 |
INTRODUCTION |
Aminoacyl-tRNA synthetases are a structurally diverse group of
enzymes divided into two classes based on the topography of their
adenylate binding sites and their modes of tRNA binding (1-3). The
active sites of class I enzymes have a classic Rossman fold formed from
two
-
-
elements resulting in a structure of four parallel beta
strands. The active sites of class II enzymes consist of a unique fold
formed from an antiparallel seven-stranded sheet element first
identified in seryl-tRNA synthetase (4). A structure similar to the
active site of class II enzymes exists in the biotin
synthetase/repressor protein (5) and the asparagine synthetase (6),
both of which have an adenylate intermediate on their reaction
pathways. The two aminoacyl-tRNA synthetase classes have different
modes of nucleotide binding and approach tRNA from opposite sides of
the polynucleotide. The differences in primary structure and modes of
ligand binding suggest that the two classes arose independently
(7-9).
Glycyl-tRNA synthetases are unusual in that there are two distinct
enzyme types that are not closely related. They have developed modes of
tRNA recognition that differ with respect to the discriminator base and
other base pairs in the stem (10, 11). The type exemplified by the
Escherichia coli enzyme is found in Gram-negative and
Gram-positive bacteria, whereas a second enzyme is found in other
eubacteria such as Mycobacteria sp. and
Mycoplasma sp., organisms classified as thermotoga
(e.g. Thermus thermophilus), the archaea
(e.g. Methanococcus jannashii), and in all
eukaryotic organisms. Examination of the crystal structure of T. thermophilus glycyl-tRNA synthetase revealed an atypical motif 1 (12) that was not identified in earlier alignments of glycyl-tRNA
synthetase (13, 14). Although the T. thermophilus enzyme
aligns with glycyl-tRNA synthetases from eukaryotic organisms, the
eukaryotic enzymes have elements that are absent in the T. thermophilus enzyme. One such element in glycyl-tRNA synthetase
from Bombyx mori, Homo sapiens,
Caenorhabditis elegans, and Arabadopsis thalinia
is a 50- to 60-residue N-terminal structure present in a number of
other eukaryotic aminoacyl-tRNA synthetases, including the
glutamyl-prolyl-, histidyl-, tryptophanyl-, and methionyl-tRNA
synthetases. Although the physiological function of this structure is
unclear, because it is apparently not required for amino acid
activation or tRNA binding, some studies suggest it may have a role in
binding to polynucleotides (15, 16) or in mediating interaction with
other proteins (17, 18). One study of human histidyl-tRNA synthetase
(19) indicated that removal of this element from the N terminus
resulted in a drastic reduction in enzymatic activity, although the
removal of the corresponding structure in B. mori
glycyl-tRNA synthetase does not have the same effect (15). Studies by
Raben et al. (19) indicated that the structure has a high
content of
-helix and may form a coiled-coil, similar to the
N-terminal domain of the E. coli (4, 20) and T. thermophilus (21, 22) seryl-tRNA synthetases.
We have examined the unfolding of the B. mori glycyl-tRNA
synthetase to determine how the N-terminal structure interacts with other elements of the protein and to determine the unfolding pathway for this dimeric enzyme. Our studies indicate that the enzyme dissociates into relatively native monomers prior to effects on spectroscopic signals sensitive to changes in conformation; the monomers unfold through a multistate process. The first 55 residues of
the protein have a high content of
-helix and unfold independently of the rest of the structure, suggesting that it constitutes a domain
that is separate from the core catalytic domains.
Apart from our interest in aminoacyl-tRNA synthetase structure and
mechanism, the present studies are of general interest as an example of
a complex unfolding pathway. Most protein folding studies have focused
on simple two-state systems, whereas many functionally interesting
proteins are likely to show more complex behavior. The techniques and
analytical methods for coping with the added complexity need to evolve,
and the present studies are a step in this direction.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Wild type B. mori glycyl-tRNA
synthetase and a mutant lacking the first 55 N-terminal residues (N55)
were expressed in E. coli BL21DE3pLysS transformed with
plasmids encoding these proteins (pNADA and pNADAN55, described
previously (15)). The proteins were purified by successive
chromatography on Q-Sepharose, hydroxylapatite, and Sephacryl S200
(15). Stock solutions of the purified proteins at 15-25 mg
ml
1 in 50 mM potassium phosphate (pH 7.5),
20% (v/v) glycerol, 0.5 mM EDTA, 0.5 mM
dithiothreitol were stored as small aliquots at
80 °C until use.
Protein concentrations were determined from absorbance at 280 nm using
extinction coefficients (based on the absorbance of a 1% solution of
the protein) of 9.1 for the wild type enzyme and 10 for the N55
deletion mutant (Ref. 23 and the present study). Guanidine-HCl was
obtained from Heico (Delaware Watergap, PA) and ultrapure urea was from
Life Technologies, Inc. Concentrations of urea and guanidine-HCl
solutions were calculated from a refractive index (24).
1-Anilinonapthalene-8-sulfonic acid
(ANS)1 was obtained from the
Sigma Chemical Co.
