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(Received for publication, October
2, 1995; and in revised form, December 1, 1995) From the
Previous studies showed that during the in vitro folding of Escherichia coli serine
hydroxymethyltransferase at 4 °C, both monomer and dimer
intermediates accumulated and were stable for periods of minutes to
hours (Cai, K., Schirch, D., and Schirch, V.(1995) J. Biol. Chem. 270, 19294-19299). To obtain structural information on these
intermediates, two of the three Trp residues in the protein were
changed to Phe to generate a set of three single Trp mutant enzymes.
These mutant enzymes were purified and characterized and shown to
retain essentially all of the properties of the wild-type enzyme. The
fluorescence and circular dichroism measurements of each mutant enzyme
were studied under unfolding-refolding equilibrium conditions and
during refolding. In addition, the sensitivity of the protein to
digestion by subtilisin during refolding was investigated. The results
of these studies show that the unfolded enzyme has two domains that
rapidly fold to form a monomer in which the first 55 amino acids and a
segment between residues 225 and 276 remain in a largely disordered
form. This partially folded enzyme can form dimers and slowly undergoes
a rate determining conformational change in which the unstructured
segments assume their native state.
Escherichia coli serine hydroxymethyltransferase
(eSHMT) ( In a previous study we demonstrated that eSHMT
could be reversibly refolded from 8 M urea after a 10-fold
dilution at concentrations as high as 1 mg/ml of protein(2) .
At 30 °C refolding was essentially complete in 6 min. This study
also provided evidence for several intermediates and a mechanism as
shown in . The most important information was obtained with
refolding done at 4 °C, where distinct kinetic intermediates could
be shown to exist for periods of several hours and complete refolding
took as long as 20 h. U is the unfolded enzyme and rapidly
folds in a few seconds to form a monomer M. This monomer forms
dimers (labeled as D`) during 20 min at 4 °C. The dimer D` does not bind PLP but undergoes a slow rate-determining
conformational change to a dimer (apoD) that can bind PLP to form
native holoD. At 30 °C the process of converting D` to
apoD takes a few minutes, but at 4 °C it takes many hours.
The purpose of this study was to obtain structural information
about M, D`, and apoD. Because these intermediates
exist for minutes to hours at 4 °C, a variety of physical
measurements can be made to aid in elucidation of their structure.
Important information can be obtained on the structure of intermediates
by monitoring the fluorescence properties of Trp residues. To simplify
the interpretation of results with eSHMT, we made single Trp mutants by
changing the remaining two Trp residues in this enzyme to Phe. We also
used protease digestion to determine what regions of the protein in
each intermediate had been folded into a protease-resistant form. The
results provide evidence for the rapid formation of two domains with an
NH
K Thermograms for the denaturation of each
protein were obtained with an MC-2 scanning calorimeter from Microcal,
Inc. (Amherst, MA). Protein samples were dialyzed for 24 h against
several changes of either 20 mM Tris-HCl, pH 7.5, 5 mM 2-mercaptoethanol, and 1 mM EDTA or the same buffer with
20 mM potassium phosphate replacing the Tris-HCl. Protein
concentrations were near 3.0 mg/ml for all samples. Data were analyzed
using the software supplied with the instrument. The T
In this equation Q is acrylamide concentration, F CD spectra were taken on a Jasco J-500A spectropolarimeter
as described previously(1) . The concentration of apo-eSHMT was
0.15 mg/ml for both fluorescence and CD experiments except where
otherwise indicated.
Y The equilibrium
unfolding-refolding plots were fit to either a two-state or three-state
model. In a three-state model an intermediate (I) accumulates
as shown in . For a two-state model only U and N are present. The equations used to fit each model are as
described by Matthews and Crisanti (9) .
K The solvent exposure of the Trp residue in each
single Trp mutant apoenzyme was estimated by analyzing its fluorescence
spectra(7) . The Trp fluorescence emission maxima at both 4 and
30 °C for the Trp
Figure 1:
Fluorescence emission spectra of apo
forms of wild-type and single Trp mutant eSHMTs. Each enzyme was 0.15
mg/ml in Tris buffer, pH 7.5. Excitation wavelength was 290 nm. The dashed line labeled calculated is the sum of the
three single Trp forms of eSHMT in comparison with the emission
spectrum of the wild-type enzyme.
Figure 2:
Equilibrium unfolding-refolding curves for
apo forms of wild-type and single Trp mutants of eSHMT as monitored by
CD and fluorescence. Symbols for enzyme forms for both A and B are: filled circles, wild type; open
diamonds, Trp
Fig. 2B compares the fluorescence signals of Trp residues at equilibrium
as a function of urea concentration. The data points were fit to either
a two-state or a three-state model and are represented by the solid
lines. A three-state model suggests that an intermediate (I) exists at a significant concentration between the unfolded
state U and the folded state N as shown in . The wild-type apoenzyme (solid circles) is fit
by a three-state model, suggesting that the fluorescence of Trp
residues in the intermediate are different from either the fully folded
or the unfolded state. Fig. 3shows how the relative
concentrations of N, I, and U vary with
increasing urea concentration. In general, between 1 and 2 M urea the predominate species are N (solid
circles) and I (triangles), and between 2 and 4 M urea the predominate species are I and U (open circles). I is the predominate protein
species at 2 M urea. An estimate of the free energy changes
for N to I and I to U () in the absence of urea was calculated by fitting
the three-state equilibrium curve for the wild-type
apoenzyme(9) . These values are recorded in Table 2.
