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(Received for publication, July 10, 1995; and in revised form, November 17, 1995) From the
A set of protein fragments from yeast phosphoglycerate kinase
were produced by chemical cleavage at a unique cysteinyl residue
previously introduced by site-directed mutagenesis. Cross-linking
experiments showed that the fragments corresponding to incomplete
N-terminal domain form stable oligomeric species. Transient oligomeric
species were also observed by both cross-linking and light scattering
experiments during the folding process of the whole protein. These
transient oligomeric species are formed during the fast folding phase
and dissociate during the slow folding phase to produce the monomeric
active protein. The multimeric species are not required for the protein
to fold correctly. Unexpectedly, the distribution of oligomeric species
is not dependent on protein concentration during the folding process. A
kinetic competition mechanism is proposed as a possible solution to
this paradox. These results provide direct evidence that the
polypeptide chain can explore nonnative interactions during the folding
process.
Recent advances in rapid methods of isotope exchange monitored
by NMR, site-directed mutagenesis, or stopped-flow circular dichroism,
have led to important developments in protein folding
research(1) . Until recently, the protein folding process was
often described as a sequential series of steps(2) , with
secondary structure being formed in early steps, then the molten
globule (3) and, in a late rate determining event, the native
structure. In contrast to other possible models(4) , this model
supposes a single folding pathway rather than several parallel
pathways, and it implies that the interactions existing in folding
intermediates still persist in the native structure, i.e. that
nonnative interactions are not usually present in the folding
intermediates. However, it is widely accepted that this scheme is
further complicated by the possible, although not general, occurrence
of slow and fast folding species related to proline isomerization in
the unfolded forms(5) . It is important to keep in mind that
methods commonly used to monitor folding, such as circular dichroism or
fluorescence, give an average picture of the molecular species present
at a given folding time. Few methodologies are able to resolve
heterogeneous populations. A classical example is the determination of
disulfide bond formation that, many years ago, had suggested that early
folding events in bovine pancreatic trypsin inhibitor could lead to a
set of diverse one-disulfide intermediates(6) . These rapidly
formed a limited set of two-disulfide intermediates, including a low
proportion of molecules (7) containing a nonnative disulfide
bond. Recent results obtained by isotope exchange and mass
spectrometry have also demonstrated that protein folding can give rise
to a heterogeneous population of intermediates or proceeds via multiple
pathways(8) . Similarly, labeling kinetics of cysteinyl
residues genetically introduced in phosphoglycerate kinase revealed
that an ``intermediate'' produced in the fast folding phases
is in fact a population of two distinct molecular species(9) . Folding studies are often focused on the folding process of small
proteins(2) . It is, however, clear that the folding process of
larger ones is frequently associated with unproductive refolding
pathways leading to nonnative multimeric
species(10, 11) . These species are generated through
wrong intermolecular pairing of local structures present in folding
intermediates; the association acts as a kinetic trap and results in
irreversibly aggregated forms. These side reactions are frequently, but
not exclusively, observed with oligomeric proteins and are greatly
favored at high protein concentrations. This paper describes
experiments conducted with yeast phosphoglycerate kinase.
Phosphoglycerate kinase is a classic example of a two-domain monomeric
protein, whose folding process has been intensively studied by several
groups(9, 12, 13, 14, 15, 16) .
Phosphoglycerate kinase unfolded by high concentration of urea or
guanidinium chloride can completely refold in vitro into its
original monomeric active form. The isolated domains have been produced
by genetic engineering and shown to refold independently both in
vivo and in vitro(17, 18) . The two
folded isolated domains do not reassociate upon mixing and therefore do
not regenerate a complemented and active protein. Two other pairs of
complementary fragments were produced by chemical cleavage of two
mutant phosphoglycerate kinases, in which a unique cysteinyl residue
was previously introduced by site-directed mutagenesis(19) .
For these two pairs of fragments, the cleavage point is located within
the N- or the C-domain. These studies have shown that for these two
pairs of fragments, the fragment smaller than a domain has a rather low
degree of structure and cooperativity. These two pairs of fragments did
regenerate a partial enzymatic activity upon mixing. One could
intuitively expect that complementation is related to the folding
autonomy of the isolated fragments. In contrast, these observations
actually suggest that correctly folded domains do not necessarily
reassociate and that it is not necessary for protein fragments to
correspond to protein domains, or to fold autonomously, to be able to
reassociate functionally. We have recently extended this study to
other pairs of complementary fragments derived from phosphoglycerate
kinase. This paper shows that the associated multimeric species
observed with one of the previously studied fragments (19) are
also observed with a set of other fragments corresponding to incomplete
N-domain. Furthermore, this paper shows that transient multimeric
species are present during the folding of this monomeric protein.
