From the Institut de Biologia Fonamental and Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Received for publication, August 30, 2000, and in revised form, December 28, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A comparative study of the oxidative refolding
for nine selected potato carboxypeptidase inhibitor (PCI) mutants was
carried out using the disulfide quenching approach. The mutations were performed at the N- and C-terminal tails of PCI outside its disulfide stabilized central core. The differences between the refolding of wild
type and mutant proteins were observed in the second phase of the
refolding process, the reshuffling of disulfide bridges, although the
first phase, nonspecific packing, was not greatly affected by
the mutations. Point mutations at the C-tail or deletion of up to three
C-terminal residues of PCI resulted in a lower efficiency of the
reshuffling process. In the case of the mutants lacking five N-terminal
or four or five C-terminal residues, no "native-like" form was
observed after the refolding process. On the other hand, the
double mutant G35P/P36G did not attain a native-like form
either, although one slightly more stable species was observed after
being submitted to refolding. The disulfide pairing of this species is
different from that of the wtPCI native form. The differences between
the refolding process of wild type and mutant forms are interpreted in
the light of the new view of protein folding. The results of the
present study support the hypothesis that the refolding of this small
disulfide-rich protein, and others, is driven by noncovalent
interactions at the reshuffling stage. It is also shown that the
interactions established between the N- and C-tail residues and the
core of PCI are important for the proper refolding of the protein.
Small proteins that are rich in disulfide bridges are good models
for studying the folding process using the disulfide quenching method,
where the folding intermediates that form during their oxidative
refolding are trapped at low pH, separated on
HPLC,1 and analyzed (1, 2).
Among the first and more thoroughly studied proteins using this
technique was bovine pancreatic trypsin inhibitor (BPTI). Initially,
mixed intermediates with native and non-native disulfide bridges were
detected along the folding process (3). In later studies of BPTI
folding (4), all of the well populated intermediates were found to
contain only native disulfide bonds. Such results would support the
idea of a single pathway for BPTI folding. Recently, it has been shown
that BPTI also unfolds through a unique mechanism (5).
In contrast, the studies of hirudin (6), potato carboxypeptidase
inhibitor (PCI) (7), epidermal growth factor (EGF) (8), and tick
anticoagulant peptide (9) showed a high heterogeneity of folding
intermediates containing both native and non-native disulfide bonds. In
these proteins, the folding of the fully reduced protein to the native
form occurs in a first phase as a flow of equilibrated 1-disulfide
intermediates through equilibrated 2-disulfide intermediates to reach
the equilibrated 3-disulfide scrambled species. In the second phase,
the rate-limiting step, the scrambled species are reshuffled to finally
form the native species. Moreover, the folding process seems to follow
multiple parallel pathways. This mechanism would be consistent with the
new view of protein folding processes describing folding funnels and
energy landscapes (10, 11), i.e. parallel folding events,
rather than the classical model of sequential folding via unique routes.
Further studies to characterize in depth the folding/unfolding process
of the above mentioned or other disulfide rich proteins, based on such
disulfide-quenching method, could help to clarify why and how multiple
pathways take place not only in those particular proteins but also in
other protein types, allowing comparisons with those lacking
disulfides. With this purpose in mind, we have concentrated our
attention on PCI.
PCI is a 39-residue globular protein that competitively inhibits
several metallocarboxypeptidases with a Ki in the nanomolar range (12). Its three-dimensional structure is known in
aqueous solution (13) and in crystal state in complex with carboxypeptidase A (CPA) (14). The 27-residue core, reinforced by three
disulfide bridges, forms a T-knot scaffold, also found in other
proteins with different functions such as serine protease inhibitors
from the squash family, the Previous studies on the effect of denaturants on the folding pathway of
PCI (7) suggested that specific noncovalent interactions mainly direct
the reshuffling stage of the process. From the results of oxidative
folding and reductive unfolding of hirudin, the intertwining dependence
of the disulfide bonds and noncovalent forces was also concluded (20).
Thus, in the proposed model for the folding of these small
disulfide-rich proteins (6-9), until the scrambled species are formed
the noncovalent interactions would not direct the folding, whereas the
reshuffling step would be driven by noncovalent forces.
We have also recently characterized the unfolding pathway of PCI (21),
showing that the bead-form scrambled isomer (presumably the most
extended) is a significant intermediate in the process and that, under
physiological conditions, scrambled isomers of PCI exist in equilibrium
with the native form.
In the course of preparing recombinant PCI mutants constructed for
functional studies, we found that some of the mutants were obtained in
very low yield when expressed in Escherichia coli, even when
the sequence changes did not affect the globular core of the protein.
These observations prompted us to perform a comparative study of the
oxidative refolding of selected mutants. The results of the study
presented here show that the residues in the N- and C-tails of PCI are
important for its folding, by affecting the reshuffling stage of the
process to a different extent. The knowledge on the involvement of the
noncovalent interactions between PCI tails and central core in its
folding can be of importance in the future redesign of this small
protein for pharmaceutical or other biotechnological purposes.