Analytical Ultracentrifugation--
Ultracentrifugation
experiments were performed in a Beckman Optima XLA analytical
ultracentrifuge employing absorbance optics and using an An60Ti rotor.
Temperature calibration was performed as described previously (25);
experiments were performed at 20 °C. Sedimentation velocity studies
were performed at 40,000 rpm in a charcoal-filled Epon double-sector
centerpiece. Density and viscosity of the solvents were estimated using
the program SEDNTERP. The partial specific volume was calculated by the
method of Cohn and Edsall (26-28). Velocity data were collected at 280 nm at 0.002-cm intervals with one average in a continuous scan mode.
Sedimentation data was analyzed using DCDT+ (29) and SVEDBERG (version
6.37) as detailed previously (25). Samples were prepared for
sedimentation studies by chromatography on a column of Sephadex G-50
medium equilibrated in 50 mM potassium phosphate (pH 7.2), 0.1 mM EDTA containing 0.05 mM DTT and the
indicated concentrations of urea. Samples were prepared 4 h prior
to the experiment; protein concentrations were adjusted to 0.2 mg
ml
1.
Size Exclusion Chromatography--
Stokes radii of wild type and
N55 glycyl-tRNA synthetases were determined on a Superdex 200 column in
50 mM potassium phosphate (pH 7.2), 0.1 mM
EDTA, and 0.05 mM DTT and different concentrations of urea.
Standards (thyroglobulin (8.0 nm), immunoglobulin G (5.0 nm), ovalbumin
(3.5 nm), and myoglobin (2.0 nm)) were run in the absence of urea.
Samples were prepared 4 h before application to the column, and
chromatography was performed at room temperature (23 ± 0.5 °C).
Unfolding Experiments--
Samples for spectroscopic
measurements were diluted at least 100-fold into solutions containing
50 mM potassium phosphate (pH 7.2), 0.1 mM
EDTA, 0.05 mM DTT and the indicated concentration of
guanidine-HCl or urea. Corresponding blanks were prepared using enzyme
storage buffer. All spectral measurements were taken at 20 °C.
Sample solutions were prepared at least 4 h prior to taking measurements. Preliminary experiments revealed a
time-dependent change in fluorescence intensity after
preparation of the sample. Most of the fluorescence change occurred in
the first hour and occurred more rapidly in samples containing urea or
guanidine-HCl; there was no detectable difference in measurements taken
at 4 and 16 h. Measurements of fluorescence were taken in an ISS,
Inc. GREG 200 spectrofluorometer using a 10- by 2-mm quartz cell.
Excitation was at 290 nm and emission was measured from 300 to 400 nm
at 1-nm intervals. In some experiments, measurements were taken at a
fixed wavelength as indicated in the figure legend. Circular dichroism
was measured using a Jasco J500 spectropolarimeter at 20 °C.
Measurements in the far ultraviolet (185-240 nm) were taken in a 0.1- or 1-mm cell on solutions of 0.05-0.2 mg ml
1 and in the
near ultraviolet (240-300 nm) in a 1-mm cell on solutions at 0.2-0.4
mg ml
1. Concentrations in specific experiments are
indicated in the figure legends. Samples were prepared for CD at least
4 h before taking the measurement; no significant differences were
detected in spectra taken between 4 and 16 h. Spectra were
recorded by taking measurements at 0.2-nm intervals at a scan rate of 1 nm/min at a 1-nm band pass and a time constant of 64. Appropriate blank spectra were recorded on the buffer components and
subtracted from spectra obtained on protein solutions. In experiments
examining unfolding as a function of denaturant, data were collected at a fixed wavelength and averaged over 2 min. Fluorescence spectra in
guanidine-HCl or urea were analyzed by singular value decomposition (30, 31) using the program MATLAB 5.3 (The MathWorks Inc.). Singular
value decomposition converts the data matrix into the product of a U
matrix (basis spectra), an S matrix (singular values), and a V matrix
(amplitude vectors). These three matrices allow for the determination
of the number of spectrally significant species over a range of
guanidine-HCl or urea. Singular values of <0.017 and autocorrelation
values of <0.8 are taken as indicative of a random nonsignificant
component. Fitting of models of unfolding to data was performed by the
nonlinear least squares algorithm of Marquart (32) using the program
FitAll (MTR Software). Intensity averaged emission wavelength was
calculated,
|
(Eq. 1)
|
where Ii is the intensity measured at
i.
Refolding Experiments--
Protein solutions were mixed with
concentrated urea to a final concentration of 7 M and then
diluted to different concentrations of urea for refolding; other buffer
components were 50 mM potassium phosphate (pH 7.2), 0.1 mM EDTA. and 0.05 mM DTT. Spectral measurements were made after 20 h.
Models of Unfolding--
Spectroscopic data obtained in
unfolding experiments were analyzed taking into consideration four
schemes for unfolding. A simple two-state model (33) with the native
protein (N) and the unfolded species (U) is described by,
|
(Eq. 2)
|
where Sobs is the observed spectroscopic
signal, SN and SU are the
signals for the native and unfolded species, respectively,
G is the free energy of unfolding in the
absence of denaturant, m is the partial derivative of
G with respect to denaturant, and d
is the concentration of denaturant. Sobs and
d are the dependent and independent variables,
SN is treated as a constant, and
SU,
G, and m
are treated as fitting parameters.
