 |
INTRODUCTION |
Numerous investigations over the last 40 years have focused on the
folding and structural determinants that govern how a polypeptide adopts its native structure. Most of our knowledge on protein folding
derives from studies on small monomeric proteins. However, the majority
of native proteins are more complex structures, composed of several
subunits that in turn consist of domains. The extrapolation of results
obtained in the study of small proteins to larger ones is not always
appropriate, and it is therefore important to investigate proteins
composed of more than one subunit.
Human glutathione transferase P1-1
(hGSTP1-1),1 a homodimeric
enzyme and thus representing the simplest type of oligomeric structure,
has been the subject of recent studies on protein folding (1-3). The
aim of these investigations was to identify sequence patterns of
importance to folding kinetics and/or structure of the final native
state. hGSTP1-1 is a Pi class member of a multifunctional superfamily
of enzymes, the GSTs. The role of GSTs is considered to be the
detoxication of a large number of hydrophobic compounds by catalyzing
the conjugation to glutathione (GSH) and thus increase their water
solubility (4). The cytosolic GSTs have been grouped into a number of
different evolutionary classes denoted by the names of Greek letters
such as Alpha, Mu, Pi, and so on (4, 5). The classification was
originally based on primary structure similarities, substrate
specificities, and immunologic properties (5), but sequence
similarities are currently the overriding criterion. The mass of the
dimeric GSTs is ~50 kDa, and subunits within the same class can
combine to form either homo- or heterodimers (6). Each subunit contains
an active site, and two domains form the subunit. The smaller
N-terminal domain (domain I) adopts an
/
topology and provides
most of the contacts with GSH. The larger C-terminal domain (domain II)
is completely helical and contains most of the residues that form the
hydrophobic binding site (10-13). Alignment of all known GST
structures (more than 100) shows that only 6-7 residues, which is less
than 5% of the entire polypeptide chain, are strictly conserved.
Despite this limited sequence similarity, all GSTs adopt the same
native fold. GST structure analysis with special emphasis on the few
conserved residues led to the identification of two local structural
motifs in a characteristic position, the N-terminal region of the
6-helix (1). The N-capping box ((S/T)XXD), and the
hydrophobic staple motif (14-19) in which two hydrophobic residues
flanking the N-capping box are present in all GSTs known today (1-3,
20). Previous investigations demonstrated that single mutations of
residues forming the capping box ((S/T)XXD) and the
hydrophobic staple motifs have a dramatic effect on the protein
stability (1-3). The same amino acid substitutions also have
significant effects on protein folding, generating
temperature-sensitive folding mutants unable to refold at the
physiological temperature 37 °C (1-3). Mutations corresponding to
those made in hGSTP1-1 have also been constructed in hGSTA1-1, a member
of the Alpha class showing 31.6% sequence identity with hGSTP1-1 (21).
The results obtained were in accordance with those found for hGSTP1-1,
further emphasizing that the highly conserved N-capping box and
hydrophobic staple motifs play critical and universal roles in GST
folding and stability. The
6-helix and preceding loop form a
substructure, named GST motif II, which is conserved in the core of all
GSTs and related proteins. The analysis of the protein crystal
structure indicates that GST motif II is stabilized by a buried network
of eight hydrogen bonds, half of which involve water-mediated contacts
(23). Crystallographic studies of the capping mutants, expressed at the
permissive temperature of 25 °C, indicated that these amino acid
substitutions locally destabilize GST motif II through a partial or
complete loss of the hydrogen bond network (23). All these results
indicate that a local destabilization of GST motif II has a critical
effect on the overall protein stability and strongly support the
hypothesis that this buried region might be involved in the nucleation
mechanism of protein folding.
A strictly conserved glycine residue, Gly-146, in hGSTP1-1 is located
four residues before Ser or Thr of the Ncap motif in all known GSTs
(1). In particular Gly-146 is part of a buried local sequence
GXXh(T/S)XXDh (X is any residue and h
is a hydrophobic residue), which is maintained in all GSTs and, as a
more general folding module, in other proteins such as EF1
and URE2.
Its role has until now remained unexplored. This amino acid residue is located in a bend of the long loop preceding the
6-helix and does
not make any specific contacts with other structural parts of the
molecule but only with neighboring residues in the polypeptide. Its
strict conservation through evolution implicates the absolute necessity
of a small amino acid residue in this position. The present
investigation addresses the function of the strictly conserved Gly-146
in folding, and its significance for structural stability. By a
combination of protein engineering and x-ray structure-function analysis we have obtained evidence that Gly-146 is part of a conserved folding module. As a universal motif it plays a critical role in the
refolding and stability of all GSTs and, probably, of other structurally related proteins such as EF1
and URE2.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Wild-type human GSTP1-1 was obtained by
expression of a cloned cDNA in Escherichia coli XL-1
Blue (Stratagene, La Jolla, CA) as previously described (24). GSH and
1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Sigma.
Oligonucleotides and dNTPs were obtained from Amersham Biosciences.
Construction of GSTP1-1 Mutants--
Site-directed mutagenesis
was employed to generate single-point mutants. The following
oligonucleotides were used: 5'-CC TTC ATT GTG A/GC/TA GAC
CAG-3' and 5'-TCT TGC CTC CCT GGT TCT G-3'. The altered codon is
underlined. The oligonucleotides were phosphorylated and then used in
inverted polymerase chain reaction. The expression clone pKHP1 (24) was
used as a template. The polymerase chain reaction mixture contained 0.8 µM each primer, 0.2 mM dNTPs, 2.5 units of
Pfu DNA polymerase (Stratagene, La Jolla, CA); the buffer supplied with the enzyme and various amounts of DNA template. The
temperature program started at 94 °C for 10 min and was followed by
25 cycles of 94 °C for 1 min, 70 °C for 1 min, and 72 °C for 9 min. The program terminated with a reaction at 72 °C for 30 min.