Production and Purification of the Mutant PCI Forms--
The
Y37G, P36G, G35P/P36G,
Refolding Experiments--
100-µg aliquots of lyophilized of
proteins were used in each folding experiment. The protein was
dissolved in 0.5 ml Tris-HCl (0.5 M, pH 8.5) containing 5 M GdnCl and 30 mM dithiothreitol. After 2 h at 25 °C the reduced and denatured protein was passed through a
PD-10 (Amersham Pharmacia Biotech) gel filtration column equilibrated
with Tris-HCl buffer (0.1 M, pH 8.5). The protein was
eluted in 1.2 ml of the same buffer and split in two parts that were
diluted to a final concentration of 60 µg/ml with 0.1 M
Tris-HCl buffer, pH 8.5, and the same buffer containing 1 mM cysteine, respectively. Samples from both solutions
containing 5 µg of protein were collected in a time course manner for
up to 3 days, and were acid-trapped by mixing with an equal volume of
1% trifluoroacetic acid. They were analyzed by HPLC on a Nova-Pak C-18
column (Waters), 3.9 × 150 mm, 4 µm, under the following conditions: solvent A was water containing 0.1% trifluoroacetic acid,
solvent B was acetonitrile containing 0.1% trifluoroacetic acid, and
the gradient was linear, 20-40% solvent B in 30 min.
Mass Spectrometry--
MALDI-TOF mass spectrometry was performed
on a Bruker-Biflex spectrometer. Ionization was accomplished with a 337 nm pulsed nitrogen laser. Spectra were acquired in linear or reflectron positive ion mode, using a 19 kV acceleration voltage. Samples were
prepared by mixing equal volumes of a 1-10 µM solution
of the protein and a saturated solution of Circular Dichroism Spectroscopy--
Samples for CD spectroscopy
were prepared by dissolving the lyophilized aliquots to a final
concentration of 100 µg/ml in 0.1% trifluoroacetic acid or buffer (1 mM sodium citrate, 1 mM sodium borate, 1 mM sodium phosphate, 25 mM NaCl, pH 2, 7, or 11) for the pH dependence spectra. The far-UV circular dichroism spectra were collected on a Jasco spectrometer at 25 °C using a cell
of 2 mm path length.
Assignment of Disulfide Bond Pairing--
The disulfide bond
pairing of the most stable species of the G35P/P36G PCI mutant was
assigned by the partial reduction method (26). 50 µg of lyophilized
protein was denatured in 10 µl of 0.1 M citrate buffer,
pH 3, containing 6 M GdnCl for 30 min at 25 °C. Then the
protein was partially reduced by 90 nM
tris(2-carboxyethyl)phosphine for 15 min at 25 °C and cyanylated by
4.8 µM 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate for 15 min. The reaction mixture was subjected to
reverse-phase HPLC and the three cyanylated isomers resulting from
reduction of a single disulfide bond, as identified by MALDI-TOF mass
spectrometry, were isolated. They were subjected to cleavage of the
peptide bonds on the N-terminal side of the cyanylated cysteines for
one h at 37 °C in a solution of 1 M ammonium hydroxide containing 6 M GdnCl, and finally the remaining disulfide
bonds were reduced by 0.1 M tris(2-carboxyethyl)phosphine
for 30 min at 37 °C. The resulting mixtures of peptides were
analyzed by MALDI-TOF mass spectrometry, and the disulfide bonds were
deduced from the obtained mass maps.
Protease Digestions--
Selected PCI scrambled species,
scrambled A form of wtPCI (defined in Ref. 7), the G35P/P36G PCI most
stable form, or scrambled species of mutants
CPA Inhibitory Assays--
The inhibitory activities of the
different mutant PCI forms were assayed according to Hass and Ryan
(12). Benzoyl-glycyl-L-phenylalanine was used as a
substrate at 0.1 M concentration, and the enzyme (bovine
carboxypeptidase A) was at 42.5 nM. The assays were
performed at pH 7.5.
Refolding Behavior--
The oxidative refolding in
vitro of different N- and C-tail mutant forms of PCI was studied
by reverse-phase HPLC analysis of the acid-trapped disulfide
intermediates present in samples collected at different times during
the refolding processes, these processes being followed either in the
absence or presence of an external thiol added (cysteine) (see
"Experimental Procedures"). The assays were always performed in
parallel to a control refolding experiment of wtPCI under the same
conditions. The refolding conditions used in this study
(i.e. protein concentration, buffer conditions, the type of
thiol reagent, and its concentration) were selected so that the
qualitative or quantitative differences in the refolding process
between the wild type and mutant PCI forms could be distinguished (number and type of folding intermediates, kinetics of the process) within a few hours' time span, being slightly different from those of
a previous paper (7).
The typical results observed for the refolding of wtPCI under the
selected conditions are shown in Fig. 1.
The protein was initially reduced and denatured in the presence of
excess dithiothreitol and 5 M GdnCl and then quickly
brought to the refolding buffer by gel filtration on a small PD-10
column. The HPLC profile of a sample collected immediately after the
gel filtration (t = 0, Fig. 1) mainly contained
a mixture of 3-disulfide paired scrambled species, as identified
according to their chromatographic behavior, well characterized in
previous studies on the disulfide refolding of PCI (7). Under the above
conditions, it was observed (Fig. 1) that the first stage of
nonspecific packing of wtPCI to reach 3-disulfide paired species was
very quick and that the second stage of disulfide reshuffling of
scrambled intermediates was rate-limiting. The evolution of these
intermediates thereafter was very slow in the buffer without added
cysteine (Fig. 1, left), probably being catalyzed only by
the small amount of protein free thiols still present and leading to a
final trapped mixture with less than 10% of the native species. In
contrast, when 1 mM cysteine was added, a much faster
reshuffling of the scrambled species led to a mixture in which more
than 80% of the protein had the native disulfide pairing (Fig. 1,
right).
When the study was performed on the N- and C-tail mutant forms of PCI,
all of the mutants analyzed showed a first stage of refolding as fast
as wtPCI under the same conditions. In the chromatograms obtained
immediately after passing the samples through the PD-10 column, the
main peaks corresponded to 3-disulfide scrambled species (results not
shown), the subsequent evolution being very small when free cysteine
was lacking in the buffer (see Figs. 2,
3, and 4 for the patterns observed after 3 h).