A more complicated three-state model (Reaction II, described in Ref.
34), which includes an intermediate species is given by,
|
(Eq. 3)
|
where Sobs, SN,
SU, and d have the same definitions
as for the two-state model. The terms
G1,
G2, m1, and
m2 are the free energies and m values
for the N
I and I
U transitions.
G1,
G2, m1,
m2, SI, and
SU are treated as fitting parameters, and SN is treated as a constant based on the value
of the observed signal in the absence of denaturant; however, when
SN was treated as a fitting parameter, similar
results were obtained. A linear term
(mU[d]) was included as a fitting
parameter to account for the post-transition baseline (34-36). This
model was used to fit CD data obtained for the unfolding of the N55 mutant.
A variant of the three-state model was derived, which includes a third
parallel transition to account for an unfolding event that occurs
independently of the other transitions (Reactions III and IV
below). This model was considered to account for the difference
observed between wild type glycyl-tRNA synthetase and the N55 mutant
when CD was monitored during unfolding,
|
(Eq. 4)
|
where the terms
G1,
G2, m1,
m2, and SI are defined in
the same manner as for the three-state model; SN
is the signal of the native species in the three state unfolding in
Reaction III. The terms
Gp
mp, SNp, and
SUp are used in the second term in Eq. 4 that
describes the independent (or parallel) unfolding event given in
Reaction IV; we have interpreted signals SNp and
SUp as arising from the native and unfolded
N-terminal element. Linear terms
(mN[d] and
mU[d]) were included in the
equation as fitting parameters to account for the dependence of the
post-transition baseline on denaturant concentration (34-36). However,
owing to the complexity of the models, including these terms had little effect on the result.
An expression based on a four state model of unfolding was derived to
analyze the fluorescence emission employing the same approach used for
the three-state model.
The thermodynamic parameters describing the N to Ia
and Ia to Ib transitions are indicated by a and
b subscripts to avoid the implication that they correspond to
transitions described by
G1 and
G2 in Eqs. 3 and 4. The term
G2 is the same in all three
expressions to indicate that it corresponds to the same unfolding
event.
|
(Eq. 5)
|
Fluorescence Quenching Experiments--
Quenching of
intrinsic tryptophan fluorescence by iodide or acrylamide (37-39) was
examined in 50 mM potassium phosphate buffer, 0.1 mM EDTA, 0.05 mM DTT, and the indicated
concentrations of urea or guanidine-HCl. Excitation was at 290 nm, and
emission was measured from 300 to 400 nm or by measuring emission at a fixed wavelength. Fluorescence in acrylamide quenching experiments was
corrected for the inner filter effect due to the absorbance of
acrylamide at 290 nm using the relationship F = Fobs10e0.5
cl,
where
is the molar extinction coefficient, c is molar
concentration, and l is the path length (1 cm). In
experiments employing KI as a quencher, ionic strength was kept
constant by the addition of KCl. Estimates of the dynamic and static
quenching constants for acrylamide were obtained by fitting the
hyperbolic form of the Stern-Volmer equation to the data, which
includes an exponential term to account for static quenching
(38),
|
(Eq. 6)
|
where KSV is the Stern-Volmer constant
for collisional quenching and
is the static quenching constant.
Data are presented using the inverse form of the Stern-Volmer equation
as F0/F versus [Quencher]. Because iodide quenching did not have a significant static component, the exponential term was not included in fits of the
equation to these data.
Fluorescence Lifetime Measurements--
Fluorescence lifetime
data were collected using an I.S.S. K2 multifrequency cross-correction
phase and modulation fluorometer with a xenon arc lamp. A scattering
solution of glycogen (0.8 mg/ml aqueous solution) was used as a
reference. Excitation was at 290 nm, and emission was measured on
emitted light that passed through a 305-nm cutoff filter to eliminate
scattered radiation. Lifetimes were determined at 15 frequencies in the
1- to 200-MHz range. Data were collected until the S.D. from each
measurement of phase and modulation was at most 0.2 and 0.004, respectively.
ANS Binding--
ANS (1-anilinonapthalen-8-sulfonic acid)
binding was detected by collecting fluorescence spectra in the
presence of 10 µM dye for the wild type glycyl-tRNA
synthetase and the N55 mutant in the presence of varying concentrations
of guanidine-HCl or urea. Excitation was at 380 nm, and emission was
measured from 400 to 600 nm. Stock solutions of ANS were prepared in
methanol and diluted into the samples such that the methanol
concentration was less than 0.05% (v/v). Concentration of the dye was
determined using an extinction coefficient of 8 × 103
M
1 cm
1 at 372 nm. The presence
of urea or guanidine-HCl in the absence of protein had no significant
effect on the fluorescence of the dye.
 |
RESULTS |
To examine the unfolding of glycyl-tRNA synthetase and the
interaction of the N-terminal structure with other elements of the
protein, we examined the urea and guanidine-HCl unfolding process by
multiple spectroscopic techniques (40). We also examined the oligomeric
state of the protein under different solvent conditions.