The EGS was dissolved
in Me Finally, the
cross-linked material was precipitated with trichloroacetic acid (50%
v/v), pelleted at 10,000 All of the refolding and
cross-linking experiments were performed at 20 °C.
Using the same methods, we produced and studied a more
extensive set of fragments as described previously(19) . These
fragments were obtained by chemical cleavage at the unique cysteinyl
residue previously introduced in several mutants by site-directed
mutagenesis. The results of cross-linking analysis are shown in Fig. 1. A distribution of oligomeric species, as previously
observed with fragment 1-96, is also clearly present with
fragments 1-123, 1-139, and 1-152. All of these
fragments correspond to the N region relative to cleavage points
located within the N-terminal domain, and therefore, these fragments
correspond to incomplete N-terminal domains. This behavior was no
longer observed when the cleavage point approaches the limit between
the domains; neither fragment 1-172 nor fragment 1-184,
which corresponds exactly to the complete N-domain, gives rise to the
pattern observed with the shorter N fragments. When the cleavage point
was located beyond the limit of the domain, the N-side fragments
1-280 (Fig. 1) recovered a tendency to form dimers, but in
a much lower proportion than the incomplete N domain fragments.
Figure 1:
Multimeric forms of
PGK fragments. The fragments were cross-linked by glutaraldehyde in
native conditions and analyzed by SDS-PAGE. The concentration of each
fragment was 2 µM. N(1-184) and C(185-415) correspond to the N- and C-terminal domains,
respectively.
C-terminal fragments or complete C-domain (185-415) formed a
low fraction of dimeric species in this range of concentrations. The
fragments corresponding to the whole C-domain plus an extra N segment
124-415, 140-415, 153-415 were also essentially
monomeric, although a very faint band indicated that a small fraction
of fragments 124-415 was dimeric. From these results, it is
clear that the tendency to form a set of multimeric species is mainly
due to a subset of the sequence located in the N-terminal part of the
polypeptide chain. The shortest N-fragment, and therefore the minimal
known region susceptible to give rise to associations, is located
between residues 1 and 96. This phenomenon appears to be correlated
with the folding autonomy of the fragments. When the sequence is long
enough to form the stable tertiary structure of the N domain, the
associated forms are not observed. Indeed, the N fragments 1-184
and 1-172 have been shown to form a relatively stable structure,
while fragment 1-96 was shown to be not as well structured and to
unfold with a lower
cooperativity(18, 19, 25) . A much weaker
tendency to produce associated forms was also observed with some
C-terminal fragments, including the whole folded C-domain. This
suggests that a secondary, but weaker, contribution to association
could be related to a stretch of the C-terminal sequence.