Although there is considerable error in these estimations, the results
suggest that the free energy of denaturation for this protein consists
of two similar transitions.
Figure 3:
Analysis of the equilibrium unfolding of
apo-eSHMT by urea gradient gel electrophoresis and equilibrium
fluorescence. Upper panel, urea gradient gel of apoeSHMT (75
µg) electrophorized for 2 h in a polyacrylamide gel made with a
0-8 M linear urea gradient. Lower panel,
analysis of the equilibrium unfolding-refolding fluorescence data for
wild-type eSHMT as shown in Fig. 2(closed circles).
The data points were fit to a three-state equation (). The solid circles represent the fraction of native enzyme N; the open circles represent the fraction of
unfolded enzyme U; and the open triangles represent
the fraction of the intermediate I.
The equilibrium fluorescence properties
of the three single Trp mutant apoenzymes are very different (Fig. 2B). Because these mutant apoproteins all have
similar stabilities, the differences in Trp fluorescence with
increasing urea concentration are not the result of differences in the
stability of the intermediate (I) for each mutant apoenzyme
but reflect the differences in the fluorescence properties of the
individual Trp residue in the intermediate. Inspection of the curves
suggests that most of the change in fluorescence for the Trp Each refolding curve was determined over a 10-fold concentration
range of enzyme. Only the Trp The multimeric state and the size of
a protein as a function of urea concentration can be investigated by
electrophoresis in a urea gradient gel. The photograph of a urea
gradient gel with wild-type apo-eSHMT is shown in Fig. 3. The
native dimer migrates only slightly faster at 0 M urea than
the fully unfolded monomer at 6-8 M urea. In the region
of 1-4 M urea, there is an increase in size with a sharp
band that runs considerably slower than the native dimer. There is also
a background smear of protein that occurs between the two bands. A
possible interpretation of this pattern is given under
``Discussion.'' No reproducible differences could be found
between wild-type and single Trp mutant apo-eSHMTs in these urea
gradient gels, further supporting the conclusion that the mutations
have not significantly altered the unfolding-refolding mechanism.
As shown previously, the rate of refolding at 4 °C is
dramatically different than at 30 °C. For the wild-type enzyme less
than 10% of the catalytic activity returns within 100 min after
initiation of refolding. Furthermore, the previous study showed that
for wild-type apo-eSHMT refolded at 4 °C, all of the secondary
structure had returned within 2 min and that the enzyme had completely
formed dimers after about 100 min(2) . This dimer could not
bind PLP and is listed as structure D` in . When
the single Trp mutant enzymes were tested for refolding at 4 °C,
the return of activity was the same slow rate as observed for the
wild-type enzyme. Although we did not check for the rate of dimer
formation, the results of urea gradient PAGE suggests that the
mutations have not altered the unfolding and separation of dimers. We
assume that each mutant enzyme forms dimers at close to the same rate
as observed previously for the wild-type enzyme(2) . These
results suggest that the rate-controlling step in each single Trp
mutant enzyme is the conversion of D` to apoD as shown in . The rates of change in fluorescence was investigated
with each of the mutant apoenzymes at 30 °C after initiation of
refolding. Both the Trp
Figure 4:
Rate of return of fluorescence emission
during refolding of apo forms of wild-type and single Trp mutants of
eSHMT. Excitation was at 290 nm, and emission was monitored at 335 nm.
Each enzyme was diluted 10-fold from an 8 M urea solution into
Tris buffer, pH 7.5. The solid lines are for data obtained at
30 °C, and the dashed lines are for data obtained at 4
°C. The lines do not conform to any model. Panels
A-C are the single Trp mutant eSHMTs and panel D is
the wild-type enzyme with 3 Trp residues.
The fluorescence emission properties for each apo-eSHMT during
refolding at 4 °C are shown as dashed lines in Fig. 4. For wild-type apo-eSHMT, refolding at 4 °C in the
presence of PLP results in almost no active enzyme being formed in the
1000-s time period recorded in this figure. For the Trp We had previously shown that in the refolding of
wild-type apo-eSHMT the CD spectrum returned rapidly even at 4
°C(2) . In this study, we determined the CD spectrum from
200 to 240 nm after initiation of refolding for each mutant apo-eSHMT
at 4 °C. Within the 120 s it took to record the spectra, the CD
signals were the same as for the native apoenzymes, confirming that for
each mutant enzyme the secondary structure returns very rapidly (data
not shown). To further characterize the environments of the three
Trp residues during refolding at 4 °C, the susceptibility of each
mutant enzyme to acrylamide quenching was investigated (Fig. 5).