Examination of Protein Unfolding by Intrinsic Tryptophan
Fluorescence--
B. mori glycyl-tRNA synthetase has four
tryptophans that are conserved in all eukaryotic glycine enzymes.
Insofar as tryptophanyl residues are sensitive to protein conformation
and local environment (41) they are potential probes of conformational
change. We examined fluorescence emission spectra for the wild type
enzyme and the N55 deletion mutant under native conditions and in the presence of different concentrations of urea (panel A) or
guanidine-HCl (panel B) as shown in Fig.
1 for the wild type enzyme; results with
the N55 mutant were similar (not shown). With both chaotropic agents
there was an increase in fluorescence intensity at lower concentrations
of denaturant followed by a red shift in the emission wavelength and a
reduction in fluorescence intensity at higher concentrations of
denaturant. Both mutant and wild type proteins exhibit a transition at
high concentrations of guanidine-HCl (at 2.7 M) or urea
(6.1 M). The change in fluorescence intensity at a single
wavelength as a function of guanidine-HCl (panel A) or urea
(panel B) for both wild type and mutant enzymes is shown in
Fig. 2. The addition of KCl to 1 M in the absence of urea or guanidine-HCl did not induce an
increase in fluorescence intensity as was observed at 1 M
guanidine-HCl, indicating that the effect at this concentration of
guanidine-HCl is not related to ionic strength or the presence of
chloride. The similarity of the results obtained with urea and
guanidine-HCl also suggests that the spectral changes reflect general
features of the unfolding process and not properties of the agents used
to induce unfolding.

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Fig. 1.
Fluorescence emission spectra of wild type
glycyl-tRNA synthetase. Spectra were recorded at 20 °C in the
presence of urea (A) or guanidine-HCl (B).
Protein concentration was 0.2 mg/ml.
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Fig. 2.
Change in fluorescence emission at 327 nm at
different concentrations of urea (A) or guanidine-HCl
(B). Protein concentration was 0.2 mg/ml.
Closed symbols, wild type enzyme; open symbols,
N55 mutant enzyme.
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|
The weighted basis spectra (expressed as the product of the U and S
matrices) were obtained by singular value decomposition of the emission
spectra from guanidine-HCl or urea induced denaturation (shown in Fig.
3) for the wild type enzyme; similar
results were obtained for the N55 mutant. Singular values and values
for the autocorrelation function for the first 10 basis spectra are
summarized in Table I. The analysis
indicates that two singular values make the largest contribution to the
spectra with a third component making a negligible contribution;
similarity of the basis spectra suggests that the variables are the
same for the two denaturants for the mutant and wild type enzymes.
Based on the magnitude of the singular values and the values of the
autocorrelation function (values greater than 0.8 indicating
significance), only the first four or five basis spectra make a
contribution to the spectra. The existence of multiple species in the
unfolding pathway is suggested by singular value decomposition of the
intensity data. Although the basis spectra are not necessarily those of
the intermediates, they suggest that there are species with spectral
properties intermediate between the native and denatured forms of the
enzyme. The red shift and the intensity changes associated with
unfolding indicate that the tryptophans are buried and at least
partially shielded from solvent (37, 41), a conclusion also supported
by iodide and acrylamide quenching studies (presented later). The
increase in fluorescence intensity suggests quenching of fluorescence
of one or more of the tryptophanyl residues by proximity to another group is relieved by a conformational change induced by low
concentrations of denaturant. No concentration dependence was observed
in unfolding experiments, where fluorescence was observable over a
range of 0.05-0.2 mg ml
1; the propensity of the protein
to adsorb to glass and plastic below 0.05 mg ml
1
precluded examination of lower protein concentrations.

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Fig. 3.
Basis spectra derived from singular value
decomposition of fluorescence emission spectra. Spectra are the
product of the U and V matrices for the first four basis spectra. Only
the first (1) and second (2) basis spectra are
labeled. Spectra were obtained during unfolding of wild type
glycyl-tRNA synthetase in urea (A) or guanidine-HCl
(B).
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Table I
Singular values and values for the autocorrelation function derived
from singular value decomposition of fluorescence spectra of
glycyl-tRNA synthetase obtained from urea and guanidine-HCl-induced
unfolding
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|
The red shift in the emission spectrum, shown as the intensity-averaged
emission wavelength in Fig. 4, was used
to monitor urea-induced (Fig. 4, A and B) and
guanidine-HCl-induced (C and D) unfolding.
Multiple transitions reflected in fluorescence changes are inconsistent
with a simple two-state model (Reaction I and Eq. 2), suggesting a more
complex scheme is required to describe the unfolding process. Although
the change in emission wavelength in Fig. 4 can be fit using a
three-state model, fits to the intensity changes shown in Fig. 2 and
the emission wavelength in Fig. 4 showed a nonrandom distribution of
residual values (Fig. 4, B and D). A better fit
was obtained with a model with two intermediate species (Reaction V and
Eq. 5); values for the F-statistic for different experiments
ranged from 4 to 10 placing the fit to the four-state model well above
the 95% interval. The values for
G and
m for the transition occurring at high denaturant
concentration are given in Table I for the wild type enzyme.