After electrophoresis the DNA product from the reaction was recovered
from the agarose gel. The DNA was ligated and used for transformation
of competent E. coli XL-1 Blue cells. The cDNAs encoding
the isolated GSTP1-1 mutants were sequenced in their entirety to verify
that no undesired mutations had been introduced in the polymerase chain reaction.
Protein Expression and Stability As a Function of Growth
Temperature of the Host Cells--
Cultures of E. coli XL-1
Blue-containing plasmids were grown in 300 ml of LB broth in a 1-liter
Erlenmeyer flask at 37 °C. At an OD555 of 0.35, isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.2 mM. From the time of addition the culture was grown for about 20 h at 25 or 37 °C. The
subsequent purification of wild-type and mutant proteins was performed
as described previously (24) with the only modification that the enzymes were purified on a GSH-Sepharose affinity column (25).
Kinetic and Structural Studies of Wild-type hGSTP1-1 and Gly-146
Mutants Expressed at 25 °C--
The kinetic parameters,
kcat and Km, were determined
at 25 °C as previously described (26). The concentrations of GSH and
CDNB were 2 and 1 mM, respectively. Spectroscopic
properties of the mutants and the wild-type enzyme were also studied. A
Jasco-600 spectropolarimeter was used for CD measurements in the
far-ultraviolet region from 200 to 250 nm. Spectra were recorded using
a protein concentration of 0.3 mg/ml with cuvettes of 0.1 cm path
length in a thermostat-controlled cell holder. Intrinsic fluorescence emission spectra were measured with a Spex (model Fluoromax)
spectrofluorometer. The excitation wavelength was 280 nm and the
max and all emission spectra were analyzed at the same
protein concentration (0.1 mg/ml).
Heat Inactivation Assays--
Enzyme was incubated at each
temperature for 10 min at a protein concentration of 0.05 mg/ml in 0.01 M potassium phosphate (pH 7.0) containing 1 mM
EDTA and 5 mM dithiothreitol to prevent oxidative
inactivation. The enzyme was heat-inactivated in sealed Eppendorf
tubes, and the temperature was monitored with a Cryson telethermometer.
The inactivation time courses were determined by withdrawing suitable
aliquots at different time points from the denaturation mixture for
assay of remaining activity. The activity was assayed in 0.1 M potassium phosphate (pH 6.5) with 2 mM GSH
and 1 mM CDNB at 25 °C. Lowering the temperature of
incubation could not reverse the thermal inactivation for any of the proteins.
Kinetics of Thermal Denaturation--
The denaturation of the
wild-type and glycine mutants was monitored at different temperatures.
The enzymes were incubated in 10 mM potassium phosphate, pH
7.0, 1 mM EDTA, 5 mM dithiothreitol, and their
activity was monitored for 120 min taking the first value as 100%
native protein. An equation describing a single exponential decay with
a rate constant of thermal unfolding ku was
fitted to the data according to Equation 1.
|
(Eq. 1)
|
The free energy of activation of thermal unfolding
(
Gu) was calculated according to Eyring
theory (27) as Equation 2,
|
(Eq. 2)
|
where kb is the Boltzmann constant; T, the absolute
temperature in Kelvin; h, Plank's constant; R, the gas
constant; and K is the transmission factor, which was set to
unity. The difference of free energy of activation of thermal
denaturation between wild-type and each mutant protein
(
Gu) was calculated according to Equation 3.
|
(Eq. 3)
|
Substitution of Equation 4,
|
(Eq. 4)
|
into Equation 2 yields Equation 5.
|
(Eq. 5)
|
Both activation enthalpy
Hu and
entropy
Su were determined from the
temperature dependence of ku.
Temperature Dependence of Refolding in Vitro for Wild-type and
Gly-146 Mutants--
When the refolding of human GSTP1-1 and its
mutants was to be monitored, 10 µM enzyme was first
denatured in 4 M guanidinium chloride (0.2 M
potassium phosphate, 1 mM EDTA, 5 mM
dithiothreitol, pH 7.0) at 25, 33, and 40 °C for 30 min and then
diluted (defining time 0) 1:40 into renaturation buffer (0.2 M potassium phosphate, 1 mM EDTA, 5 mM dithiothreitol, pH 7.0) at the same temperature. The
final guanidinium chloride concentration was 0.1 M during refolding. All refolding experiments were carried out by rapid addition
of the denatured enzyme to renaturation buffer. Activity recovered as a
function of time was monitored by withdrawal of appropriate aliquots of
the renaturation mixture followed immediately by dilution into 2.0 ml
of assay buffer. The kinetic parameters of refolding were determined by
non-linear regression analysis by fitting equations with one or two
exponentials to the experimental data using the KaleidaGraph 3.0.5 program (Abelbek Software). The values reported in this study represent
the means of at least three different experimental data sets.
Refolding Yields at Different Protein Concentrations--
The
influence of concentration on folding was analyzed by refolding the
wild-type and Gly-146 mutants as described in the previous section. Six
different protein concentrations were used for denaturation ranging
from 0.2 to 6.0 mg/ml. Refolding was monitored as described above by
assaying for recovered activity after 2 h.
Effect of a Second Dilution on Refolding Yields--
A second
dilution during refolding was performed at different time points after
the initiation of reactivation. Enzymes at an optimized concentration
(1 mg/ml) were denatured and allowed to start to refold as described
above. After 1 min (wild-type) and after 5 and 20 min (all enzymes),
the refolding mixture was diluted 10-fold and analyzed for reactivation
by measuring increase of activity with time.
