100 and 0.5 nM,
respectively (16, 17). Our results show that most of the enzyme
molecules should be dimeric at these total concentrations (99% of the
molecules at 100 nM and 82% at 0.5 nM).
Moreover, high concentrations of tyrosine and ATP favor dimerization
(30).
Cooperativity of Unfolding--
Myers et al. (31) have
studied the relationship between the denaturant m value,
defined by Equation 8 in Table I, and the variation of accessible
surface area (
ASA) between the folded and unfolded states for a
series of 45 monomeric and dimeric proteins with known crystal
structures. The value of
ASA is strongly correlated with the number
of residues in a protein and with the value of m. The
application of these relationships to the dimer of TyrRS(
1) (which
comprises 2 × 320 residues) and to its monomeric intermediate (320 residues) gave predicted values of m and
m2. The predicted and measured values of
m (6.8 and 5.9 ± 0.2 kcal·mol
1·M
1, respectively)
were in reasonable agreement if one notes that the above correlations
were established with proteins that were shorter than TyrRS(
1)
(
415 residues), mainly monomeric, and unfolded according to a
two-state mechanism. Comparison of the predicted and measured values of
m2 (3.5 and 2.5 ± 0.1 kcal·mol
1·M
1, respectively)
(Table IV) showed that the unfolding of the monomeric intermediate
increased the exposure of the polypeptide to the solvent slightly less
than expected for a native monomeric protein of the same length. The
30% difference between the predicted and measured values could have
several causes if it was significant. For example, the accessible
surface area of the monomeric intermediate could be slightly larger
than the areas for native monomeric proteins of the same length. Some
loops at the surface of the protein, whose crystallographic
B factors are high (11), could be unfolded in the
intermediate. However, the hydrophobic core of the TyrRS(
1) subunits
was not exposed to the solvent in the monomeric intermediate because we
found that it did not bind ANS and had its tryptophan residues
partially buried. We tried to apply the correlation between m and
ASA to the dissociation of the TyrRS(
1) dimer.
This dissociation exposes 1520 Å2 of accessible surface
area on each subunit and thus a total of 3040 Å2 (13). The
corresponding value of m1 would be 0.70 kcal·mol
1·M
1. This
predicted value was in good agreement with the measured value,
0.90 ± 0.06 kcal·mol
1·M
1 (Table IV). We
found that the value of m1 was 2.8 times lower than the value of m2. This finding indicated
that the dissociation of the TyrRS(
1) dimer increased the protein
surface area exposed to the solvent much less than the unfolding of the
monomeric intermediate. It was compatible with the existence of the
monomeric intermediate in a folded compact state.
Comparison with the C-terminal Domain--
In a previous work
(15), we studied the unfolding of the disordered C-terminal domain
(TyrRS(
3), residues 320-419) of tyrosyl-tRNA synthetase from
B. stearothermophilus by urea under equilibrium conditions.
We analyzed its unfolding profile with a two-state model and found the
following values for its thermodynamic parameters:
G(H2O) = 4.3 ± 0.4 kcal·mol
1, m = 0.65 ± 0.08 kcal·mol
1·M
1, and
fu
1(0.5) = 6.65 M urea
(where the errors correspond to S.D. values in the fitting of a global
equation to the fluorescence data). These data showed that the
concentration of half-unfolding for TyrRS(
3) was higher than the
value for the monomeric intermediate of TyrRS(
1). Thus, TyrRS(
3)
was at least as resistant to unfolding by urea as TyrRS(
1). However,
the
G(H2O) stability of TyrRS(
3) was much
lower than the stability of the monomeric intermediate of TyrRS(
1),
because of its lower m value. The m value for
TyrRS(
3) was only half of the value that could be predicted from its
number of residues, 1.29 kcal·mol
1·M
1 (31). This
comparison suggested that the unfolding of TyrRS(
3) by urea was less
cooperative than expected for a monomeric protein of the same length
(15).