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Fig. 4.
Intensity averaged emission wavelength at
different concentrations of denaturant. The value for
  EI was determined at different concentrations of
urea (A and residuals in B) or guanidine-HCl
(C and residuals in D). Closed
symbols, wild type; open symbols, N55 mutant.
E, unfolding (closed symbols) and refolding
(open symbols) of wild type glycyl-tRNA synthetase
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Fig. 4E shows the results of a refolding experiment with
wild type glycyl-tRNA synthetase where fluorescence was examined. The
emission spectra of the species seen in the unfolding and refolding
were similar at relatively high concentrations of denaturant (3-6
M urea) showing a characteristic increase in fluorescence and a blue shift in the emission spectrum. At lower concentrations (<2.5 M) the signal of the refolded protein does not
return to that of the native protein and retains the higher emission
intensity and slight red shift in the emission spectrum. Similar
results were obtained with the N55 mutant. These experiments indicate that transitions occurring at higher concentrations of denaturant are
readily reversible, whereas the transitions at lower concentrations are not.
Examination of the Effect of Unfolding on Tryptophan Fluorescence
Lifetimes--
The fluorescent properties of tryptophans in
glycyl-tRNA synthetase during guanidine-HCl-induced denaturation was
examined by fluorescence lifetime measurements (see Fig. 1s in the
Supplemental Material). Two classes of lifetimes were detected with one
class accounting for 70-80% of the fluorescence. Consistent with the steady-state measurements, there was an increase in the lifetime of the
larger class of fluorophors with increasing denaturant concentration
followed by a decrease as the protein unfolds at high concentrations;
this result is seen in both the wild type enzyme and N55 mutant.
Examination of Tryptophan Exposure by Fluorescence
Quenching--
To corroborate the results of the unfolding studies
indicating that the tryptophans in the native proteins are at least
partially shielded from the solvent, we examined iodide and acrylamide
quenching of tryptophan fluorescence. Both wild type and mutant gave
linear Stern-Volmer plots with iodide, whose slopes increased with
increasing concentrations of urea or guanidine-HCl (shown in Fig. 2s,
A and B, in the Supplemental Material). Quenching
by acrylamide gave a similar pattern of increasing quenching upon
addition of urea or guanidine-HCl (see Fig. 2s, C and
D, in the Supplemental Material) but exhibited both static
and dynamic components as indicated by the upward curvature in the
Stern-Volmer plots. The quenching studies, summarized in Table
II, indicate that the tryptophans in
native glycyl-tRNA synthetase are inaccessible to a polar quencher (KI)
and only slightly more accessible to a more hydrophobic quencher (acrylamide), consistent with most of the four tryptophans being shielded from solvent in the protein interior. That both the wild type
and N55 mutant exhibited similar behavior qualitatively and quantitatively is consistent with the steady-state fluorescence results
indicating that the tryptophans are buried. Perturbation of the
structure of both enzymes with a relatively low concentration of
denaturant resulted in the same extent of exposure of tryptophanyl residues to solvent. As expected for unfolded proteins, both wild type
and mutant showed the same susceptibility to iodide and acrylamide quenching when treated with high concentrations of urea (6.4 M) or guanidine-HCl (5.8 M).
Examination of Unfolding by Circular Dichroism--
We employed
circular dichroism in the analysis of the unfolding of glycyl-tRNA
synthetase as a spectroscopic probe that is sensitive to protein
secondary structure. The CD spectra of the wild type and N55 deletion
mutant in Fig. 5 revealed greater
negative ellipticity for the wild type enzyme in the region of 210-230 nm (Fig. 5A), a region with bands characteristic of
-helix. The difference spectrum derived from the wild type and
mutant proteins has features characteristic of
-helix (not shown).
There was a small, though significant, difference between wild type and mutant in the CD spectra recorded in the near-UV range (panel B). The far-UV CD spectra of the native and the unfolded/refolded wild type and N55 mutant proteins were also examined as shown in Fig. 5
(C and D, respectively). The native
(C, closed symbols) and unfolded/refolded
(C, open symbols) wild type enzyme were similar,
indicating that unfolding of a significant fraction of secondary
structure is reversible. The CD spectrum of the unfolded/refolded N55
mutant (D, open symbols) is also similar to the
native form of the protein (D, closed symbols).
We examined the change in ellipticity at 222 nm at different
concentrations of urea for the wild type and N55 mutant proteins as
shown in Fig. 6A. The wild
type enzyme showed significantly higher ellipticity than did the
mutant, which declined with a transition with a midpoint at ~2.7-3
M urea; this transition was absent or at least far less pronounced in the mutant. At higher concentrations of urea, the behavior of the mutant and wild type proteins were similar, both showing a relatively noncooperative decrease in ellipticity between 3 and 5.5 M urea followed by a cooperative transition
centered at 6-6.1 M urea. The latter transition occurs at
the same concentration of urea as the transition seen when fluorescence
was examined and appears to correspond to the global unfolding of the
protein. When guanidine-HCl was employed to induce unfolding (Fig.