However, significant differences between mutants were observed in the
second step of the refolding process, i.e. in the
reshuffling step, when 1 mM cysteine was present. According
to the evolution of their chromatographic pattern, the mutants were
classified in three groups. Mutants of a first group (Group I)
resembled wtPCI in their behavior (Fig. 2.). These included point
mutations in the C-terminal tail (P36G and Y37G) and consecutive
deletions of up to three residues of the C-tail. It seems clear that
the isomers marked "N" are what could be called native-like forms of each mutant, i.e. they possess the same disulfide
bridging as wtPCI native form and presumably have a compact stable
structure, as supported by the following findings: they all maintained
CPA inhibitory activity, their retention time in the HPLC column was almost identical to that of the wtPCI form, and their relative amounts
in the refolded mixtures increased in the course of refolding, so they
seemed to be the most stable forms. As in the case of wtPCI, there was
a remarkable difference between the mixtures with and without cysteine
added. Little or no native-like form could be observed in the samples
refolded in the buffer alone, whereas a significant amount of this form
was seen in the mixtures with cysteine. The percentages of native-like
forms in
A second group was represented by mutants with a very different
refolding behavior and which are not able to reach a native-like conformation (Group II). Their chromatograms after 3 h of
refolding are shown in Fig. 3. Their
mutations represent the largest changes made in the sequence of PCI
tails (although they do not affect the amino acid residues of the core
stabilized by the disulfide bonds), being the result of the deletion of
four or five residues in the C-terminal tail or the deletion of five
residues in the N-terminal tail of PCI. In this Group II no differences
in the chromatographic profile could be observed between the 3-h
trapped mixtures with and without cysteine. In none of these mixtures was a significant peak that could be considered a native-like form
seen.
It is worth mentioning that the
It is not obvious whether some of the disulfide isomers of the mutants
lacking four or five amino acids in the C-tail of PCI have the same
disulfide pairing as the native PCI. As these mutants do not contain
the residues that constitute the primary binding site to CPA, the
activity assay does not give information about the conformation of the
proteins. Besides, the HPLC retention times of the "correctly
folded" forms of these mutants are not known. However, as we observe
no evolution of the "scrambled" forms to one HPLC peak during
refolding in the presence of cysteine, it seems likely that these
mutations cause the inability of PCI to form a native-like stable
conformation. The species present in the final refolding mixtures of
these two mutants were isolated by HPLC and assayed for susceptibility
to protease digestion. All of the species were found to be extensively
degraded by elastase or thermolysin under conditions in which the
native form of wtPCI is resistant to digestion. This behavior is also
consistent with these species not having a native-like compact conformation.
For all three mutants of Group II attempts to obtain the correctly
folded proteins in the presence of higher concentrations of reducing
agent were carried out. The formation of a native-like form was not
detected even in the presence of 4 mM cysteine, conditions under which wtPCI forms more than 90% of native species in less than
1 h (data not shown).
The refolding process and characteristics of the G35P/P36G double
mutant are different, and therefore it was included in a third group
(Group III). The chromatograms of its 3-h trapped refolding mixtures
are shown in Fig. 4. A clear difference
is observed between the chromatograms of the trapped mixtures in the
absence or presence of added cysteine in a way similar to that observed
for wtPCI or the mutants of Group I. In this case, the relative amount
of one species (peak labeled S in Fig. 4) also increased
when cysteine was added to the refolding buffer. Although the overall
amino acid content of this double mutant is the same as that of wtPCI,
the chromatographic behavior on the reverse-phase HPLC of this species
(S) is very different from that of the wtPCI native form and
resembles that of the scrambled species A of wtPCI (7), suggesting that
such a species S can have a disulfide bond pairing different from the
native form of wtPCI. To confirm this possibility, the disulfide
bond pairing of such a most stable form of G35P/P36G (peak S) was
determined by a partial reduction-mass spectrometry method (see
"Experimental Procedures"). Disulfide bridges between cysteines
I-V, II-III, and IV-VI (see Fig. 4) were found, confirming a
different pairing from that of the native wtPCI. Interestingly, the
same disulfide pairing has also been determined for the scrambled
species A of wtPCI.2
Unlike results observed for the mutants of Group I, when refolding was
attempted under more favorable conditions (higher cysteine concentrations) or longer times, no further evolution of the mixture of
intermediates was observed for the G35P/P36G mutant. This seems to
indicate that the species S, which increases in the refolding mixture
with cysteine, is not actually native-like, in the sense that its
thermodynamic stability is not much higher than that of the other
coexisting 3-disulfide species. Susceptibility to elastase hydrolysis
was used as an additional tool to check whether this species S has a
compact, stable structure, the hydrolysis being followed in parallel
with wtPCI native and scrambled A forms. After a 1.5-h incubation, only
Gly39 was cut from the wtPCI native form, whereas the
scrambled A and S species were degraded to fragments of less than 1000 Da, as observed by MALDI-TOF mass spectrometry.
In conclusion, the G35P/P36G mutant seems not to be able to refold into
a native, compact structure, just as the mutants of Group II. However,
unlike what was observed for the mutants of Group I or II, in
this case the formation of one of the scrambled species, S, has a
kinetic barrier greater than the rest of the scrambled species, and,
accordingly, its proportion in the mixture of trapped intermediates
depends on the addition of thiols to the refolding buffer (see
"Discussion").