The results are presented as the collisional quenching constant (K
Figure 5:
Acrylamide quenching curves for apo forms
of single Trp mutants of eSHMT at 4 °C as a function of refolding
time. Each mutant enzyme was diluted 10-fold from an 8 M urea
solution into 2 ml of Tris buffer, pH 7.5. 10-µl aliquots of an 8 M acrylamide solution were added, and the fluorescence was
monitored at 335 nm at various times after initiation of refolding. It
took about 5 min to record each quenching curve. The times on each
graph represents the time at which the quenching curve had been
started. The symbols are as follows: squares, Trp
The
quenching by acrylamide was used to follow each Trp residue during
refolding at 4 °C to determine what the status of each Trp residue
was during the formation of intermediates M, D`, and
apoD (). Quenching by acrylamide was followed for each
single Trp mutant enzyme as a function of time after initiation of
refolding and compared with the quenching properties of the native
mutant enzymes as shown in Fig. 5D. For the Trp
Figure 6:
SDS-PAGE of proteolytic digestion during
the refolding of wild-type apo-eSHMT. Unfolded enzyme was diluted
10-fold into Tris buffer at 4 °C, and at various times aliquots
were removed and incubated with subtilisin for 2 min at 4 °C. The
digestion was stopped by the addition of phenylmethanesulfonyl
fluoride. These digestions were then submitted to SDS-PAGE. Lane 1 is subtilisin. Lanes 2-6 are refolding solutions
that were incubated from 0, 0.17, 1.7, 17, and 60 min, respectively,
before incubation with subtilisin. Lane 7 is a sample that had
been refolded for 60 min at 4 °C, and then the temperature was
increased to 30 °C for 10 min before protease digestion. Lane 8 is native apoenzyme and subtilisin incubated at 4 °C for 2
min. Lane 9 is native enzyme without protease. Lane 10 shows the molecular mass markers.
The major bands at 23 and 17 kDa were eluted
from the gel and analyzed for their amino-terminal and
carboxyl-terminal sequences. The 17-kDa band showed a NH Several other researchers have cited the many advantages of
using single Trp mutants to study the mechanism of protein
folding(12, 13, 14, 15) . E.
coli SHMT offers several unique advantages for using single Trp
mutants. First, this enzyme has three Trp residues that are widely
spaced in the sequence of the enzyme. There is no conservation of any
of the Trp residues in a list of 14 different SHMT sequences from a
variety of sources, suggesting that they do not play a critical
catalytic role in this enzyme(16) . Second, eSHMT refolds
rapidly at high protein concentration. Third, monomeric and dimeric
intermediates accumulate at 4 °C for periods that allow physical
and chemical probes to be used. The single Trp mutant forms of the
enzyme used in this study appear to fold by the same mechanism and to
form essentially the same structure as previously observed for the
wild-type enzyme. Both equilibrium and kinetic folding studies suggest
that at 4 °C intermediates accumulate on the refolding pathway.
Kinetic studies suggest that upon dilution of unfolded enzyme at 4
°C, two domains rapidly fold to structures characteristic of the
native state. These are listed as domains 1 and 2 in Fig. 7.
This rapid folding is supported by the fluorescence studies that show
that Trp
Figure 7:
Model showing the structure of an early
intermediate in the folding of apo-eSHMT. Protease digestion
experiments show that folding intermediates representing both monomer
and dimer forms of apo-eSHMT are digested at Tyr
Kinetic studies show that Trp Protease digestion studies also suggest that an
amino acid segment between domains 1 and 2 is unordered, being
accessible to protease digestion during the period when D` is
the dominant intermediate. This segment includes the active site
Lys Equilibrium
unfolding-refolding studies are in agreement with the model developed
from the kinetic refolding studies as shown in Fig. 7.
Equilibrium fluorescence studies of the wild-type enzyme containing all
three Trp residues suggest that an intermediate structure I dominates the population of protein molecules at 2 M urea (Fig. 3). Equilibrium fluorescence studies of the single Trp
mutants show that Trp
However, it is clear that in I the amino-terminal
segment is largely disordered and Trp Protease digestion by subtilisin gives some evidence
about the structure of D`. Protease digestion fails to
distinguish between M and D` as evidenced by the same
pattern of forming 17- and 23-kDa fragments from the first few seconds
of refolding until 1000 s after refolding. By 1000 s the apoenzyme is a
dimer(2) . These results suggest that the dimer D` is
greatly expanded, having disordered segments at the amino terminus and
between domains 1 and 2. For most oligomeric proteins, forming dimers
occurs only after the formation of nearly native
monomers(18, 19) . This does not seem to be the case
with apo-eSHMT. Subtilisin will probably not digest the full length
of the amino acid residues between domains 1 and 2. Only the portion of
the sequence that is readily accessible to the protease and meets its
specificity requirements will be digested. It is likely that the
unfolded segment starts at the sequence Several studies have
suggested that eSHMT has the same fold as several other PLP enzymes,
including aspartate aminotransferase(16, 20) . One of
these studies aligned the amino acid sequence of eSHMT with aspartate
aminotransferase(16) . The three-dimensional structure of
aspartate aminotransferase has been solved and shown to consist of two
domains. These two domains coincide with domains 1 and 2 of eSHMT as
determined from the protease digestion studies. In mitochondrial
aspartate aminotransferase the large domain starts at residue 47 and
the small domain starts at residue 329. These are only a few residues
different than the digestion sites found for our subtilisin digestion
of eSHMT. It has been shown previously that the large domain of
aspartate aminotransferase can be expressed and folded independently of
the small domain(21) . This large domain also bound PLP but did
not form dimers and showed no catalytic activity. Our studies suggest
that the small domain (domain 2 in Fig. 7) may also fold by
itself.