6B), a similar result was obtained; a transition at low
concentrations of denaturant was present in the wild type enzyme but
absent in the mutant and both exhibited a cooperative transition at 2.7 M guanidine-HCl. Despite the difference between the wild
type and mutant proteins below 3 M urea in unfolding
experiments, both could be fit to a three-state unfolding model; the
result may be a reflection of the small amplitude of the spectral
change in the mutant and the transitions in the wild type occurring
coincidentally over a similar range of denaturant. Although application
of the F-statistic to the fit of four-state models to the
data for N55 indicated that these complex models could not be
justified, the wild type enzyme gave a better fit to the four-state
model (F = 5.8, 95% confidence interval) described by
Eq. 5. A more appropriate model (Reactions III and IV and Eq. 4)
for the wild type enzyme includes a three-state unfolding process that
describes features of both the wild type and mutant and an independent,
parallel unfolding process that accounts for the transition seen in the wild type enzyme at low denaturant concentration, and it was this model
that was used for analysis of the wild type enzyme. Although the fit to
the four-state scheme described by Eq. 5 was better than to the
three-state model (F = 11, with p greater
than 95%), it was almost the same as the
fit obtained to Eq. 4. Tables III and IV
summarize the thermodynamic parameters (m and
G values) derived from urea- and
guanidine-HCl-induced unfolding experiments. Discrepancies for free
energies between urea and thermal unfolding and guanidine-HCl-induced
unfolding noted previously (24) have been attributed to the stabilizing
effect of guanidine-HCl on some structures (34, 42). Makhatadze (43)
has attributed some of the effects of guanidine-HCl on protein
stability to the nature of the anion and suggests that guanidine-HCl is
unsuitable for determining thermodynamic parameters of protein
unfolding using the commonly employed linear extrapolation method. The
work of Smith and Scholtz (44) indicates that m values show
a clear dependence on ionic strength such that guanidine-HCl and urea are equally effective in denaturation experiments performed at constant
ionic strength. Despite these cautions with respect to the use of
guanidine-HCl, the unfolding of glycyl-tRNA synthetase with the two
denaturants was quite similar. The similarity in the values of
G2 where fluorescence or CD were
observable suggests that they reflect the same unfolding process.

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Fig. 5.
Circular dichroic spectra of wild type and
the N55 mutant glycyl-tRNA synthetases. A, spectra in
the far-UV range were recorded at 20 °C in a 0.1-mm path cell at a
protein concentration of 0.2 mg/ml. B, spectra in the
near-UV range were recorded in a 1-mm path cell at a protein
concentration of 2.5 mg/ml. In A and B,
solid symbols represent wild type and open
symbols represent the N55 mutant. C, refolding of wild
type glycyl-tRNA synthetase. D, refolding of N55 mutant
glycyl-tRNA synthetase. In C and D, solid
symbols represent native protein and open symbols
represent unfolded/refolded protein.
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Fig. 6.
Change in ellipticity at 222 nm at different
concentrations of urea (A and residuals in
B) or guanidine-HCl (C).
Open symbols, wild type glycyl-tRNA synthetase; closed
symbols, N55 mutant glycyl-tRNA synthetase. Protein concentration
was 0.1 mg/ml. Measurements were taken in a 1-mm cell.
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Table III
Thermodynamic parameters for urea and guanidine-HCl-induced unfolding
of glycyl-tRNA synthetase monitored by CD
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Table IV
Thermodynamic parameters for urea and guanidine-HCl-induced unfolding
of glycyl-tRNA synthetase monitored by fluorescence
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Examination of ANS Binding--
ANS binding was used as a probe of
the extent of exposure of hydrophobic regions of the protein during
unfolding. Glycyl-tRNA synthetase bound ANS in the absence of
denaturant (Fig. 3s in the Supplemental Material). The fluorescence of
the dye in the presence of the protein increased with increasing urea
(Fig. 3A) or guanidine-HCl (Fig. 3B) and then
declined at higher concentrations. At 4-6 M guanidine-HCl
or 6-8 M urea the fluorescence of the dye in the presence
of protein was the same as that of the free dye. Essentially the same
results were obtained with the N55 mutant (data not shown). With both
urea and guanidine-HCl, ANS fluorescence increased significantly at
denaturant concentrations (1 M urea or 0.4 M
guanidine-HCl) that caused little change in either intrinsic tryptophan
fluorescence or CD at 222 nm. The results are consistent with increased
exposure of hydrophobic residues as the protein unfolds with increasing
denaturant concentration.
Examination of the Oligomeric State of Glycyl-tRNA Synthetase
during Unfolding--
Protein denaturation through a dimeric
intermediate confers a concentration dependence on the unfolding
process and is described by different expressions than for proteins,
which denature through monomeric intermediates (45, 46). Because
glycyl-tRNA synthetase is a dimer, such an intermediate must be
considered. To determine the extent to which dissociation of the dimer
contributes to changes in the spectroscopic signals, we examined the
sedimentation constant at concentrations of urea (shown in Fig. 4s,
A, of the Supplemental Material) associated with
changes in both the CD and fluorescence spectra. In the absence of
denaturant the wild type protein had a weighted average value of
S20,w of 6.91, consistent with previous
determinations of this parameter (15, 47) and consistent with a dimer.