Cross-linking
experiments can be conducted in a sufficiently short time to freeze the
states of association during the refolding process(26) . The
refolding kinetics monitored by cross-linking are reported in Fig. 2. In addition to the bands corresponding to the monomeric
form, three bands corresponding to a distribution of dimeric, trimeric,
and tetrameric forms are present. These multimeric forms are formed
rapidly and disappear progressively when the refolding time, prior to
the cross-linking step, increases; they were not observed when the
refolding time was long enough to produce mainly refolded protein. The
disappearance of the multimeric forms is concomitant with the formation
of the native protein during the slow refolding phase. Upon
cross-linking, the monomeric protein displays an electrophoretic
pattern that is dependent on its conformational state. When the protein
is internally cross-linked in its native form, the band corresponding
to the monomeric protein appears sharper and higher in the gel than the
protein internally cross-linked in its unfolded form. This unexpected
peculiarity provides a kind of internal control, supporting the
observation that the dissociation of the multimeric forms occurs in
parallel with the refolding of the monomeric form. Furthermore, the
approximate rates of disappearance of the ``unfolded
monomeric'' species and of formation of the ``native
monomeric'' species (Fig. 2B), were estimated from
the scanning of the SDS-PAGE gel (Fig. 2A). These two
processes were found to have rate constants close to the rate constant
of the slow folding phase under the same conditions (k
Figure 2:
A, refolding kinetics of PGK monitored by
cross-linking. PGK, previously unfolded in 3 M guanidinium
chloride, was refolded by dilution. Protein and denaturant
concentrations were then 2 µM and 0.05 M,
respectively. Samples of the refolding protein were taken at the
indicated times and cross-linked during 30 s by glutaraldehyde. A
sample of native protein cross-linked in the same conditions is shown
as a control. B, kinetic of evolution of monomeric and
multimeric species. The electrophoresis was scanned, and the relative
intensities were determined by integration of optical density. *, sum
of the folded and unfolded monomeric species;
Figure 5:
Refolding kinetics monitored by light
scattering at 310 nm. The kinetics were initiated by dilution in
phosphate buffer of a solution of yeast PGK previously unfolded in
guanidium chloride. The scattered intensity is plotted against the
refolding time. The continuous line corresponds to a monoexponential
fit (k = 4.6
Figure 3:
Control experiments. Yeast PGK (2
µM) was cross-linked by glutaraldehyde after 20 s of
refolding in 0.08 M denaturant (A) or unfolded in 3 M guanidinium chloride (B) or in the absence of
denaturant (C). D, yeast PGK cross-linked by EGS
after 20 s of refolding in 0.08 M denaturant; E,
soluble aggregated form of horse muscle PGK cross-linked by
glutaraldehyde for 30 s. The arrow shows the band
corresponding to cross-linked aggregated material. F, soluble
aggregated form of horse muscle PGK without any cross-linking
treatment. G, horse muscle PGK cross-linked by glutaraldehyde
after 30 s of refolding in 0.08 M denaturant. M,
molecular weight standards.
Several
control experiments were conducted in order to demonstrate that the
presence of transient multimeric forms is not an artifactual
observation. The most convincing evidence against cross-linking of
unassociated species subsequently to intermolecular collisions is that
both denatured and native protein appeared as perfectly monomeric (Fig. 3) after the same cross-linking treatment. A more
difficult question is whether or not the distribution observed after
cross-linking quantitatively reflects the distribution of associated
forms originally present in the solution. Indeed, higher aggregates,
which were incompletely trapped, could give rise to this type of
electrophoretic pattern. The following experiments indicate that the
cross-linking step is likely to be complete. Control experiments show
that the apparent species distribution is not modified either by an
increase of the cross-linker concentration or by an increase of the
time-length of the cross-linking step. Other experiments (Fig. 3) showed that this transient distribution of oligomeric
species can also be trapped by cross-linking experiments conducted with
EGS, a different amino-specific cross-linker whose chemistry is better
defined than that of glutaraldehyde. Cross-linking experiments carried
out with PGK aggregated by a previous heat treatment suggest that,
after cross-linking, the protein appeared effectively as aggregated, at
the top of the gel. A convincing control is the cross-linking pattern
of PGK under conditions where the protein is known to be in a stable
soluble multimeric form. Native PGK is monomeric, but it has been shown
that when incubated in intermediate concentrations of denaturant, horse
muscle PGK forms stable soluble multimeric aggregates(32) . The
size of this aggregated form was indirectly evaluated by the order of
the reaction, which was found to be less than 10. We reproduced this
experiment and purified the multimeric species by HPLC gel-filtration.
The cross-linking pattern obtained with this species (Fig. 3)
shows only aggregated forms at the top of the gel and no evidence for
incompletely cross-linked forms. Another possible artifact could be
the disappearance from the system of the multimeric species not related
to their folding but caused by some trivial effect such as adhesion to
tube walls or formation of higher aggregates. This is obviously ruled
out by the observation that the refolding process of PGK is fully
reversible, while the multimeric species initially represented about a
third of the overall protein population (Fig. 2B). The
reversibility of the folding process has been demonstrated for yeast
PGK and horse muscle PGK by various signals such as far UV CD,
tryptophan fluorescence, fluorescence intensity of AEDANS, UV
difference spectrophotometry, and accessibility of thiol groups in
different
laboratories(9, 16, 18, 23, 25) .