Molecular Graphics Analysis and Computer Search for Structural
Motifs--
Coordinates of GST x-ray structures were derived from the
Protein Data Bank (www.rcsb.org/pdb/) via the anonymous file-transfer protocol. The crystal structures were analyzed by using Hyperchem (Autodesk, Sausalito, CA) and MolView 1.4.6 (Purdue University) programs. The figures were generated using the RasMol (version 2.6)
program. A PHI-BLAST (28) (www.ncbi.nlm.nih.gov/blast/psiblast.cgi) search was performed using the BLOSUM62 matrix. The pattern used as a
query was GX(2)-[LIVY]-[ST]-X(2)-[D]-[LYIVA] and the threshold value was set to 0.001.
Crystallization--
Crystallization was performed by the
hanging drop vapor diffusion method as described elsewhere (29).
Briefly, a 2-µl drop of a protein solution containing the GST mutant
(4.2 mg/ml for G146V, 2 mg/ml for G146A) in 1 mM EDTA, 1 mM dithiothreitol, and 10 mM HEPES buffer (pH
7.0) was mixed with an equal volume of reservoir solution, which
consisted of 15-25% (w/v) polyethylene glycol (PEG) 8000, 20 mM CaCl2, 1 mM GSH, 10 mM dithiothreitol, and 100 mM MES buffer (pH
range 5.2-5.8). All trials were carried out at a constant temperature
of 22 °C. Crystals took between 3 and 5 days to appear and grew to
their final size within 2 weeks. The x-ray diffraction data were
collected using a MARResearch area detector with CuK
X-rays generated by a Rigaku RU-200 rotating anode x-ray generator. The
data were collected at 100 K. The diffraction data were processed and
analyzed using programs in the HKL (30) and CCP4 suites (31). The G146A
and G146V mutants crystallized in the same space group and cell as wild type.
Structure Determination--
For both mutants the refinement
began with wild-type Pi class GST in the C2 space group (10GS; Ref. 29)
that had inhibitor and water molecules omitted. In addition residues
between 144 and 150 in each monomer were removed from the starting
model. Rigid body refinement in CNS (32) was used to compensate for any
possible changes in crystal packing. As the asymmetric unit of the
crystal contained two GST monomers, use was made of the non-crystallographic symmetry (NCS) restraints on all non-hydrogen atoms throughout the course of the positional refinements. The 2Fobs
Fcalc map of the
G146A mutant showed clear and continuous density for residues between
144 and 150 that had been omitted from the search model. These residues
were then built into the map. Among the significant features in the
subsequent Fobs
Fcalc map were positive peaks (close to 4× the r.m.s. error of the map) within 2 Å of the C
of Gly-146, thus confirming the mutation. The
residue at position 146 was changed from glycine to alanine and the
model refined. The 2Fobs
Fcalc and Fobs
Fcalc maps of the G146V mutant clearly indicated
a change in the backbone conformation of the region between residues
144 and 146. After the backbone alterations were made, there was
additional density next to the C
position of residue 146 sufficient
to accommodate a valine side-chain (close to 3× the r.m.s. error of
the Fobs
Fcalc map),
and hence the glycine residue was changed to a valine residue. These
residues were then built into the map of the G146V mutant. For both
mutants, a number of rounds of positional refinement were performed
followed by model building and then by rounds of positional and
individual NCS-restrained B-factor refinement. In the final stages of
refinement a bulk solvent correction was employed. The correctness of
the final structures in the regions around the mutation were confirmed
by calculating omit maps between residues 144 and 148. A stereochemical
analysis of the refined structure with the program PROCHECK (33) gave
values either similar or better than expected for structures refined at
similar resolutions. The coordinates for the G146A and G146V models
have been deposited in the Protein Data Bank with accession numbers 1MD3 and 1MD4, respectively.
 |
RESULTS |
Expression and Purification of Wild-type and Gly-146 Mutants of
Human GSTP1-1--
To investigate the role of the buried and conserved
glycine residue (Gly-146, in hGSTP1-1) this amino acid was replaced
with alanine and valine by oligonucleotide-directed mutagenesis
producing the mutants G146A and G146V. Mutant and wild-type enzymes
were expressed in E. coli XL-1 Blue and purified by affinity
chromatography on immobilized GSH. The purified enzymes gave a single
band on SDS-PAGE (not shown). 65-90% of the total activity was
recovered showing that the affinity for GSH-Sepharose was essentially
unaffected by the mutations. Considering that the above substitutions
could represent temperature-sensitive mutations, protein expression was
performed both at 25 and 37 °C of host cell growth. No dependence of
protein yield on temperature could be observed (not shown). The yields
of the G146A and G146V mutants, in percentage of total cytosolic
proteins, were 2.4 and 1.7% respectively after purification, both
slightly lower than that (4.0%) of the wild-type enzyme.
Kinetic and Structural Characterization of Wild-type Human GSTP1-1
and Gly-146 Mutants--
Table I
summarizes the kinetic parameters for the conjugation of CDNB to GSH
catalyzed by the wild-type hGSTP1-1 and Gly-146 mutants. The
replacement of Gly-146 with either alanine or valine significantly
increases the kcat values of the corresponding
mutants as compared with that of the parental enzyme. The
Km values of the mutants were 1.5-2-fold higher for
both substrates than those of the wild type. This suggests that the
above substitutions slightly but significantly decrease the affinity
for GSH and CDNB in both mutants. The physical properties of the
Gly-146 mutants and the wild-type enzyme were very similar. The far-UV
CD spectra as well as their gel filtration retention times (not shown)
were the same, suggesting that all enzyme variants are, in terms of secondary structure and dimeric state of the molecule, essentially identical. The
max values of the intrinsic fluorescence
spectra were 338, 338, and 339 nm for the wild-type enzyme and the
G146V and G146A mutants, respectively (not shown), suggesting that a similar polarity characterizes the environment of the tryptophan residues of all enzyme variants. The normalized intensities of the
fluorescence of the mutants were slightly higher than that of the
wild-type enzyme. This indicates that limited conformational changes
distinguish the final structure of the mutants from that of the parent
enzyme. These differences involve the environment of one or both
tryptophan residues located in GST domain I, far from the mutation
site.