At 1.5 M urea the weighted average
S20,w was 5.75. The latter value is
consistent with other data indicating that the protein is a dimer of
subunits of Mr 76,919 (15, 47) and that it
dissociates reversibly into monomers (23). The sedimentation data
collected in the presence of 1.5 M urea is accounted for by
a monomeric species with an S20,w of
4.57 constituting 76% of the material with the remaining material
reflecting an early stage of aggregation. The presence of the larger
material skews the weighted average for
S20,w to 5.75. At 4 M urea
the protein is aggregated and polydisperse. The transition from
dimer to monomer occurs at a concentration of urea where there is no
significant change in the fluorescence emission spectrum or the CD
spectrum of the protein. The result indicates that dissociation of the
dimer into monomers occurs prior to conformational changes reflected in
fluorescence and CD spectra and that the monomers are probably similar
to the native structure. In experiments not shown we examined the
effect of guanidine-HCl on the oligomeric state of the wild type and
N55 mutant enzymes by cross-linking the proteins with
succinimidyl-suberate under conditions that we had previously
established for cross-linking the enzymes (15). In the absence of
guanidine-HCl we observed a species with a mobility expected for the
dimer. At low concentrations of guanidine-HCl (up to 1 M)
there was a reduction in cross-linking. At higher concentrations of
denaturant (up to 2.2 M), the cross-linked species just
entered the gel migrating at a position expected for cross-linked aggregates. Above 2.5 M the protein migrated at a position
expected for the monomer. We also examined the wild type and N55 mutant enzymes by size exclusion chromatography (shown in Fig. 4s,
B, of the Supplemental Material). The native proteins eluted
from Superose 12 at a retention time corresponding to the dimer
(Rs 5.5 nm for wild type, 5.3 for N55), whereas
protein in the presence of 1.5 M urea eluted at a retention
time characteristic of the monomer (Rs 4.9 nm
for wild type, 4.7 nm for N55); at 4 M urea the protein
eluted at a retention time indicating a size greater than thyroglobulin
(>8 nm) and suggesting that it was aggregated. These results indicate
that the wild type and mutant enzymes dissociate into monomers at low
concentrations of denaturant, aggregate at intermediate
concentrations, and then dissociate to unfolded monomers at high
denaturant concentration. Because the dissociation of dimers to
monomers is not reflected in spectral changes at low denaturant and
there is no detectable concentration dependence to the unfolding, the
data can be fit to a model which need only account for unfolding of the monomer.
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DISCUSSION |
Unfolding of Glycyl-tRNA Synthetase--
Examination of the
denaturation of glycyl-tRNA synthetase by urea or guanidine-HCl using
spectroscopic techniques sensitive to protein conformation reveals a
complex unfolding pathway expected of a multidomain, dimeric enzyme
(48). Depending on the spectroscopic signal examined, unfolding of the
wild type enzyme can be described by a three-state (for CD) or a
four-state (fluorescence) model. Although a three-state model can be
satisfactorily fitted to the CD data obtained for the wild type enzyme,
analysis of the deletion mutant lacking the first 55 residues suggests
that an additional parallel unfolding process is required to describe
the unfolding of the N terminus. The results with the two proteins
suggest that the N-terminal element constitutes an independent folding
entity. This may also be the case for E. coli seryl-tRNA
synthetase (49) where the unfolding of the N-terminal coiled-coil has
been examined, although this element was examined while separated from
the rest of the protein. Based on the difference in the CD spectra of
the wild type and N55 mutant, the N-terminal element of glycyl-tRNA synthetase appears to be largely
-helix, and conforms to the 7-amino
acid repeat (a through g with hydrophobic residues at a and d and
charged residues usually at positions e and g) (50) expected for a
typical coiled-coil. Although we do not have a structure of the
N-terminal element of glycyl-tRNA synthetase, an NMR structure reported
recently (51) for a homologous element from hamster
glutamyl-prolyl-tRNA synthetase demonstrates that this structure is an
antiparallel coiled-coil; the structure binds to polynucleotides as
originally suggested from studies of glycyl-tRNA synthetase (15). The
structure appears to fold independently and its removal from
glycyl-tRNA synthetase does not alter the environment of tryptophanyl
residues as detected by examination of unfolding by fluorescence and by
susceptibility of the tryptophanyl residues to quenching by iodide or
acrylamide. The independent folding properties of this structure are in
accord with its being present in the eukaryotic glycyl-tRNA synthetases
and absent in the prokaryotic enzyme and the fact that the N-terminal
element is not required for amino acid activation or tRNA
aminoacylation. This structure is present in a number of aminoacyl-tRNA
synthetases of both classes, in the N terminus (tryptophanyl-,
histidyl-, and glycyl-), in the C terminus (methionyl-), and as
multiple copies between two tRNA synthetase domains
(glutamyl-prolyl-).