CD Spectroscopy--
Although the wtPCI native form does not have
regular secondary structures, except for a short 5-residue helix, we
found that it presents features that have made the far-UV CD
spectroscopy helpful as a tool to indicate the folding state of the
mutants. The far-UV CD spectrum of wtPCI showed a characteristic
positive ellipticity band at 228 nm and a minimum at 204 nm. The
positive band at 228 nm was not found in some PCI mutants (Fig.
5). For instance, the so-called
native-like refolded form of the Y37G mutant, lacking the
Tyr37 residue, which presents a strong CPA inhibitory
activity (Ki, 3.5 nM) and hence probably
has a three-dimensional structure very similar to the wtPCI native
form, does not display the characteristic positive band on its CD
spectra. Therefore, Tyr37 side chain seems to be related to
the presence of this band. It is known that aromatic residues may cause
optically active bands in the far-UV region, their environment
affecting the position of CD bands of proteins (27). When the CD
spectrum of wtPCI was recorded at different pH values (pH 2, 9, and 11)
it was found that the maximum at 228 nm disappears at pH 11 (Fig.
5a), probably when the tyrosine hydroxyl groups are ionized,
in agreement with the potential involvement of Tyr37 in
such a CD band. Interestingly, the scrambled forms of wtPCI, which
posses all the residues of the native form but are folded in different
ways, do not display either the maximum at 228 nm (not shown). Hence,
the environment of Tyr37 in correctly folded PCI is
probably responsible for the characteristic maximum at 228 nm, and so
the PCI forms lacking the Tyr37 residue or having a
different three-dimensional structure would not display the
characteristic CD spectrum.
Indeed, all of the main protein species found in the HPLC peaks of the
final refolding mixtures for the mutants from the first folding group
(that is, peaks N), except for Y37G and
All of the mutations performed in this study alter the PCI
sequence out of its central globular core. The substitution of one
residue at the C-tail at its boundary with the core (in mutants P36G
and Y37G) and deletion of up to three residues from the C terminus
significantly affected the PCI behavior during refolding but did not
prevent the attainment of a native-like state. As in the case of wtPCI,
in the presence of cysteine, the equilibrated mixture of scrambled
forms in such mutants decreased to give rise to the native-like species
(although the molar proportion between the scrambled forms is
maintained along the refolding process). Yet, the yield of a
native-like form in the final mixture of species was lowered in such
mutants in relation to wtPCI. All of these mutations therefore affect
the refolding process by diminishing the efficiency of the reshuffling step.
The mutations that have the strongest influence on the folding of PCI
are the deletion of five residues at the N terminus or four or five
residues at the C terminus, giving rise to a "trimmed" core with
2/5, 7/1, and 7/0 residues at the N/C tails, respectively. These
mutants also quickly reached the equilibrium of scrambled species in
the first stage of folding, but no reshuffling occurred in the second
stage even in the presence of 1 mM cysteine. These results
show that the reshuffling step is clearly influenced by the residues of
the C- and N-terminal tails of PCI. It is worth mentioning that the
first three residues at the PCI N terminus are disordered in the NMR
structure (13), no interactions being observed between them and the PCI
core. Only the fourth residue, alanine, forms a backbone hydrogen bond
with the Cys8 residue, the one that establishes the
boundary with the core, as observed in the crystal structure of the
PCI·CPA complex (14). In addition, three main-chain/side-chain
hydrogen bonds involving the N-terminal residues of PCI are found in
the crystal structure of its complex with CPA:
Ala4-Trp22,
Ile7-Asp5, and
Phe23-Asp5. None of these interactions is
supposed to be important for the PCI inhibitory activity, because when
five N-terminal residues are cut off from an already folded protein,
the trimmed PCI is still fully active (25). However, the present
results show that this N-tail could play a role in the PCI folding process.
As mentioned above, when up to three residues from the PCI C terminus
are removed, the yield of the native-like form in the refolded mixture
of species is lowered, in the conditions used in this study. If the
fourth residue of the C terminus, Pro36, is also deleted,
no formation of the native-like species is detected. Both in the NMR
and x-ray structure of PCI, a hydrogen bond between the carbonyl oxygen
of Pro36 (in the tail) and the amide nitrogen of
Trp28 (in the core) is observed. Our results seem to
indicate that this hydrogen bond, which could be formed by any residue
in position 36, could participate significantly in directing the native
disulfide pairing of PCI. When no residue is present in position 36, that is when the C-tail only has one residue (Gly35), no
native-like form is seen in the course of refolding. Substitution of
Pro36 by glycine in the wild type form (in P36G mutant)
causes only the formation of a lower relative amount of the native-like
form in the final mixture of species.
The results observed in the refolding of the G35P/P36G double mutant
emphasize the participation of the Gly35 and
Pro36 residues in driving the native PCI folding process.
The substitution of Gly35 by proline probably influences
the orientation of the PCI C-tail and thus prevents the formation of
the hydrogen bond between Trp28 (in the core) and the new
residue at position 36 in the double mutant, a glycine. It is important
to mention that Gly35 in wtPCI establishes two hydrogen
bonds with Ala26,
Ala26(N)-Gly35(O) and
Gly35(N)-Ala26(O). These two backbone hydrogen
bonds are observed both in the NMR structure of wtPCI in solution and
in the crystal structure of its complex with CPA. The substitution of
Gly35 by proline causes suppression of at least one of
these hydrogen bonds, as proline lacks the amide hydrogen. In the
course of refolding of the G35P/P36G double mutant no native-like
species is formed. On the other hand, formation of a slightly more
stable scrambled species that differs from the native form of wtPCI is
observed. The importance of Gly35 and its two hydrogen
bonds with Ala26 is reflected also in the fact that
Gly35 and Ala26 are two of the eight PCI
residues with less than 10 Å2 of accessible surface area
(14). The other six residues buried in the wtPCI core are
Ala21 and five cysteines forming the disulfide bridges.