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Table I
Kinetic parameters of wild-type and glycine mutants of hGSTP1-1
heterologously expressed at 25 °C in E. coli
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Structure of Mutant G146A--
The structure of helix
6 and the
preceding loop is well defined in the wild-type 1.9 Å resolution
electron density maps (29). The region encompassing the loop (residues
141-149) and the N-terminal end of the helix is characterized by a
network of eight hydrogen bonds between the two, half of which
involving a water-mediated contact (Fig.
1A). In addition to these
contacts, other significant polar contacts involving the loop include
the backbone carbonyl moiety of Gly-146 forming hydrogen-bonding
interactions with the side-chains of Asn-137 and Thr-142 and the
side-chain of Gln-148 forming water-mediated contacts with the backbone
of Ile-149, Gly-78, and Gly-81. Gly-146 adopts
/
angles of 70°,
163° lying within an allowed region for glycine residues in the
Ramachandran plot (33). Statistical data generated from the structure
determination of the G146A mutant are presented in Tables
II and III. The G146A structure is
essentially identical to the wild-type
structure with an r.m.s. deviation on superposition of
-carbon atoms
of 0.2 Å and no deviations greater than 0.6 Å. Ala-146 adopts
/
angles of 56°,
143° that lie within the additionally allowed region of the Ramachandran plot (33). The extensive network of
interactions seen in the wild-type structure in the region of the loop
is maintained in the mutant structure (Fig. 1B). Hence the
only obvious difference between the two structures is that the mutated
residue lies in a higher energy conformation than wild type as judged
by its location in the Ramachandran plot.

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Fig. 1.
Stereoviews of the region in GST P1-1 about
the sites of mutation. A, wild-type structure (10GS;
Ref. 29); B, G146A; and C, G146V. Only key
side-chains are shown for reasons of clarity. These figures were
produced using BOBSCRIPT (41).
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|
Structure of Mutant G146V--
Statistical data generated from the
structure determination of the G146V mutant are presented in Tables II
and III. Most of the G146V structure superimposes closely on the
wild-type structure (r.m.s. deviation on C
positions of about 0.3 Å). However, within the region of the mutation (residues 144-147) the
r.m.s. deviation rises to 0.6 Å (Fig. 1C). In particular
there are shifts of about 1 Å for residues 145 and 146. Also
deviations between 0.5 and 0.8 Å occur in the region encompassing the
first turn of helix
4 (residues 82-86), which abuts onto the long
loop of GST motif II. The origin of these shifts can be traced to
changes in the backbone conformation of the mutated residue. In the
mutant structure the backbone conformation of residue 146 has changed
considerably with the introduction of the valine side-chain. The
/
angles for Val-146 are
124°,
177° (compared to wild
type of 70°,
163°) which falls in the additionally allowed region
of the Ramachandran plot (33). The other significant change occurs at
Val-145 where the angles in the mutant structure are
144°,
25°
compared with the wild-type structure where they are
141°, 124°.
These changes have led to some changes in protein contacts. The
critical water molecule in the loop region has lost three potential
hydrogen-bonding contacts with Val-145 and Ser-150 although it has
gained a new hydrogen-bonding interaction with the main-chain of
Gln-148. The loss of contact with the side-chain of Ser-150 appears to
be due to a movement of the water molecule away from this residue
caused by loss of contacts with Val-145. Surprisingly, the backbone
carbonyl moiety of Val-146 is still in hydrogen-bonding contact with
Asn-137 (but not with Thr-142) despite the change in backbone
conformation. Despite all these changes the temperature factors of the
loop are not significantly higher than for the wild-type protein.
Thermal Stability of the Wild-type and Mutant Enzymes--
Heat
inactivation of wild-type and Gly-146 mutants, expressed at 25 °C,
was investigated. The results indicate that single Gly-146
substitutions with hydrophobic residues cause very large effects on the
catalytic competence even though the active site is situated far from
the location of the mutations. Both mutants were significantly
destabilized compared with the wild-type enzyme, by being partially
inactivated already at subphysiological temperatures (Fig.
2). Furthermore, the G146V mutant was
much more unstable than the wild type and the G146A mutant (Fig. 2). It
is important to note that the mutants became unstable at a lower
temperature than the wild-type; at 32 and 37 °C the mutants were
unstable, and their inactivation rates were dependent on the protein
concentration (not shown). Different experiments, performed by
gel-filtration and electrophoresis techniques, made to clarify whether
glycine mutations favor the dissociation into monomers during the
thermal inactivation of mutants failed because of the formation of
protein aggregates under the experimental conditions used. In all
experiments 0.25 µM of each enzyme was incubated at the
different temperatures, and this was also found to be the optimal
protein concentration for reversible refolding (Fig. 4). Thermal
denaturation of all variants was irreversible implying that
inactivation kinetics cannot be used to determine thermodynamic
parameters at equilibrium. However making use of the temperature
dependence of the unfolding rate, the application of Eyring formalism
provides the thermodynamic parameters of the activation barrier of the
thermal denaturation. As shown in Fig. 3
the heat inactivation of all variants is described by straight lines in
the Eyring plot. This indicates that the temperature dependence of both
the unfolding activation enthalpy
H, and activation
entropy
S is negligible. The temperature dependence of
G is reflected in the slope of the linear fit, dependent
on the
H. For the G146A mutant (Table
IV) the large energetic change compared
with the wild type (
H 95 kJ/mol) is almost completely compensated by an accompanying reduction of
S. This
corresponds, at 40 °C, to an activation barrier for the G146A mutant
about 9 kJ/mol lower than that of the wild type. An even lower value of
the unfolding free energy (Table IV) was observed for the G146V mutant
(
G 13.69 kJ/mol). However this is the result of an
unfolding
H value (226 kJ/mol) for the G146V mutant
similar to that of the wild-type protein (228 kJ/mol), while the
S determination (0.341 kJ/mol·K) was found to be higher
than the corresponding value for the parent enzyme (0.304 kJ/mol·K).