Furthermore, there are about 35% of multimeric species formed at the
end of the fast folding phase (Fig. 2B), ruling out a
simple aggregation mechanism. Indeed, no evidence for aggregated
protein can be observed at the top of the gel obtained by monitoring
the folding process by cross-linking (Fig. 2A), while
the cross-linking reaction was able to detect high aggregates (Fig. 3). A further control experiment was performed. A
sample of refolded and native protein was prepared at exactly the same
concentration by dilutions of the same protein stock solution in
appropriate buffers. These experiments were performed under the same
conditions as used for the cross-linking experiments. The two samples
were analyzed by HPLC gel filtration in order to quantify the recovery
of the monomeric species in the sample subjected to an
unfolding-refolding cycle relative to the native sample. The refolded
protein was eluted in a single peak corresponding to the elution volume
of the native protein with a peak area equal to 95% of that obtained
with the native sample. Therefore, the multimeric species are
transformed into monomeric species during the folding process. There
are not many other well adapted methods to monitor the state of
association of transient species at low protein concentrations. The
multimeric forms of PGK cannot be analyzed by HPLC gel filtration
because of their transient nature, but we have conducted gel filtration
experiments on the stable oligomeric forms obtained with the fragments
(data not shown). The profiles obtained with the N fragments clearly
indicate a mixture of different forms. The permanently associated
forms observed with the fragments by cross-linking were also observed
by analytical equilibrium ultracentrifugation. A sample of the
monomeric N-terminal domain (1-184), which was analyzed in
parallel with fragment 1-280 gave rise, as assessed by
cross-linking, to a small fraction of dimer. This fragment was chosen
in spite of its less pronounced susceptibility to oligomerization
relative to other shorter N fragments, because it allows a more
sensitive detection and therefore an analysis at a lower protein
concentration than smaller fragments. These fragments were not
cross-linked before the ultracentrifugation analysis. The results
presented in Fig. 4show that the N domain behaves as a
monomeric protein, while the sample of fragment 1-280 contains
both monomeric and dimeric species.
Figure 4:
Ultracentrifugation analysis of fragments
1-280 (A) and 1-184 (B). The fragments
were in native conditions and were not cross-linked before the
experiments. For fragment 1-280 (molecular mass expected, 30.7
kDa), the best fit was obtained using a model where the sample is a
mixture of monomer and dimer (molecular mass observed, 30.8 and 62.1
kDa, respectively), the fraction of higher multimeric species being
negligible. For fragment 1-184 (molecular mass expected, 20.3
kDa), the best fit was obtained using a model where the protein is
essentially monomeric (molecular mass observed, 19.5 kDa) and the
fraction of multimeric species is negligible. Semilogarithmic plots (Ln Abs. versus r
Recent dynamic light-scattering
experiments have shown that species with large Stokes radius are
observed transiently during the refolding of yeast PGK(15) .
This observation was originally interpreted as resulting from expanded
monomeric species but may be related to the presence of transient
oligomeric species. A further indication that multimeric species do
not result from some unknown cross-linking artifact is provided by
measurements of light scattering during the folding process (Fig. 5). The scattered light intensity monitored during the
refolding of the protein decreases with a rate corresponding to that of
the slow folding phase under the same conditions.
``Double jump'' experiments have
indicated that the two phases observed during the refolding of PGK did
not result from the presence of different unfolded forms due to proline
isomerization(9, 31) . The same observation also holds
for the presence of oligomeric forms. The associated forms are observed
even when the denaturation step was too short (31) to allow
proline isomerization in the unfolded states (data not shown). The
multimeric forms are no more related to cysteine pairing since they
were observed, even in the presence of dithiothreitol, with the
wild-type protein, which has only one cysteine, and with fragments,
which have no cysteine. The same distribution of transient oligomeric
forms can also be observed if the protein previously denatured by urea
is refolded by dilution (data not shown) indicating that this
phenomenon was not dependent upon the nature of the denaturant. In
order to evaluate the affinity between the protomers in the transient
oligomers, a set of cross-linking experiments was conducted with
different protein concentrations. The results are reported in Fig. 6. The important, but completely unexpected result of this
experiment is that the distribution of the multimeric species is not
affected by the protein concentration in a range of more than 2 orders
of magnitude. If this is not due to incomplete cross-linking, as
suggested by the control experiments reported above, the inescapable
conclusion is that the different species, monomers, dimers, trimers,
and tetramers, are not in equilibrium during the lifetime of the
transient species.