As previously reported, the cystine knot (or T-knot) structural motif
gives rise to compact structures found in several proteins with
different functions, such as EGF-like molecules, On the other hand, the The conservation of a glycine residue after the last cysteine of the
cystine-knot core in squash inhibitors, the higher yield of correctly
folded The results of the present study show that residues at the tails of a
protein can be important for the folding process. Related results have
been reported in the study of the folding of chymotrypsin inhibitor-2,
where formation of the native structure was observed in a set of
fragments growing from the N terminus (35). It was shown that only when
the penultimate residue, Val63, was added, was a full,
compactly folded structure of chymotrypsin inhibitor-2 obtained.
The present study supports the hypothesis that the noncovalent forces
guide the folding of disulfide-rich proteins at the reshuffling step.
The loss of the interactions established by the N- and C-terminal tail
amino acid residues of PCI has clearly been shown to cause changes in
the reshuffling of the disulfide bridges, whereas the first step of
their nonspecific formation is not affected.
The oxidative refolding processes of the PCI and PCI mutants observed
in the present study, and those of other disulfide-rich proteins shown
in previous studies (6-9), could be interpreted according to the
theory of folding funnels and energy landscapes (10, 11), by bumpy
energy landscapes with kinetic traps and energy barriers, where the
protein folds to the native state via multiple pathways in the
conformational space. Oxidative folding would proceed from the reduced
state through progressively more stable species, thermodynamically
speaking: 1-disulfide, 2-disulfide, and 3-disulfide intermediates
(scrambled species). For wtPCI and most mutants, the energy barriers
between the intermediates of each group are small, so that they can
interconvert freely, being essentially in equilibrium. Thus, a
very high number of intermediates can exist along the pathways to the
native state. The scrambled species would represent kinetic
traps, which can be overcome by the addition of reducing agents to
allow their reshuffling to the thermodynamically favored native state.
The results presented here indicate that the energy landscape has some
differences for the various mutants. The mutants of Group I have an
energy barrier to the native state that is higher than that of wtPCI, a
fact that results in the observed slower kinetics for the reshuffling
step of these mutants or the need of higher concentrations of reducing
agent to reach the native state efficiently. In the case of mutants of
Group II, their inability to reach a native state can be explained in
two ways. On one hand, the mutations introduced can cause a situation
in which no conformation possesses a far higher thermodynamic stability
than the rest, i.e. no truly native state exists. On the
other hand, in the case that a hypothetical native state does exist,
the "uphill step" necessary to leave the trap would be
energetically too unfavorable, and therefore even at high
concentrations of reducing agent the native state would not be
obtained. In the case of the The inability of these mutants to reach a native-like state could
result from the formation of aggregates of the misfolded forms.
However, the scrambled forms of these proteins show a behavior in
reverse-phase HPLC very similar to wtPCI scrambled species, which would
indicate a similar degree of exposure of the hydrophobic regions and
consequently a similar tendency to aggregation as wtPCI. Moreover, when
the refolding of these mutants was performed in more diluted solutions,
no native form could be observed either (data not shown). Therefore it
does not seem probable that aggregation plays a major role in the
misfolding of these proteins, although it cannot be ruled out based
on the present evidence.
For all proteins studied here, with the unique exception of the
G35P/P36G mutant, the energy barriers between the different scrambled
species seem to be much lower than those leading to the native form,
and therefore the equilibrium proportions of the different forms are
maintained throughout the folding process regardless of the addition of
external thiols. In the case of G35P/P36G PCI, the scrambled S form
presents a higher energy barrier, and its equilibrium proportion can
only be attained in the presence of added thiol.
Given that in the last few years PCI has become a molecule with
biomedical potentialities, having been suggested as a drug for blood
fibrinolysis (36) and for antitumoral strategies (18) and given that
its N- and C-tails seem to be involved in such potentialities, it is
interesting to fully characterize the influence of these tails in the
folding of the protein and, therefore, in its recombinant production
and redesign. The same applies to other topologically related
molecules, such as EGF, transforming growth factor-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxins from snake venoms, or the
EGF superfamily of growth factors (15). PCI has 7 amino acid residues
at the N-tail of the hydrophobic central core and a 5-residue C-tail,
which is the primary binding site of PCI to carboxypeptidase A. The energetic contribution of each residue of the C-tail in the
PCI·CPA complex has been evaluated by site-directed
mutagenesis studies (16, 17). The biological function of PCI is
probably the inhibition of insect digestive metallocarboxypeptidases as
part of the defense system of the potato plant against insect attack
(12). Recently, we have shown that PCI possesses interesting
anti-tumoral properties that are related to its structural similarity
to EGF and other related growth factors (18), all of them having the
so-called T-knot topology (19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Val38-Gly39,
Tyr37-Gly39,
Pro36-Gly39, and
Gly35-Gly39 PCI mutant genes were
constructed by site-directed mutagenesis of wild type PCI synthetic
gene (22) cloned in PINIII-OmpA3 vector (23). The proteins were
expressed in E. coli and purified from the culture medium by
ion exchange fast protein liquid chromatography and reverse-phase HPLC.
Details regarding the construction of the mutant vectors and
purification of the expressed proteins have been published elsewhere
(17).