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Fig. 2.
Thermal stability of wild-type
(A), G146A (B), and G146V
(C) mutants at different temperatures. Each
enzyme (10 µM) was incubated at the various temperatures
for 120 min in 10 mM potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM dithiothreitol. Appropriate
aliquots from this incubation mixture were assayed at 25 °C to
monitored the residual activity. The lines represent linear
fits according to Equation 1, as reported under "Experimental
Procedures."
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Fig. 3.
Eyring plots of wild-type ( ), G146A ( ),
and G146V ( ). The lines represent linear fits
according to Equation 5, as reported under "Experimental
Procedures."
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Table IV
Kinetic and thermodynamic parameters for the activation barrier of
thermal denaturation for wild-type-GSTP1-1 and glycine mutants
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Effect of Protein Concentration on the Final Recovery of Native GST
after Refolding from 4 M Guanidinium Chloride--
To
identify the optimal protein concentration for reversible refolding
studies, a series of different concentrations were tested (Fig.
4). At low protein concentration (< 5 µg/ml), both wild-type and mutant enzymes appeared to be unstable
since the final yield was significantly reduced. At protein
concentration above 25 µg/ml, the yield was compromised, presumably
due to aggregation, a phenomenon that has been reported for other
proteins. Maximal refolding yields of 75-90% were observed at protein
concentrations in the range of 5-15 µg/ml.

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Fig. 4.
Effect of hGSTP1-1 concentration on the final
yield of active enzyme following dilution from 4 M
guanidinium chloride. hGSTP1-1 was permitted to refold at the
concentration indicated for 2 h at 25 °C after rapid 40-fold
dilution from 4 M guanidium chloride into renaturation
buffer (0.2 M potassium phosphate, 5 mM
dithiothreitol, pH 7.0). The different symbols represent the mean of
three different experiments and correspond to wild-type ( ), G146A
( ), and G146V ( ). Percent recovery is expressed relative to the
activity of the native control sample at each concentration diluted
into the same renaturation buffer with 0.1 M guanidinium
chloride added, and incubated for the same period of time.
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Reactivation Yields at Different Temperatures--
Replacing
Gly-146 with amino acids containing larger hydrophobic side-chains
could result in temperature-sensitive mutants with impaired folding
properties. To test this hypothesis, we have investigated the
reactivation yields of hGSTP1-1 and its mutants at different
temperatures of refolding. 10 µM enzyme, expressed at
25 °C, was denatured in 4 M guanidinium chloride for 30 min. This denaturant concentration was sufficient to completely unfold
the protein as indicated by the loss of their CD signal at 222 nm (data
not shown). The unfolded enzyme was diluted 40-fold with phosphate
buffer at pH 7.0 to a final guanidinium chloride concentration of 0.1 M. Appropriate aliquots from this incubation mixture were
immediately assayed for activity at 25 °C. Fig.
5 shows that the reactivation yields of
the Gly-146 mutants were very different from that of the wild-type
protein at increasing temperatures of refolding. While the reactivation
yield of the wild-type enzyme was essentially unaffected by
temperatures in the 25-40 °C range, the yields of the mutants,
albeit to different extents, decreased markedly with temperatures
increasing toward physiological values. At 25 °C, when the specific
activity recovered by both mutants was significantly higher than that
of the wild type, all enzyme variants were characterized by a similar
reactivation yield (80-90% of initial activity). Both mutants showed
a lower refolding yield than the wild-type enzyme already at 33 °C.
At 40 °C the mutants, in contrast to the wild-type enzyme, displayed very poor or complete lack of refolding capacity even at the very beginning of the refolding reaction. It should be noted that during the
time course of reactivation competing inactivation reactions also take
place. However, most of the activity is regained in a few minutes when
the inactivation of both mutants is negligible. Thus, these results
seems to suggest that single substitutions of Gly-146 also destabilize
the folding of GSTP1-1. This possibility is further supported by the
analysis of the formation of aggregates (Fig.
6), as followed by measuring the apparent
absorbance (turbidity) at 360 nm. During the reactivation at 37 °C
of the G146V mutant, detrimental aggregation reactions are observed
even at the very beginning of the refolding reaction. In contrast, the
thermal inactivation of this variant, at the same temperature,
generated large aggregates only at extended times of incubation (30 min). This means that the destabilization of a folding intermediate causes faster aggregation than during the unfolding of the final structure. Thus, the results could suggest that substitution of the
conserved Gly-146 not only affects the stability of the final folded
protein but, to a larger extent, also destabilizes a productive intermediate of folding.

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Fig. 5.