Figure 6:
Effect of protein concentration on the
distribution of the transient multimeric forms. SDS-PAGE of yeast PGK
cross-linked after 20 s of refolding. The protein concentrations during
the refolding step are indicated at the top of the
gel.
The results described in this paper demonstrate that a set of
oligomeric species is rapidly formed during the folding of a monomeric
protein. The tendency of incorrectly or incompletely folded species to
aggregate is a well documented property of
proteins(11, 33) . We have observed transient
oligomeric species that have several features not previously reported,
indicating that these oligomers are distinct from the frequently
observed ``wrong aggregates.'' These species are formed at a
very low protein concentration, and the extent of oligomerization does
not depend on protein concentration. Wrong aggregation is frequently
related to illicit hydrophobic interactions and is therefore strongly
temperature-dependent, while the transient oligomeric species described
here are also observed to the same extent at 4 °C (data not shown).
Finally, the most unusual property of these transient oligomers is
precisely that they are transient, while wrong aggregation is generally
irreversible. The oligomers were first observed as stable species
with the fragments corresponding to the incomplete N-domain and later
as transient species during the folding of the whole protein. These
observations suggest that these oligomers are due to somewhat
sequence-specific interactions originating in the N-domain sequence.
The illicit interactions between monomers is certainly related to the
formation of partial or incorrect structure in the fragments. Indeed,
the stable oligomers produced by the N-fragments are not observed in
the presence of denaturant. Moreover, they are no longer observed when
the sequence is long enough to fold cooperatively as a
``correct'' domain. Finally, the formation of the native-like
structure of the protein during the refolding process dissociates the
oligomers. These results directly demonstrate that during the
folding of phosphoglycerate kinase, an heterogeneous population of
intermediates is present and that, in these intermediates, interactions
are involved that do not persist in the final native structure. The
disappearance of transient multimeric species is obviously not due to
an irreversible aggregation or adhesion to the reaction vessels; these
transient species are converted into native monomeric species. A
possible mechanism that could explain the paradoxical absence of a
concentration dependence of a reaction leading to multimeric species is
suggested in Fig. 7. Although several aspects of this scheme are
purely hypothetical, its essential feature is that the oligomer
distribution is explained by the existence of a kinetic competition
between two monomolecular steps (or between two bimolecular steps). In
this qualitative model, the observed oligomer distribution results from
the relative rates of two processes. One process leading to a monomeric
state able to initiate the formation of oligomers or to extend the
already formed oligomers by a unidirectional, fast, and tight
association, and the other process leading to a different monomeric
state incompetent to produce an expandable association. The relative
distribution of oligomeric forms actually observed indicates that the
formation of these two types of states must have rate constants of the
same order of magnitude. The overall mechanism must take place during
the fast folding phase.
Figure 7:
A
hypothetical model for the formation of the multimeric species. The
refolding of the protein produces two types of conformers. One of the
conformers can be directionally extended either by association with the
same type of monomer or by association with the other type of monomer.
In the latter case, the association cannot be extended further. The
distribution results from a competition between two kinetic processes
of the same order and therefore is not dependent on protein
concentration.
The scheme proposed in Fig. 7is
formally analogous to an irreversible reaction of copolymerization
between two types of monomers. The incorporation of the first type of
monomers produces an association that will be further extended, while
the incorporation of the second one produces a dead-end association. A
simulation of this model shows that the distribution finally obtained
is directly dependent on the ratio between the two types of monomers,
but does not depend on the total concentration of the two species. The
initial ratio of the two species is directly dependent on the ratio of
the rate constants k This model supposes that the association step
is essentially irreversible during the fast folding phase. It is,
however, clear that during the time range of the slow folding phase,
the tight association observed between the monomers is reversed by a
folding step producing a monomeric folded protein. Alternative
mechanisms are probably possible for the formation of these oligomeric
species, such as the formation of cyclic oligomers, but should explain
the following experimental observations: (i) the oligomer distribution
does not depend on protein concentration; (ii) the association is
sufficiently tight to take place for protein concentrations as low as
0.05 µM (and may be lower); and (iii) the association
distribution is established at the end of the fast folding phase. When a kinetic intermediate is detected, it is important to know
whether this intermediate is on or off pathway. The observation that
the species are not in equilibrium rules out the possibility that these
species result from a side reaction such as a reversible association of
an intermediate preceding the rate-limiting step. In other words, the
step that leads to the dissociation of the multimeric species is not
simply the reverse of the step that leads to their association. Their
dissociation is probably induced by a folding step occurring in the
multimeric species. These oligomers are formed rapidly and disappear
with a rate similar to the slow folding phase. From these criteria, the
oligomeric species are kinetically productive and could be considered
to be on the pathway, or more correctly, on some of the pathways. However, it is essential to note that a large fraction of the
protein remains monomeric and is not in equilibrium with the oligomeric
forms. This result suggests that this fraction of protein does fold
without going through any oligomeric forms. Therefore, these
intermolecular nonnative interactions cannot be considered as necessary
for the protein to fold correctly. Previous kinetic studies on PGK
folding have shown that the rate constant of the slow refolding phase
increases, as expected, when the denaturant concentration decreases.