GIy39PCI was obtained by the incubation of wtPCI with
CPA at 1:1 molar ratio for 4 h followed by purification on a Vydac
C-4 HPLC column. CPA is known to cleave the Gly39 residue
of PCI rapidly after the enzyme-inhibitor complex is formed (24).
Glu1-Asp5 PCI was obtained by acid
hydrolysis of wtPCI as described previously (25). The trimmed form was
separated from the intact wtPCI on a Vydac C-4 HPLC column, and its
N-terminal sequence was checked on a Beckman LF3000 automatic
sequencer. All of the purified mutant proteins were characterized by
analytical reverse-phase HPLC and MALDI-TOF mass spectrometry.
-cyano-4-hydroxycinnamic acid, used as a matrix, in aqueous 30% acetonitrile with 0.1% trifluoroacetic acid. 1 µl of this mixture was spotted on the sample
slide and allowed to evaporate to dryness
Pro36-Gly39 and
Gly35-Gly39) were isolated by HPLC on a
Vydac C-18 column from the corresponding mixture of refolding
intermediates, obtained as detailed under "Refolding Experiments."
1 µg of the purified scrambled species, or the native wtPCI form as a
control, were incubated for 1 h with 1 µg of elastase in 0.1 M Tris-Cl, pH 8.8, buffer or with 0.05 µg of thermolysin
in 50 mM N-ethylmorpholine, pH 6.5, buffer. The
resulting digests were analyzed by MALDI-TOF mass spectrometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
[in a new window]
Fig. 1.
Control refolding experiments of wtPCI in the
absence and presence of an external thiol. Reduced and denatured
PCI was allowed to refold in the absence (left) or presence
of 1 mM cysteine (right). Samples of
acid-trapped intermediates were analyzed at the noted times by reversed
phase HPLC as detailed under "Experimental Procedures." The peaks
corresponding to native PCI (N) and reduced PCI
(R) are indicated.
View larger version (18K):
[in a new window]
Fig. 2.
Folding behavior of PCI mutants able
to form a native-like structure (Group I). Refolding experiments
on wild type (wt) and mutant PCI forms were performed as
described in the legend to Fig. 1. Chromatograms corresponding to the
3-h refolding mixtures (that is, at the end of the second refolding
stage) in the absence ( Cys) or presence (+Cys)
of 1 mM cysteine are shown for each protein. The elution
positions of native-like and fully reduced species are indicated by
N and R, respectively. The graphics
above the chromatograms show the percentage of native-like species
in each mixture after 3 h of refolding, calculated from the peak
areas in the corresponding chromatograms.
Gly39,
Val38-Gly39,
and Y37G mutants are close to wtPCI (more than 55%), whereas the
trapped mixtures of P36G and
Tyr37-Gly39
mutants after 3 h contained only about 30% of such form. The percentages of native-like forms obtained for each protein under these
conditions indicate a different kinetic efficiency for their reshuffling process. If a higher concentration of reducing agent is
present in the refolding buffer, more than 90% of native-like species
is formed for each of the mutants from this Group I (data not shown).
View larger version (11K):
[in a new window]
Fig. 3.
Folding behavior of PCI mutants unable to
form a native-like structure (Group II). Refolding experiments on
wild type (wt) and mutant PCI forms were performed as in
Fig. 1. The chromatograms corresponding to the 3-h refolding mixtures
in the absence ( Cys) or presence (+Cys) of 1 mM cysteine are shown for each protein. The elution
positions of native-like and fully reduced species are indicated by
N and R, respectively.
Glu1-Asp5
mutant was initially prepared by direct mild acid hydrolysis from
native wtPCI, leading to a protein that maintained the disulfide
pairing and CPA inhibitory activity. Therefore, the HPLC elution time
of such an N-terminal truncated form can be considered that
which corresponds to the actual native-like species. However,
when this truncated protein was reduced and allowed to refold, none of
the resulting "refolded" chromatographic forms showed either
a peak at the former elution time (Fig. 3) or any inhibitory activity.
Thus, this protein variant lacking the N-terminal tail, once reduced,
is not able to refold to its original native-like conformation.
View larger version (11K):
[in a new window]
Fig. 4.
Folding behavior of G35P/P36G PCI double
mutant (Group III). Top panel, chromatograms
corresponding to 3-h refolding mixtures of the wtPCI and
G35P/P36G mutant without ( Cys) and with (+Cys)
1 mM cysteine added. The refolding experiments were
performed as described in Fig. 1. The peaks are marked as follows:
N, the native wtPCI form; A, the
scrambled A wtPCI species; and S, the more stable
G35P/P36G mutant species. The elution position of the fully reduced
species for the double mutant is indicated by R. Bottom panel, disulfide bond pairing of the S
species of the G35P/P36G mutant compared with that of the native
species of wtPCI. The disulfide bond pairing was determined by using a
partial reduction method and mass spectrometry as described under
"Experimental Procedures." The boxes mark the residues
changed by the mutation.
View larger version (24K):
[in a new window]
Fig. 5.
Far-UV CD spectra of wild type and mutant PCI
forms. The spectra were collected at 25 °C using a cell of 2-mm
path length with the protein concentration being 100 µg/ml.
a, far-UV CD spectra of wtPCI at pH 2, 9, and 11;
b, far-UV CD spectra of native forms of wtPCI and group I
mutants measured at pH 2; c, far-UV CD spectra of wtPCI
native species and the G35P/P36G most stable double mutant species
(S) measured at pH 2.