Kinetics of the reactivation of
wild-type hGSTP1-1 ( ), G146A ( ), and G146V ( ) during the
refolding at different temperatures. Purified enzyme (10 µM), heterologously expressed at 25 °C, was first
denatured in 4 M guanidinium chloride at 25, 33, and
40 °C respectively for 30 min. This denaturant concentration was
sufficient to completely unfold the proteins, as indicated by the loss
of their CD signal at 222 nm (not shown). Successively each unfolded
enzyme was diluted (defining time 0) 1:40 into renaturation buffer (0.2 M potassium phosphate, pH 7.0, 5 mM
dithiothreitol) at the same temperature as during denaturation. The
final guanidinium chloride concentration was 0.1 M during
refolding. Appropriate aliquots from this incubation mixture were
immediately assayed for catalytic activity at 25 °C.
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Fig. 6.
Aggregation of the wild-type
( ), G146A mutant ( ), and G146V mutant
( ) during refolding. The proteins were incubated in
10 mM potassium phosphate pH 7.0, 1 mM EDTA at
36 °C for 60 min, and the aggregation was determined by monitoring
the energy loss of the transmitted light (apparent absorbance) at 360 nm.
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Kinetics of Reactivation at 25 °C--
A single-exponential
equation could be fitted to the kinetic reactivation curve of the
wild-type enzyme at 25 °C (Fig. 7). In
contrast, as shown in Table V, the
refolding kinetics for the G146A and G146V mutants were better
described by a double-exponential equation. The attempt to fit a
single-exponential equation to the reactivation data of the mutants
resulted in higher chi-square values of at least one order of
magnitude. The initial part of the refolding curve for the mutants
corresponds to an exponential phase with rate constants of 0.29 min
1 and 0.31 min
1 for the G146V and G146A
mutants, respectively, which are lower than the single-exponential rate
constant of the wild-type enzyme (0.43 min
1). Their
relative amplitudes were 73 and 75% for the G146V and G146A variants,
respectively. Rate constants of 0.015 min
1 and 0.010 min
1 and relative amplitudes of 27 and 25% characterized
the slower phase of the reactivation for the G146V and G146A mutants,
respectively. The reactivation kinetics were also analyzed by following
the recovery of the intrinsic fluorescence during refolding of wild type and the G146V mutant. As shown in Fig.
8, a blue-shift and concomitant quenching
characterized the spectra of both variants at the beginning of
refolding. However, while no changes in the spectra of the wild-type
protein could be observed after 10 min of refolding, significant
changes in the intrinsic fluorescence of the G146V mutant was seen even
after 120 min. These results are in accordance with the reactivation
kinetics and clearly indicate that the substitution of Gly-146 by
valine causes the formation of a second refolding step. In order to
better identify the molecular species characterizing this slower phase
of refolding another approach was tried. 20 min after the initiation of
refolding, when the contribution of the first rapid phase was
negligible, a second 10-fold dilution of the protein was performed.
Rate and yield of refolding were essentially identical to that obtained during refolding without secondary dilution for all protein variants. In contrast to the results with the wild-type enzyme, the final refolding yield of the mutants was significantly reduced when the
second dilution was performed after 5 min (Fig. 7). This is in
accordance with the results of the concentration-dependence analysis
(Fig. 4) in which a lower protein concentration results in a lower
overall yield of reactivation of mutants as compared with the wild
type. In addition, because of the higher refolding rate constant, more
than 90% refolding is reached by the wild type at 5 min of
reactivation. The results also show that the refolding rates were
unaffected by the secondary dilution.

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Fig. 7.
Secondary 10-fold dilution of wild-type
hGSTP1-1 (A), G146A (B), and G146V
(C) during refolding. All hGSTP1-1 variants were
diluted 40-fold from 10 µM of enzyme in 4 M
guanidinium chloride to 0.25 µM in the renaturation
buffer at time 0. After 5 and 20 min of refolding, an aliquot was
further diluted 10-fold into recovery buffer to yield 0.025 µM of enzyme. The time course of activity recovery after
the first dilution ( ) and 5 min ( ) and 20 min ( ) after the
second dilution were monitored by removal of an appropriate aliquot
from each incubation mixture and immediately assayed for catalytic
activity at 25 °C.
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Table V
Kinetics of the reactivation of the wild-type and Gly146 mutants during
the refolding at 25 °C
A single-exponential (wild-type) and double-exponential (G146A and
G146V) equation could be fitted to the kinetic reactivation curve.
k1ref and k2ref, A1 and
A2 represent the rate constants and their relative amplitudes
of the faster and the lower reactivation phase, respectively.
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Fig. 8.
The refolding of wild-type
(A) and G146V mutant (B) monitored by
fluorescence measurements. Purified enzyme (10 µM)
was first denatured in 4 M guanidinium chloride at 25 °C
for 30 min. Successively each unfolded enzyme was diluted (defining
time 0) 1:40 into renaturation buffer (0.2 M potassium
phosphate, pH 7.0, 5 mM dithiothreitol) at the same
temperature as during denaturation. The refolding was monitored by
fluorescence measurements at an excitation wavelength of 280 nm. For
each spectrum, the acquisition time was 15 s.
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Molecular Graphics Analysis and Computer Searches for Local
Motifs--
Previous sequence analysis showed that only two residues
are strictly conserved in domain II (C-terminal domain) in all GSTs. These are Asp-153 and Gly-146, both located in the hydrophobic core of
the molecule distant from the active site. Structure analysis showed
that Asp-153 belongs to a local motif ((S/T)XXD) named the
N-capping box that is strictly conserved at the N terminus of the
6-helix in GSTs. Studies performed by the peptide approach (1) and
site-directed mutagenesis (2) verified that this motif has a crucial
role in folding and stability of hGSTP1-1. The sequence analysis also
revealed the presence of a second motif at the N terminus of the
6-helix (1). This consists of a specific interaction made by two
hydrophobic residues flanking the residues that constitute the capping
box (hydrophobic staple motif). Recent work shows that this specific
hydrophobic interaction serves to enhance the rate of protein folding
and to define the folding pathway (3). The fourth residue preceding the
Ncap, a glycine residue, is conserved in all GST sequences (Gly-146 in
hGSTP1-1). This residue is possibly playing a joint role with the above
described structural motifs in forming a folding nucleus that initiates the folding process. Gly-146 is partially buried (solvent accessible surface, 9.0 Å2), and it is located in a bend of the long
loop preceding the
6-helix (Fig. 9). A
PHI-BLAST search (28) was performed by using the conserved structural
motif GXXh(S/T)XXDh (X is any residue and h is a hydrophobic residue) as a query. It was found that this
sequence is strictly conserved in all GSTs (Fig.