Unfolding Mechanism--
We monitored the unfolding of TyrRS(
1)
by urea using several signals. The intensity of fluorescence at 330 nm
allowed us to show the existence of an equilibrium in the presence of
urea among the native dimeric state of TyrRS(
1), a monomeric
intermediate state, and the unfolded state. The monomeric intermediate
had a 5-fold lower molar fluorescence intensity in the absence of urea
than one subunit of native dimeric TyrRS(
1). This variation of
intensity could be due to global or local structural changes around
some tryptophan residues or to the loss of fluorescence transfer
between the tyrosines and tryptophans of one subunit and those of the
other subunit upon dissociation. The ratio of the fluorescence
intensities at 330 and 350 nm showed that the mean environment of the
tryptophan residues became cooperatively more polar during the
unfolding of the monomeric intermediate. Therefore, the monomeric
intermediate had its tryptophan residues partially buried. It possessed
a large part of the secondary structure of the native subunit,
according to the CD ellipticity. It was compact, according to the
partition coefficient in fast-SEC. It was not in a molten globular
state, according to the lack of ANS binding. Finally, the activity of
pyrophosphate exchange showed that the inactivation of TyrRS(
1)
occurred at a much lower concentration of urea than the dissociation of
its subunits. This inactivation occurred before any of the structural
signals tested, and therefore, our results did not allow us to
determine its structural cause. Thus, our results showed that urea led
to the dissociation of the TyrRS(
1) dimer into a monomeric
intermediate that had its tryptophan residues partially buried, had
secondary structure, was compact, and was not a molten globule. The
extrapolation of these results suggested that these different
conformational states could also exist in equilibrium in the absence of
urea.
Comparison with the Properties of an Interface Mutation--
In
TyrRS, the two symmetrical copies of Phe-164 interact with each other
across the subunit interface. The side chain of Phe-164 has been
changed into Asp. The mutant enzyme TyrRS (F164D) is monomeric and
compact in fast-SEC experiments at high pH, which favors the ionization
of the aspartate residues at position 164 and disfavors dimerization.
The free monomer, unlike the dimer, does not bind tyrosine and is
enzymatically inactive (30). Our work showed that wild-type TyrRS(
1)
could adopt a folded compact monomeric conformation. The results were
thus compatible with the data on TyrRS (F164D). A comparison between
wild-type TyrRS(
1) and a F164D derivative in experiments of
unfolding by urea could determine whether the mutation F164D stabilizes
the monomeric intermediate by introducing a charged residue in the
hydrophobic interface, which becomes exposed to the solvent upon
dissociation of the subunits.
Up to now, it has never been possible to detect and thus measure the
dissociation of the TyrRS and TyrRS(
1) dimers under native
conditions (26, 32). Therefore, our experiments of unfolding by urea,
monitored by spectrofluorometry, resulted in the first estimation of
the dissociation constant K1(H2O)
for the wild-type dimer (84 pM). The dissociation constant
Kd for TyrRS (F164D) and other mutants of Phe-164
has been measured by enzyme kinetics methods (32). At low pH, which
disfavors the ionization of the aspartate in position 164 and favors
dimerization, the Kd for the TyrRS (F164D) dimer is
equal to 30 mM and thus 3 × 105 times
higher than the K1(H2O) for
wild-type TyrRS(
1). At high pH, which disfavors dimerization,
Kd is equal to 100 mM and thus
~106 times higher than
K1(H2O). The corresponding
variations of the free energy of dissociation (
G), on
going from the wild type to mutant F164D, would be equal to 7.5 kcal·mol
1 at low pH and 8.2 kcal·mol
1
at high pH. It seems that the mutation F164D does not completely abolish the dimerization of TyrRS because the association between the
subunits of the wild-type dimer is particularly strong. Similar experiments with the dimer of the gene V protein of bacteriophage M13,
whose Kd is 1.25 µM, led to the
monomerization of the mutant protein F68D under all the pH conditions
tested (33).
We thank Valérie Guez for valuable
advice, Alain Chaffotte for help with the circular dichroism
experiments, and Michel E. Goldberg for constant interest.