Tyr37-Gly39 lacking the Tyr37,
show the maximum at 228 nm on their CD spectra (Fig. 5b). In contrast, none of those bands in the HPLC peaks of the final refolded mixtures of the mutants from the second group displayed the 228 nm
maximum (data not shown). In the case of
Pro36-Gly39 and
Gly35-Gly39 mutants, which have lost most
or all of the C-tail and do not contain the Tyr37 residue,
the maximum at 228 nm would not be expected even if the correctly
folded protein (at the globular core) was present. Also, in the most
stable form of the G35P/P36G mutant (species S), the
Tyr37 residue is present but, as noted above, the
conformation of this form is different from that of the native wtPCI,
and, accordingly, the positive band at 228 nm is not present (Fig.
5c).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-toxins, and
proteinase inhibitors from plants (15, 19). Some of these T-knots
structures are included as domains within multidomain proteins, but
many of them, such as the serine protease inhibitors from the squash
plants, PCI, or the
-conotoxins from snake venom are found in nature
as isolated monodomain small disulfide-rich proteins. The question of
whether these proteins need the tails for correct refolding, as does
PCI, is an interesting issue. The trypsin squash inhibitors have
a very similar structural topology to PCI and the same disulfide bridge
pattern (28). The sequence alignment of a large number of the squash
inhibitors shows that the cysteine-reinforced core contains 25 amino
acids (27 in PCI); there are zero to five amino acids at the N terminus
and, in most cases, only one glycine residue at the C terminus, after
the last cysteine of the T-knot core. This glycine is conserved among
all of the 41 known members of the squash inhibitor family (28). In the
crystal structure of the Cucurbita maxima trypsin
inhibitor-I (CMTI-I), in complex with trypsin, such a glycine forms two
internal backbone hydrogen bonds with Val21, analogously to
PCI. Refolding of the chemically synthesized proteins is a common
procedure used to obtain squash inhibitors and their variants (29).
Some of these variants lacked any amino acid out of the core enclosed
by the disulfide bridges. However, the yield of correctly folded forms
during in vitro refolding ranged between 5 and 10%, both
for wild type and mutant proteins, independently of the presence of the
tail residues. These low yields may be explained by the possible
requirement of pro-sequences for the correct folding of the trypsin
squash inhibitors.
-conotoxins have all of their 23-26 amino
acids enclosed by three disulfide bridges. Refolding studies in
vitro revealed that under optimal conditions the yield of the correctly folded proteins is between 16 and 50%. The presence of an
additional glycine residue at the C terminus of
-conotoxin MVIIA enhanced this yield to 80% (30). The
-conotoxins are synthesized in vivo with a C-terminal glycine residue that
is subsequently modified by the addition of a terminal amide group. This modification probably occurs at secretory granules after the
protein has been folded. Therefore, the C-terminal glycine residue can
be of importance for the folding of
-conotoxins.
-conotoxins in the presence of this glycine, and the
importance of Gly35 in PCI folding all support the
potentially important role of a glycine residue in such position in the
in vivo folding of disulfide-rich proteins. Other folding
helpers, such as molecular chaperones, isomerases, or pro-sequences of
the proteins that are cleaved upon protein folding (sometimes called
intramolecular chaperones) (31), are supposed to catalyze the folding
of these proteins in vivo, as the rate of folding in
vitro is very low. The pro-sequences present in the genes of PCI
(32) or the homologous protein from tomato (33) of the
-conotoxins
(30) and of the squash inhibitors (34) could theoretically play the
role of intramolecular chaperones. Yet, the in vitro folding
studies of
-conotoxins showed that their pro-sequences do not
accelerate the in vitro folding process (30). Currently, the
role of the PCI pro-sequence in the folding process in vitro
is being studied in our laboratory.
Glu1-Asp5 N
terminus trimmed protein, the existence of a native form is known,
because the protein, obtained by hydrolysis of wtPCI, presents all of
the structural characteristics (CD, inhibitory activity, resistance to
proteolysis) of the compactly folded form of wtPCI. Thus, at least for
this protein, the inability to refold to the native state is because of
kinetic rather than thermodynamic reasons.
, toxins,
and defensins (19), several of them having clear biotechnological applications.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Miquel Pons (Universitat de Barcelona) for kindly helping us in the CD studies and to Dr. R. J.-Y. Chang (Institute of Molecular Medicine, Houston) for helpful discussions. The support and advice of Drs. Cristina Marino and Silvana Pavia are also gratefully acknowledged.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants BIO98-0362 and 2FD97-0872 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), Spain and by the Center de Referencia de Biotecnologia de la Generalitat de Catalunya (CERBA).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from CERBA.
§ To whom correspondence should be addressed. Tel.: 34-93-581 1315; Fax: 34-93-581 2011; E-mail: fx.aviles@blues.uab.es.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M007927200
2 S. Pavia, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HPLC, high performance liquid chromatography; BPTI, bovine pancreatic trypsin inhibitor; CPA, carboxypeptidase A; EGF, epidermal growth factor; GdnCl, guanidinium chloride; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; PCI, potato carboxypeptidase inhibitor; wtPCI, wild type PCI.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Creighton, T. E. (1986) Methods Enzymol. 131, 83-106[Medline] [Order article via Infotrieve] |
2. | Creighton, T. E. (ed) (1992) Protein Folding , pp. 301-351, EMBL, Heidelberg |
3. | Creighton, T. E., and Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497-526[Medline] [Order article via Infotrieve] |
4. | Weissman, J. S., and Kim, P. S. (1991) Science 253, 1386-1393[Medline] [Order article via Infotrieve] |
5. |
Chang, J. Y.,
Li, L.,
and Bulychev, A.