10). With a threshold value of 0.001, 115 hits were obtained. 104 were representing GSTs, 6 hits
represent probable GSTs or GST-related sequences and 3 encode cDNAs
for which there are no known translation products. At higher threshold
values, sequences of known GST-related proteins such as translation
elongation factor 1
, bacterial dichloromethane dehalogenase, and
lignin-degrading
-etherase were identified. These results strongly
suggest that the residues forming the above motif, which includes the
non-catalytic residues conserved in domain II in all GSTs, create a
specific pattern that serves to generate a characteristic
alpha-loop-alpha supersecondary structure strictly maintained only in
the core of GSTs.

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Fig. 9.
Ribbon picture of a monomer of human Pi class
GST. The location of GST motif II (the conserved sequence motif
consisting of helix 6 and the preceding long loop) is rendered in
dark yellow. GSH and the site of mutation are shown in
ball-and-stick. This figure was produced using
BOBSCRIPT (41).
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Fig. 10.
The conserved folding module of GST.
Sequence alignment of amino acid residues belonging to helices 5 and
6 of Alpha, Mu, Pi, Theta, and Sigma classes GST, of distant members
of GST and GST-related proteins. The highly conserved capping box and
hydrophobic staple motif residues are shown in blue and
green, respectively. The glycine residue, always located
four residues before the Ncap, is shown in red. The
conserved and buried local pattern identified by the PHI BLAST program
is also reported. X denotes any residue and h is
a hydrophobic residue. The corresponding three-dimensional model of the
conserved folding module (GST motif II) is shown in the upper
panel. The aspartic acid forms internal hydrogen bonds important
for GST motif II stabilization.
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DISCUSSION |
Gly-146 is one of few residues that are strictly conserved in the
GST superfamily. More specifically, Gly-146 is part of a buried local
sequence GXXh(T/S)XXDh, which is maintained in
all GSTs and, as a more general folding module, in other proteins such
as EF1
and URE2. Previous investigations indicated that single
mutations of capping box (T/S)XXD and hydrophobic staple (h)
residues cause similar dramatic effects on the folding and stability of
hGSTP1-1 and hGSTA1-1 (2, 3, 21, 23). Gly-146 is always located four
positions before the first Ncap residue (S/T), and its role has not
previously been investigated. In the present work the G146A and G146V
mutants were generated by site-directed mutagenesis in order to
investigate the possible function played by this strictly conserved
amino acid residue for folding and stability of human GSTP1-1.
Gly-146 is located in a hydrophobic region, and the choice of alanine
was considered the most conservative replacement. The introduction of
valine was expected to require more space and therefore could be more
disruptive. The steady-state kinetic parameters of both GST variants
were very similar at 25 °C to those of the wild-type enzyme (Table
I). This similarity was in accordance with the data obtained by CD, gel
filtration, and intrinsic fluorescence experiments. These results
indicate that the overall structure and dimeric state were the same,
suggesting that the global fold of mutants and wild-type GST were
similar. However, both mutants, as compared with the wild-type enzyme,
were characterized by a higher sensitivity toward heat inactivation.
The mutants, expressed at the permissive temperature of 25 °C, have
been crystallized and their three-dimensional structures determined by
x-ray crystallography. No structural change to the protein was caused
by the G146A mutation and the principal interactions formed between the
main-chain carbonyl group with Asn-137 and Thr-142 were preserved.
Nevertheless, the Ala-146 residue lies in a higher energy conformation
than wild type as judged by its location in the Ramachandran plot. This is sufficient to destabilize the active site at 37 °C, even though it is located far from the mutation site. Due to a large decrease in
activation enthalpy (
H 95 kJ/mol), the destabilizing
effect of the G146A mutation was less important at higher temperatures but clearly significant at subphysiological and physiological ones. In
addition, the magnitude of T
S at 40 °C (87 kJ/mol) indicates that major changes in the conformational freedom
characterizes the denaturation of the wild type compared with those
occurring during the thermal inactivation of the G146A mutant. This
means that the wild-type hGSTP1-1, because it is more rigid than the G146A mutant, may tolerate larger perturbations of its structure before
the unfolding transition state is reached. The crystallographic data
also indicate that the mutation of Gly-146 to valine, unlike the change
to alanine, had to result in a substantial change of the backbone
conformation because otherwise the valine side-chain would have
sterically clashed with Glu-86. Thermal inactivation measurements
indicate that these local structural changes, observed at 25 °C, had
destabilizing effects at temperatures as low as 32 °C. The decrease
in the unfolding free energy (14 kJ/mol) for the G146V mutant is higher
than that estimated for the G146A variant. It is important to note that
the entropic contribution of thermal inactivation of the G146V mutant
is higher than it is to the wild type and G146A mutant and is the
driving force for the denaturation of this variant. Thus the thermal
inactivation of this mutant causes perturbations of the active site
structure, which are larger than those experienced in similar unfolding
conditions by the other variants and more readily leads to a more
denatured state of the protein.