Volume 271,
Number 6,
Issue of February 9, 1996 pp. 2987-2994
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)is a 94-kDa homodimer that catalyzes the
reversible interconversion of serine and glycine with tetrahydrofolate
serving as the one carbon carrier. Each subunit of the holoenzyme
contains PLP attached to Lys as an aldimine. Removal of
the PLP generates the apoenzyme, which remains dimeric. The 417-residue
subunit contains 3 Trp residues at positions 16, 183, and
385(1) .
-terminal region and a central region of the amino acid
sequence remaining largely in a random coil until the final
rate-determining step.
Materials
Ultra pure urea, all coenzymes, amino
acids, and buffers were obtained from Sigma. A Transformer(TM)
site-directed mutagenesis kit was purchased from ClonTech (Palo Alto,
CA). Restriction enzymes were from DuPont NEN.Site-directed Mutagenesis
All mutants were made by
the unique site elimination method of Deng and Nickoloff(3) .
The oligonucleotides used to generate Trp to Phe mutants and to
introduce silent mutations for new restriction sites are as follows:
5`-pGCCGAACTGTTCCAGGCCATGGAG-3` was used to change Trp to
Phe and to introduce a NcoI site (the underlined bases are the
positions of the mismatch); 5`-pCGTGGTGGACTTCGCGAAAATGCG-3` was used to
change Trp
to Phe and to introduce a NruI site;
5`-pGCGAAAGAGCTCGCTGGCTTCATGTGTGACG-3` was used to change Trp
to Phe and to introduce a SacI site. Double and triple
mutants were made by introducing two or three primers in one mutagenic
experiment. Mutations were identified by unique restriction enzyme
digestion and by sequence analysis of the structural gene.
Expression and Purification of Mutant eSHMTs
All
mutant eSHMTs, as well as the wild-type enzyme, were expressed and
purified from E. coli strain GS1993, which is
glyA, as described previously(4) . The purity
of each protein was >98% as judged by SDS-PAGE. Apo-eSHMT was
prepared by adding L-cysteine and chromatographing on a
phenyl-Sepharose column(2) . The apoenzyme was stored at 0
°C for no more than 3 days before use.
Characterization of Mutant eSHMTs
for each holoenzyme was calculated from the amino acid
composition by the method of Gill and von Hippel(5) . The
original molar absorption coefficient for wild-type eSHMT was
determined by weighing a dried protein sample of known absorbance. This
value was in close agreement with the predicted value determined by the
method calculated from the amino acid composition and the value
determined from the Bio-Rad protein kit using bovine serum albumin as a
standard. The experimentally determined molar absorption coefficient
values for the mutant proteins were obtained only by the Bio-Rad
method.
and V
values
were determined at 30 °C with serine and tetrahydrofolate as
substrates (1) . Tetrahydrofolate was used at 0.15 mM in each assay, which is six times its K value, and L-serine was varied in concentration between
0.2 and 3.2 mM. was the average value of three individual measurements for each
sample.Fluorescence and CD Measurements
Fluorescence
spectra were taken on a Shimadzu 5000 fluorimeter with 5-nm slits for
both excitation and emission. A circulation bath was used to maintain
the desired temperature. Fluorescence quenching experiments were
performed by adding 10-µl aliquots of an 8 M solution of
acrylamide to 2 ml of the enzyme solution. Data were corrected for
dilution and inner filter effects (6) as well as any change in
fluorescence intensity of the sample during the measurement. Constants
for collisional and static quenching were calculated according to the
methods of Eftink and Ghiron (7) using the following equation.
and F are the fluorescence in the absence and the
presence of acrylamide, respectively, K is the
collisional quenching constant, and V is the static quenching
constant.
Equilibrium Unfolding
20 mM Tris-HCl, pH
7.5, 5 mM 2-mercaptoethanol, and 1 mM EDTA buffer was
used throughout the unfolding-refolding experiments and will be
referred to as the Tris buffer. Equilibrium unfolding was performed by
adding a concentrated solution of either wild-type or mutant apo-eSHMTs
to a series of urea concentrations in the Tris buffer(2) .