Although transitions seen at lower concentrations of denaturant
detected by CD are absent in the mutant, the transition at higher
denaturant concentration associated with global unfolding of the
protein occurs at the same concentration of denaturant in both mutant
and wild type and has the same amplitude. A major transition at high
denaturant detected by fluorescence corresponds to the transition
detected by CD. Caution must be exercised in interpreting the results
of experiments where the observable is fluorescence, because, as
pointed out by Eftink (41), there is no simple relationship describing
fluorescence or
max for a mixture of states; the state
with the highest quantum yield will tend to dominate the emission,
skewing the measured parameter toward that state. However, singular
value decomposition analysis (Table I and Fig. 1) of glycyl-tRNA
synthetase unfolding suggests that the fluorescence properties of
intermediates in the unfolding must be similar to the native state.
That the value for
Gb is poorly
determined by the data is in accord with this difficulty in the
analysis of fluorescence data, the complexity of the model required to
account for the unfolding of glycyl-tRNA synthetase, and the small
amplitude of the spectral change. However, the close correspondence
between the transitions occurring at high denaturant concentration
observed by fluorescence and CD suggests that they reflect the same
unfolding process. The minimal model describing the unfolding of
glycyl-tRNA synthetase requires the native state, the unfolded state,
at least two intermediate species, and an independent unfolding process
with its own native and unfolded states.
The dissociation of the dimeric enzyme into subunits is not accompanied
by changes detectable by CD and fluorescence, suggesting that a
native-like monomer is formed. This is consistent with earlier work
(23) indicating that at low protein concentrations, in the absence of
chaotropic agents, the enzyme dissociates into inactive monomers.
Similar results were obtained in studies of the homologous glycyl-tRNA
synthetase from S. cerevisiae (52), suggesting that this
enzyme dissociates reversibly to inactive monomers. Although the
dissociation to monomers must be considered as part of the overall
unfolding pathway, we have not taken it into consideration in the
analysis of our data, because there is no spectral manifestation of it
and no protein concentration dependence was seen in the unfolding
transitions. The absence of concentration dependence can be explained
by the dissociation greatly favoring the monomer conformation
even at relatively low denaturant concentrations. Presumably,
concentration dependence would be detected if sufficiently high protein
concentrations could be examined. Although analysis based solely on the
spectroscopic data will underestimate the total free energy of
unfolding, the contribution of dimer dissociation to the process
appears to be small, because it occurs at relatively low concentrations
of denaturant. The unfolding of dimeric proteins involves free energy
changes that are typically higher than monomeric proteins and roughly proportional to the number of residues (42); the relationship between
size and free energy of unfolding appears to reflect the surface area
available for interaction between subunits. It would appear that in the
case of glycyl-tRNA synthetase the dimer-dimer contacts do not make a
major contribution to the overall stability of the protein. In other
systems such as the dimeric N-terminal domain of tyrosyl-tRNA
synthetase, the dimer is converted to a monomeric intermediate (53).
Less commonly, as in the cases of luciferase (46), organophosphorus
hydrolase (54), and 3-isopropylmalate dehydrogenase (55), a native
dimer is converted to a dimeric intermediate, which is then converted
to an unfolded monomer. The Arc repressor (56, 57) unfolds directly to
monomers, whereas the remarkably stable tetrameric lac repressor (58)
forms an unfolded tetramer, which dissociates to monomers at higher
denaturant concentration.
One aspect of the unfolding we have not considered in our
analysis is the formation of the oligomeric species with the wild type
and mutant enzymes at intermediate concentrations of denaturant. Guanidine-HCl and urea can promote aggregation in unfolding studies due
to the association of an intermediate (59) or the denatured state (60).
Partially folded intermediates of staphylococcal nuclease aggregate to
form more structured species (61, 62); partially folded conformations
appear to be intermediates in the formation of most aggregates (59) and
can be mistaken for kinetic folding intermediates when they form
transiently (63). Rigorous analysis of such a system would presumably
require fitting data to a polynomial whose order would depend on the
number of monomers in the aggregate; an additional vexing problem is
the existence of multiple oligomeric states. The unfolding process
usually exhibits dependence on the concentration of the monomer as
observed with staphylococcal nuclease (61, 62). Although analysis of
the effect of oligomerization is a tractable problem when describing the unfolding of a dimer or a monomer that goes through a dimeric intermediate, analysis of a higher order polynomial where n
is indeterminate poses a significant obstacle. The absence of
concentration dependence in the unfolding experiments where
fluorescence was measured warrants some comment. One would expect both
the dissociation of a dimer and the formation of oligomeric species to
show some concentration dependence such that any expression that
accounts for the unfolding would require a mass action term(s).
Concentration dependence might be detectable at lower concentrations
than were examined, but the propensity of the protein to adsorb to
surfaces at low concentrations precluded our observing it. Although
there appears to have been no explicit treatment of aggregating systems for denaturants such as guanidine and urea, pressure-induced unfolding of higher ordered structures (64) such as erythrocruorin (65) and the
capsid protein of Brome mosaic virus (66) exhibit a lack of
concentration dependence so that such structures exhibit behavior
characteristic of macroscopic bodies.