However, for denaturant concentration lower than 0.2 M, the
folding rate constant decreases with the denaturant
concentration(31) . We suggest that this unusual effect could
be related to the presence of oligomeric species. The proportion of
transient multimeric species decreases progressively when the
denaturant concentration increases to a value higher than 0.2-0.3 M. It seems possible that for very low denaturant
concentrations the oligomeric forms are sufficiently stable to slow
down the folding process. Under these conditions, GdnHCl in
destabilizing too stable intermediates, accelerates the overall folding
process. The effects of low denaturant concentrations to destabilize
nonnative interactions have already been shown to increase the
refolding yield of proteins difficult to refold (11) or to
speed-up the formation of a disulfide-bond in native-like bovine
pancreatic trypsin inhibitor species(7) . The fundamental
observation reported in this paper, i.e. the presence of
transient multimeric species during the folding of a monomeric protein,
was certainly not anticipated, and it is currently difficult to
evaluate whether or not PGK should be considered as a rare exception.
There are very few examples of related observations. A transient
absorbance at 320 nm (where the difference spectrum is zero) was
observed during the refolding of The process leading to irreversible aggregation has been studied in
few model systems such as bovine carbonic anhydrase (36) or P22
tailspike protein(37) . It has recently been shown with P22
tailspike that during in vitro refolding experiments, a
distribution of small oligomers is rapidly formed and is the first step
toward irreversible aggregation. The irreversible formation of
aggregates is more common in large multidomain proteins than in small
proteins. Furthermore, it has been reported for some multidomain
proteins, including horse muscle PGK, that stable species observed in
intermediate concentrations of denaturant can induce some degree of
irreversibility(32) . These observations have been interpreted
as the consequence of the wrong intermolecular pairing of folded but
unpaired domains(11) . This idea was nicely illustrated by the
recent demonstration that domain swapping can indeed be experimentally
observed(38) . However, in the case of the transient multimeric
forms of PGK, fragments with only a fraction of a domain, in which
wrong pairing of associated domains is not possible, do form multimeric
species. Therefore, the associated forms are produced by the wrong
pairing of structural units smaller than a domain. An interesting
aspect of these results is that the formation of multimeric species is
under kinetic control. The multimeric species are formed in the fast
folding steps by kinetic partitioning and are not in equilibrium. It is
only on a longer time scale that these different states converge to
form the unique native protein. To summarize, this study shows that
a sequence located within the N-domain of PGK confers on protein
fragments smaller than the N-domain the ability to form stable
multimeric species. These multimeric forms are not observed as stable
species with the whole protein and with the complete, stabilized, and
folded N domain. However, they were shown to occur during the folding
of the whole protein as transient species. Thus, the distribution of
multimeric species probably results from a kinetic competition taking
place during the fast folding phase. The dissociation of the multimeric
forms occurs during the unique slow folding phase, giving rise to the
native monomeric protein. In contrast to folding models where the
folding process is described as a strictly sequential and hierarchical
process, these results provide direct evidence the polypeptide chain
can explore transiently nonnative interactions during the folding
process.