These solutions were incubated for 2 h at 30 °C or 20 h at 4 °C
before analysis by fluorescence spectrometry. The fluorescence
equilibrium unfolding data were converted to the apparent fraction of
native protein (F) and plotted versus urea concentration as defined by:
is the observed value of the
fluorescence signal at a particular urea concentration in the region
where the protein is unfolding. The fluorescence values for Y
(native apoenzyme) and Y
(unfolded apoenzyme) were obtained by linear extrapolation of the
base lines for native and unfolded protein into the region where the
protein was unfolding(8, 9) .
Refolding Kinetics of apo-eSHMT
Unfolding
experiments were performed by a 20-fold dilution of concentrated
apo-eSHMT solutions (30 mg/ml) into a solution of freshly prepared 8.4 M urea containing Tris buffer. A 10-fold dilution of unfolded
protein into Tris buffer was used throughout the study to initiate
refolding. The final protein concentration was typically 0.15 mg/ml
except where otherwise indicated. Refolding was monitored by Trp
fluorescence or the formation of functional active sites as described
previously(2) .Proteolytic Digestion
Stock solutions of
subtilisin carlsberg (1 mg/ml) in 2 mM CaCl and 100 mM phenylmethanesulfonyl fluoride in ethanol
were prepared immediately before use. Unfolded eSHMTs in 8 M urea were diluted into Tris buffer by a 10-fold dilution at 4
°C. 10 µl of the subtilisin stock solution was added to 200
µl of the refolding sample at desired times from 0 s to 60 min
after initiation of refolding and incubated for exactly 2 min. Each
digestion was terminated by the addition of 10 µl of the
phenylmethanesulfonyl fluoride stock solution. A native apo-eSHMT
control was made in 0.8 M urea with the same final protein
concentration as the refolding samples and digested by the same
procedure. All samples were concentrated 10-fold at low temperature in
a speed-vac. 20 µl of SDS-PAGE sample buffer, which had been
adjusted to pH 3 by HCl, was added to each concentrated protein. All
samples were placed in a boiling water bath for 1 min and after cooling
adjusted to neutral pH by the addition of 10 µl of 2 M NaOH. Samples were analyzed by SDS-PAGE on a 15% acrylamide
gel(10) .Urea-Gradient Gel Electrophoresis
A 0-8 M linear urea gradient polyacrylamide gel was made from stock
solutions of 50 mM Tris-HCl, pH 8.0, and 8 M urea in
the same buffer(11) . To compensate for the change in pore size
of the polyacrylamide with increasing urea a gradient of acrylamide was
made from 15 to 11%. Each form of eSHMT, 75 µg, in Tris buffer, pH
8.0, was layered on top of the gel in a 6-cm slot, and electrophoresis
was performed at 300 V for 2 h at room temperature. The gel was then
stained with Coomassie Blue R-250.Amino Acid Sequence Determination
The
amino-terminal and carboxyl-terminal sequences of proteolytic-resistant
fragments were determined as follows. Protease-treated enzyme was
analyzed by SDS-PAGE as described above and stained with Coomassie Blue
R-250. After destaining, the protein bands were cut from the gel with a
razor blade. The protein was eluted from the gel by electrophoresis.
The protein was precipitated with acetone-triethylamine acetic acid
(90:5:5) solution. The precipitated protein was centrifuged, washed
with the acetone-triethylamine acetic acid solution, dissolved in 50
mM pH 6.0, MES, and dialyzed against this buffer. After
determining the protein concentration by absorbance at 278 nm, aliquots
were subjected to amino-terminal sequencing by five cycles of
conventional Edman degradation on a G1000 A HP sequencer. Another
aliquot of eluted protein (about 0.2 mg) was incubated with 4 µg of
carboxypeptidase Y, and aliquots were removed at 10, 30, 60, 90, and
180 min. After removing protein with trichloroacetic acid and
centrifugation, the supernatant was analyzed for released amino acids
on an amino acid analyzer.
Characterization of Mutant eSHMTs
Single Trp
mutant eSHMTs were prepared by site-directed mutagenesis and analyzed
by DNA sequencing. The wild-type enzyme contains 3 Trp residues at
positions 16, 183, and 385. We will refer to the single Trp mutants by
the position of the single remaining Trp residue in each mutant, i.e. Trp, Trp
, and Trp
eSHMTs. In addition, a mutant protein was purified that contained
no Trp residues. This is referred to as the Trp
eSHMT
in Table 1. Each mutant protein was expressed and purified as
described previously for wild-type eSHMT (1) . The mutant
enzymes were analyzed for kinetic constants, thermal stability, and
absorption properties at 278 nm. These results are recorded in Table 1. For each mutant protein the determined molar absorption
coefficient values at 278 nm were very close to the predicted values
determined from the amino acid composition(5) . The T
values, determined by differential scanning
calorimetry in Tris buffer, showed that each single Trp mutant was
slightly less stable to thermal denaturation, with the Trp mutant being the least stable with a T
almost 7 °C lower than the wild-type enzyme (Table 1).