(2000)
J. Biol. Chem.
275,
8287-8299 |
6. |
Chatrenet, B.,
and Chang, J. Y.
(1993)
J. Biol. Chem.
268,
20988-20996 |
7. |
Chang, J. Y.,
Canals, F.,
Schindler, P.,
Querol, E.,
and Aviles, F. X.
(1994)
J. Biol. Chem.
269,
22087-22094 |
8. |
Chang, J. Y.,
Schindler, P.,
Ramseier, U.,
and Lai, P. H.
(1995)
J. Biol. Chem.
270,
9207-9216 |
9. | Chang, J. Y. (1996) Biochemistry 35, 11702-11709[CrossRef][Medline] [Order article via Infotrieve] |
10. | Baldwin, R. L. (1994) Nature 369, 183-184[CrossRef][Medline] [Order article via Infotrieve] |
11. | Dill, K. A., and Chan, H. S. (1997) Nat. Struct. Biol. 4, 10-19[Medline] [Order article via Infotrieve] |
12. | Hass, G. M., and Ryan, C. A. (1981) Methods Enzymol. 80, 778-791 |
13. | Clore, G. M., Gronenborn, A. M., Nilges, M., and Ryan, C. A. (1987) Biochemistry 26, 8012-8023[Medline] [Order article via Infotrieve] |
14. | Rees, D. C., and Lipscomb, W. N. (1982) J. Mol. Biol. 160, 475-498[Medline] [Order article via Infotrieve] |
15. | Lin, S. L., and Nussinov, R. (1995) Nat. Struct. Biol. 2, 835-837[Medline] [Order article via Infotrieve] |
16. |
Molina, M. A.,
Marino-Buslje, C.,
Oliva, B.,
Aviles, F. X.,
and Querol, E.
(1994)
J. Biol. Chem.
269,
21467-21472 |
17. |
Marino-Buslje, C.,
Venhudová, G.,
Molina, M. A.,
Oliva, B.,
Jorba, X.,
Canals, F.,
Aviles, F. X.,
and Querol, E.
(2000)
Eur. J. Biochem.
267,
1502-1509 |
18. |
Blanco-Aparicio, C.,
Molina, M. A.,
Fernández-Salas, E.,
Frazier, M. L.,
Mas, J. M.,
Querol, E.,
Aviles, F. X.,
and de Llorens, R.
(1998)
J. Biol. Chem.
273,
12370-12377 |
19. | Mas, J. M., Aloy, P., Martí-Renom, M. A., Oliva, B., Blanco-Aparicio, C., Molina, M. A., de Llorens, R., Querol, E., and Aviles, F. X. (1998) J. Mol. Biol. 284, 541-548[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Chang, J. Y.
(1997)
J. Biol. Chem.
272,
69-75 |
21. |
Chang, J. Y.,
Li, L.,
Canals, F.,
and Aviles, F. X.
(2000)
J. Biol. Chem.
275,
14205-14211 |
22. | Molina, M. A., Aviles, F. X., and Querol, E. (1992) Gene 116, 129-138[Medline] [Order article via Infotrieve] |
23. | Ghrayeb, J., Kimura, H., Takahara, M., Hsiung, H., Masui, Y., and Inouye, M. (1984) EMBO J. 3, 2437-2442[Abstract] |
24. | Hass, G. M., and Ryan, C. A. (1980) Biochem. Biophys. Res. Commun. 97, 1481-1486[Medline] [Order article via Infotrieve] |
25. | Hass, G. M., Ako, H., Grahn, D. T., and Neurath, H. (1976) Biochemistry 15, 93-100[Medline] [Order article via Infotrieve] |
26. |
Wu, J.,
and Watson, J. T.
(1997)
Protein Sci.
6,
391-398 |
27. | Adler, A. J., Greenfield, N. J., and Fasman, G. D. (1973) Methods Enzymol. 27, 675-735[Medline] [Order article via Infotrieve] |
28. | Otlewski, J., and Krowarsch, D. (1996) Acta Biochim. Pol. 43, 431-434[Medline] [Order article via Infotrieve] |
29. | Rolka, K., Kupryszewski, G., Rózycki, J., Ragnarsson, U., Zbyryt, T., and Otlewski, J. (1992) Biol. Chem. Hoppe-Seyler 373, 1055-1060[Medline] [Order article via Infotrieve] |
30. | Price-Carter, M., Gray, W. R., and Goldenberg, D. P. (1996) Biochemistry 35, 15547-15557[CrossRef][Medline] [Order article via Infotrieve] |
31. | Shinde, U., and Inouye, M. (1993) Trends Biochem. Sci. 18, 442-446[CrossRef][Medline] [Order article via Infotrieve] |
32. | Villanueva, J., Canals, F., Prat, S., Ludevid, D., Querol, E., and Aviles, F. X. (1998) FEBS Lett. 440, 175-182[CrossRef][Medline] [Order article via Infotrieve] |
33. | Martineau, B., McBride, K. E., and Houck, C. M. (1991) Mol. Gen. Genet. 228, 281-286[Medline] [Order article via Infotrieve] |
34. |
Ling, M. H.,
Qi, H. Y.,
and Chi, C. W.
(1993)
J. Biol. Chem.
268,
810-814 |
35. | de Prat, Gay, G., Ruiz-Sanz, J., Neira, J. L., Corrales, F. J., Otzen, D. E., Ladurner, A. G., and Fersht, A. R. (1995) J. Mol. Biol. 254, 968-979[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Klement, P.,
Liao, P.,
and Bajzar, L.
(1999)
Blood
94,
2735-2743 |