The
6-helix and preceding loop form a substructure (GST motif II),
which is conserved in the core of all GSTs. It is stabilized by a
network of eight hydrogen bonds with half involving a water-mediated contact and makes interdomain contacts with the
1-helix which is an
important structural element of the active site. Previous investigations indicated that single mutations of residues forming the
capping box ((S/T)XXD) and hydrophobic staple motif, located at the N-terminal of the
6-helix, have dramatic effect on the stability of hGSTP1-1 and hGSTA1-1. X-ray structure analysis of the
capping mutants, expressed at the permissive temperature of 25 °C,
indicated that these amino acid substitutions locally destabilize GST
motif II through a partial or complete loss of the buried hydrogen bond
network. Glycine 146 is located in the bend of the long loop (residues
141-149) preceding the
6-helix (Fig. 9). The crystallographic and
thermodynamic analyses of the G146V mutant confirm that alterations of
the buried hydrogen bond network inactivate the enzyme already at
temperature as low as 32 °C. In this region, a glycine residue must
be maintained at position 146 principally because of lack of space.
However, even the more conservative Gly/Ala substitution, which does
not alter the protein structure at 25 °C, inactivates the enzyme at
37 °C. It is important to note that the formation of a hydrogen bond
network in the interior of the protein poses geometric constraints that
have to be reconciled with the geometric constraints of the dense
packing. A major flexibility of the main-chain, due to the presence of
a glycine residue, permits the maintenance of the internal hydrogen
bond network even at higher temperatures, despite larger motions of the
side-chains. Thus the present results, in accordance to previous
findings, confirm the critical role of GST motif II substructure for
the overall protein stability. In addition they strongly support the hypothesis that GST motif II, stabilized by a characteristic hydrogen bond network, behaves as a general structural motif whose precise conformation serves to stabilize GSTs and, likely also other proteins, in the physiological range of temperature.
The in vitro refolding experiments suggest that the thermal
lability of the final structure of the mutants reflects differences in
the conformational properties of a productive intermediate of folding.
Reactivation in vitro of both mutants, in fact, was thermosensitive with the most pronounced effect for the G146V variant.
It should be noted that during the time course of reactivation competing inactivation reactions also take place. Thus, although most
of the catalytic activity is regained in a few minutes, a time period
in which the inactivation of the enzyme is negligible, it is difficult
to decide whether single substitutions of Gly-146 generate temperature
sensitive folding mutants. However this possibility is further
supported by the analysis of the formation of aggregates (Fig. 5), as
followed by measuring the apparent absorbance at 360 nm. During the
reactivation at 37 °C of the G146V mutant, large aggregates are
formed faster than those that can be observed during the thermal
inactivation of this variant at the same temperature. Thus, the results
indicate that substitution of the conserved Gly-146 not only affects
the stability of the final folded protein, but also favors detrimental
aggregation events during refolding. This also means that the fourth
position before the Ncap motif must be maintained as a glycine residue
because of its essential contribution to a productive folding pathway.
Its substitution by another residue likely alters the conformation of
an essential intermediate of folding, favoring protein aggregation at
37 °C. The fact that the Gly-146 mutations cause lesser effects on
thermal stability (Fig. 2) than on the refolding kinetics (Fig. 5) at comparable temperatures suggests that the conformation of this intermediate is more labile than that of the fully folded state. This
interpretation is confirmed by the analysis performed at a permissive
temperature (25 °C) of the refolding of mutants, as followed by
regain of activity and fluorescence. The results show that a slower
refolding step, subsequent to dimerization, becomes rate-limiting for
reactivation. It is conceivable that an altered conformation of GST
motif II during the refolding of mutants makes the formation of the
necessary interdomain interactions made by the
6-helix with the
1-helix more difficult (1-3). It has already been shown, that the
proper conformation of GST motif II is important for protein stability
and, to an even higher extent, for a productive folding pathway (2, 3).
From a more general point of view, the present results together with previous studies suggest that a well defined stereochemical code underlies the refolding of GSTs. All conserved residues forming the
local sequence G146XXh(S/T)XXDh, albeit through
different mechanisms, are critical for folding (Fig. 7). The important
role played by Gly-146 for the GST refolding is reported here. Present work supports the hypothesis that substitution of Gly-146 destabilizes a conserved loop-helix substructure that is essential during the first
refolding events. However, it cannot be completely ruled out that
substitution of Gly-146 promotes the formation of off-pathway intermediates under the in vitro refolding conditions used
here. We have previously shown, in fact, that the other conserved
residues belonging to the above local pattern form an N-capping box and a hydrophobic staple motif are important for the
6-helix nucleation and stabilization (1-3, 21). These few conserved residues are all
crucial for the formation of GST motif II that might represent a
nucleation site for the refolding of GST. It has been hypothesized that
for monomeric single domain proteins, the two-state kinetics of folding
follows a molecular growth mechanism (34-36). Buried and highly
conserved residues, making a definite set of native like contacts,
determine the formation of a specific folding nucleus that represents
the transition state of refolding (37-40). Conserved non-functional
amino acid residues in globins and in the cytochrome c
family have been implicated in the folding process of these proteins
(39-40). Certain amino acid residues are favored in a few positions
that are located far apart in the primary structure of the polypeptide
chain. Folding is initiated when these residues interact to form a
folding nucleus, which is thought to promote
-helix formation in
these particular regions from which folding then proceeds (39-40).
Thus the present work, together with our previous studies, also support
the hypothesis that even in more complex multi-domain proteins such a
specific nucleation mechanism exists. However, in GSTs, the strictly
conserved non-functional residues that play different and well defined
roles for GST folding are clustered in a linear local sequence (Fig.
10). We propose that this conserved and buried pattern represents a
general folding module that plays an important role in the refolding
and stability of GSTs and, likely, of other proteins such as EF1
and URE2.