The values for T obtained in potassium phosphate
buffer were within 1 °C of those obtained in Tris buffer for each
enzyme form. Increasing temperature irreversibly denatures eSHMT, and
the thermograms do not fit a two-state curve. The thermograms can be
deconvoluted into several two-state processes, suggesting that thermal
denaturation is a complex process of subunit separation and unfolding
of domains.
values for serine were
essentially identical for all of the single Trp mutants and wild-type
enzymes (Table 1). Each mutant enzyme exhibited considerable
catalytic activity as well as the enzyme, which had all three Trp
residues replaced by Phe. The single Trp mutants with the least
activity were the Trp and Trp
enzymes, which
exhibited relative specific activity values of 0.86. The properties of
the mutant eSHMTs, as recorded in Table 1, suggest that each
mutant enzyme has only minor structural differences compared with the
wild-type enzyme.
, Trp
, and Trp
mutant apoenzymes were 338 nm, 325 nm, and 334 nm, respectively (Fig. 1). Evidence that the individual Trp residues in the
mutant proteins are very close to their environment in the wild-type
enzyme is suggested by the combined fluorescence spectra of the mutant
enzymes. If the Trp residues in the mutant apoenzymes are in the same
environment as they are in the native apoenzyme, then their combined
emission spectra should be the same as the emission spectrum of the
wild-type apoenzyme. As shown in Fig. 1, the sum of the
fluorescence spectra for the three Trp mutant apoenzymes is about 5%
lower than the fluorescence spectrum of the wild-type apoenzyme. Errors
in calculating protein concentrations for the mutant enzymes could
easily account for the 5% difference. The sum of the three emission
spectra of the mutant apoenzymes has the same 333 nm wavelength maximum
observed for the native enzyme (Fig. 1).
Equilibrium Refolding
The equilibrium fraction
folded ratio (F) was determined in increasing
urea concentrations for mutant and wild-type apo-eSHMTs by both
fluorescence and CD spectroscopy. Fig. 2A compares the
CD
signals of the apoenzymes. The urea concentration
resulting in 50% folding is shifted from 2.5 M urea for the
wild-type enzyme to about 2.3-2.4 M urea for the three
mutant enzymes. The results suggest that with respect to secondary
structure there is little difference in the unfolding pattern between
the mutant and wild-type apoenzymes. Although each set of data was fit
to a two-state model, the data points are not of sufficient accuracy to
rule out a three-state model. These CD results are in agreement with
the thermal denaturation studies that the mutant enzymes are slightly
less stable than the wild-type enzyme.
; open triangles,
Trp
; open squares, Trp
. A,
CD signals of 0.15 mg/ml solutions of enzymes were monitored at 220 nm.
The data are plotted as the fraction folded as described under
``Experimental Procedures.'' The solid lines are the
fit of the data points to a two-state transition. B,
fluorescence emission signals of 0.15 mg/ml solutions of enzymes
monitored at 335 nm. The data are plotted as the fraction folded as
described under ``Experimental Procedures.'' The solid
lines are the fit of the data points to either a two-state (open diamonds and triangles) or three-state
transition (closed circles and open
squares).
apoenzyme occurs in the N to I transformation,
whereas for Trp
apo-eSHMT virtually all of the change in
fluorescence occurs in the I to U transition. This
shows that in the intermediate Trp
is nearly fully exposed
and Trp
is still fully buried. The Trp
fluorescence change occurs in both reactions, suggesting that in
the intermediate Trp
experiences an environment that is
different than in either the native state or the unfolded state. The
equilibrium fluorescence curve for Trp
eSHMT did not fit
either a two-state or three-state model. It is shown in Fig. 2B as a two-state fit only for visual effect.
mutant apoenzyme showed any
concentration dependence. A 25-fold increase in protein concentration
shifted the midpoint of the urea unfolding curve from 1.1 to 1.6 M urea. This suggests that changes in the fluorescence of Trp
apo-eSHMT may be associated with the formation of dimers in the
equilibrium unfolding pathway.
Kinetics of Refolding
The rate of refolding of
each mutant apoenzyme was first investigated at 30 °C by a 10-fold
dilution of apoenzyme, which had been unfolded in 8 M urea for
at least 2 h, into buffer containing excess PLP. Active holoenzyme is
formed quantitatively in about 6-10 min for each mutant enzyme at
essentially the same rate previously observed for wild-type enzyme
(data not shown). This suggests that changing two of the Trp residues
to Phe has not significantly altered the kinetics of the refolding
reaction. and Trp
mutant
apo-eSHMTs showed a rapid change in fluorescence spectra to native
values following a 10-fold dilution into refolding buffer at 30 °C (Fig. 4, A and B, solid lines).
However, the Trp
apoenzyme showed a much slower return of
the fluorescence signal, taking about 500 s to reach 90% of the native
enzyme value (Fig. 4C). The rate of return of
native-like fluorescence for the Trp
enzyme was
concentration-dependent increasing about 3-fold from 0.004 to 0.1 mg/ml
enzyme. Above this concentration there was no further increase in rate.
and Trp
mutant apoenzymes, the fluorescence signal
returned to native values by 120 s (Fig. 4, A and B, dashed lines). However, the return of native
fluorescence for the Trp
mutant eSHMT took as long as 15 h
to reach final native values. As shown in Fig. 4C about
50% of the fluorescence change has returned in 20 min after refolding.
As observed at 30 °C, only Tp
eSHMT showed any
observable concentration dependence. The fluorescence emission
properties of the wild-type eSHMT during refolding at 4 °C as shown
in Fig. 4D is a composite of the properties of the
mutant enzymes.
) and the static quenching constant (V) as recorded in Table 3. K
is
obtained from the initial slope of the quenching curve, and V is obtained by applying (7) . The collisional
quenching constant values show that each Trp is partially
solvent-exposed with values ranging from 1.6 M
for Trp
eSHMT to 5.8 M
for Trp
eSHMT, suggesting that Trp
is
the most buried and Trp
is the most solvent exposed of the
three Trp residues. The collisional quenching constant values are in
agreement with the values of the fluorescence emission maximum for each
mutant enzyme as shown in Fig. 1. In general, the shorter the
wavelength of maximum emission the more buried the Trp residue
(Trp
, 325 nm; Trp
, 334 nm; and
Trp
, 338 nm) with fully exposed Trp residues in the
unfolded state having an emission wavelength of about 354 nm.
eSHMT; triangles, Trp
eSHMT; circles, Trp
eSHMT. The panel labeled Native shows the quenching curves obtained at 4 °C for the native
single Trp mutant enzymes in 0.8 M urea.
apoenzyme, acrylamide quenching was native-like within the 5 min
it took to determine a quenching curve (Fig. 5A and Table 3). After 5 min the quenching of Trp
apo-eSHMT was close to native values, but both the values of K
and V were larger than the native
enzyme. By 5 min the CD spectra of these proteins show that all of the
secondary structure has returned and the fluorescence emission of both
Trp
and Trp
have returned. Previous studies
using size exclusion chromatography show that the enzyme is mostly the
monomer (M) after 5 min of refolding. These results suggest
that both Trp
and Trp
are close to their
native environments. However, Trp
is far from its native
value, as indicated by the large value of 0.93 for the static quenching
constant (V) (Table 3). This shows that Trp
is largely solvent exposed in structure M. After 2 h of
refolding the enzyme will be mostly D` with some apoD. Both
Trp
and Trp
apoenzymes have values for the
quenching constant that are the same as the native enzyme. Trp
apo-eSHMT may still be slightly more solvent exposed than the
native enzyme.
Protease Digestion
Structural information on the
intermediates trapped at 4 °C during refolding was also obtained by
observing if domains had become protease-resistant. A variety of
proteases were used to digest the refolding enzyme at 4 °C. The
best results were obtained with subtilisin, which did not digest the
native enzyme with a digestion period of 2 min at either 4 or 30
°C. A slightly smaller band representing about 5% of the protein
was seen in the native enzyme both with and without subtilisin during
SDS-PAGE (Fig. 6, lanes 8 and 9). We assume
this was due to a proteolytically nicked form of the enzyme. When the
unfolded enzyme was added to refolding buffer containing subtilisin, no
native enzyme was observed on analysis by SDS-PAGE. Three clear bands
of smaller size were observed, however (Fig. 6, lane
2). A doublet was seen at about 23 kDa, and two other bands were
observed at 20 and 17 kDa. If the protease was added 0.17 min (10 s)
after initiation of refolding, the 20-kDa band disappeared, but the
23-kDa doublet increased in intensity (Fig. 6, lane 3).
Still no native enzyme was observed. The appearance of native enzyme
did not appear until 17 min (Fig. 6, lane 5) after
initiation of refolding and even at 60 min only a small fraction of the
enzyme existed with native-like resistance to protease digestion (Fig. 6, lane 6). After 60 min of refolding the
temperature was rapidly increased to 30 °C and incubated a further
10 min. During this time period the enzyme would be converted to fully
active enzyme. As shown in lane 7 of Fig. 6, the enzyme
became almost completely protease-resistant except for a small amount
of the 17-kDa fragment.
terminus sequence of KEAME-, which is consistent with residues
277-281. Carboxypeptidase Y digestion released Ala, Tyr, and Val,
consistent with the sequence of the carboxyl terminus of the enzyme.
NH-terminal sequencing of the 23-kDa fragment was
consistent with the sequence AEGYP-, which suggests protease cleavage
at Tyr. Carboxypeptidase Y digestion was complex,
releasing rapidly two equivalents of Val, two equivalents of Leu, and
one equivalent each of Thr, Ile, and Tyr. These results suggest that
subtilisin had cleaved the protein about equally in at least two
positions. The major sites were Thr
and
Leu
. The origin of the single Tyr is not clear.
, which is in domain 1, is rapidly buried within
a few seconds and exhibits characteristics of fluorescence emission, CD
spectrum, and acrylamide quenching of its environment in the native
enzyme. Almost the same conclusions can be stated for
Trp
, which is a part of domain 2. Only the acrylamide
quenching studies suggest that the enzyme does not completely reach its
native environment by 5 min. Protease digestion studies show that these
two domains have become resistant to digestion immediately upon
dilution of the unfolded enzyme into refolding buffer, further
supporting the view that two domains exist in a largely condensed
state.
,
Thr
, Leu
, and Leu
. A
23-kDa domain is resistant to protease digestion. This domain
contains Trp
(circle), which is the most buried
of the three Trp residues. Also, a
17-kDa domain at the carboxyl
terminus of the protein containing Trp
is resistant to
protease digestion. Trp
remains exposed to solvent during
the early stages of refolding and appears to reach its native state
only during the rate-determining step of forming native apoenzyme.
Lys
, which is the PLP binding site, also remains
protease-sensitive until the final stages of forming native
enzyme.
is outside of
domains 1 and 2 and appears not to be part of any ordered structure
during the first stages of folding. It is removed by protease treatment
and is solvent exposed as indicated by fluorescence emission and
acrylamide quenching ( Fig. 4and Fig. 5). It appears to
reach its native state only when the enzyme reaches its catalytically
active form. The fact that the equilibrium refolding studies and the
rate of burial, as determined by fluorescence emission of this residue
in the Trp
eSHMT, both show some concentration dependence
suggests that this residue is in a different environment in the dimers D` and apoD as compared with the monomer intermediate M (). The observation that the CD signal of all mutant
eSHMTs are fully formed after a few seconds and that the equilibrium
refolding curves are nearly the same by this technique (Fig. 2)
suggests that most of the secondary structure of eSHMT is present in
domains 1 and 2.
, which binds PLP (Fig. 7). In our previous
study we concluded that the rate-determining step in forming active
eSHMT involved the formation of the active site that includes
Lys
(2) . The protease digestion studies confirm
that this section of the protein remains in a nonnative state until the
rate determining final isomerization of D` to apoD (). Unfortunately there is no Trp residue in this segment
of the molecule to monitor the environment during folding studies.
However, we have used NaCNBH
to reduce the bound PLP of
holo-eSHMT to form a stable secondary amine to Lys. This
now places a fluorescent probe in this region of the protein.
Preliminary studies show that the PLP is not buried in its native state
until late in the folding process. (
) becomes disordered at a much lower
urea concentration than the other two Trp mutants. This would suggest
that the conversion of the native form N to the intermediate I may not represent a significant energy barrier. However, as
shown in Table 2,
G
for the conversion
of N to I is significant and in the same range as the
conversion of the intermediate I to the unfolded form U that takes place at a much higher urea concentration. The
relationship of G to the concentration of urea is given
by (17) . For the conversion of the native
apoenzyme N to I, the value of m is
-3.1
kcal
molM,
and its value for the conversion of I to the unfolded form U is -1.6
kcal
molM.
When plots of
G versus [urea] are extrapolated
to zero urea concentration to obtain G, the
two transitions give nearly the same values for G (Table 2).
is solvent-exposed.
It is not clear from the fluorescent studies if the residues between
the two domains are also being exposed and disordered between 1 and 2 M urea. The urea gradient gel suggests that two distinct sizes
of molecules do exist in the range of stability where I is
formed (Fig. 3). The band at the left of the gel (0 M urea) represents the migration of the dimer apoD, and the band
above 6 M urea represents the migration of the unfolded
monomer (U). There is not much difference in the size of these
two species. In the range where the intermediate I is stable
(1.5-3.0 M urea), there is a transition between these
two extremes. In this range of urea concentration, we observed for all
apoenzyme forms a distinct band that migrated much more slowly than
either native or unfolded enzyme (Fig. 3). We suggest that this
could be an expanded dimer in which the NH
-terminal
sequence and the section between the two domains have become
disordered. The smear of protein between the upper and lower bands in
the 2-4 M urea range of the gel suggests that there is
equilibrium between species of different sizes during the 2 h it took
to run the gel.PNPVP- and
extends to -PEP
(16) . This portion of the amino
acid sequence contains Lys
, which binds PLP and 8 of the
20 Pro residues in eSHMT. The lack of native structure of this part of
the molecule helps explain why D` does not bind
PLP(2) . A highly conserved sequence in all SHMT enzymes lies
next to the active site Lys
. In almost all sequences
there are 4 Thr and 2 Val residues between Val
and
Thr
(16) . This stretch of 6
-branched amino
acids would be predicted to exist in an unordered state. The rate
determining step may involve the folding in of this stretch of amino
acids to form the active site of the apoenzyme.
))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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