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 5270-5276
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
Urea and EGS (
)were
obtained from Pierce; GdnHCl was from Life Technologies, Inc.; and
NbS
was from Sigma. All other reagents were of analytical
grade.Mutagenesis and Protein Purification
Mutants from
yeast phosphoglycerate kinase (PGK), C97A/I124C, C97A/S140C,
C97A/S153C, C97A/V281C, were obtained from a first mutant in which the
unique cysteine was replaced by an alanine residue. Site-directed
mutagenesis was performed as described previously(20) . The
coding sequences of the mutated genes were completely sequenced in
order to check the absence of other unwanted mutations. Each mutated
gene was then inserted in the expression vector (20) and
over-expressed in pgk Saccharomyces cerevisiae strain
BC3(21) , and purified according to previously published
procedures(22) . Horse muscle PGK was purified as reported
previously(23) .Production of Yeast PGK Fragments
Fragments were
obtained by cleavage at the introduced cysteinyl residue. The method of
cleavage used was adapted from Vanaman and Stark (24) and has
been described previously(19) . Fragment 1-172 was
obtained by CNBr cleavage of wild-type PGK, as described by Adams et al.(25) . Detailed purification procedures will be
published elsewhere. Purified fragments were controlled for the absence
of complementary fragments or uncleaved protein by SDS-PAGE.Production of Soluble Aggregated Form of Horse Muscle
PGK
Horse muscle PGK (42 µM) was incubated for 1 h
in 10 mM phosphate buffer, pH 7.5, containing 0.75 M GdnHCl and 1 mM dithiothreitol. A sample was
chromatographed on a Superose 12 column eluted with a 10 mM phosphate buffer, containing 1 mM dithiothreitol. Flow
rate was 0.13 cm min. The peak corresponding to the
excluded material was collected and used to check the efficiency of the
cross-linking method.
Cross-linking Experiments
The oligomerization of
fragments and uncleaved PGK was studied by cross-linking with
glutaraldehyde or EGS. Proteins in a 10 mM phosphate buffer,
pH 7.5, were cross-linked with glutaraldehyde, according to Hermann et al.(26) . The glutaraldehyde (25% v/v) was added to
samples at 1% (v/v) of the final mixture. The reaction was allowed to
occur at 20 °C, typically for 30 s unless otherwise indicated, and
was quenched by the addition of a freshly prepared 2 M NaBH
in 0.1 M NaOH.SO at 5 mM and added to the sample to give
the final concentration of 200 µM. After 30 s, the
reaction was quenched by the addition of 0.5 M Tris-HCl buffer
(final concentration, 100 mM), pH 8.
g for 15 min and then
resolubilized in electrophoresis sample buffer to give the
concentration required for electropheresis. The SDS-PAGE was performed
using conventional procedures at room temperature with a 12%
polyacrylamide slab gel. The gels were stained with Coomassie Blue and
scanned with a Desaga CD60 densitometer.Sedimentation Equilibrium
Equilibrium
ultracentrifugation analyses were conducted with two PGK fragments. The
N-terminal domain (fragment 1-184), used as a control for a
monomeric species, was solubilized in a 10 mM phosphate
buffer, pH 7.5. The fragment concentration was 22 µM.
Fragment 1-280 was previously refolded in the same phosphate
buffer. Protein and residual denaturant concentrations were 10
µM and 0.1 M GdnHCl, respectively. These samples
were centrifuged at 20 °C for 11 h at 25,000 rpm in a Beckman XL-A
analytical ultracentrifuge. After reaching the equilibrium, the
concentration profiles were recorded by monitoring the absorbance at
276 nm. The partial specific volume of fragments at 20 °C were
calculated to be 0.751 ml/g for fragment 1-280 and 0.743 ml/g for
fragment 1-184, according to Perkins(27) . Experimental
data were analyzed by nonlinear least-squares fitting, using the
SEDEQ1B program(28) .Light Scattering Experiments
A solution of fully
unfolded (29) PGK (2 M GdnHCl, 100 µM protein) was diluted 10-fold in a 10 mM phosphate buffer,
pH 7.5. Special care was taken to avoid insoluble material during these
experiments; most importantly, buffer and solutions were filtered
through a 0.22-µm filter just before use. The kinetics were
recorded with a spectrofluorimeter using the same wavelength (310 nm)
as excitation and emission settings. Two different instruments were
used: a SLM 8000C Aminco spectrofluorimeter with a 10-mm light path
cell and a SFM 3 Bio Logic stopped-flow apparatus equipped for
fluorescence measurements with a cut-off filter that stopped light at
wavelengths shorter than 310 nm. With instruments, the scattered light
intensity was monitored at 90° with respect to the incident beam.
The results were averaged from five independent measurements.
The Presence of Stable Multimeric Forms of PGK
Fragments Is Mainly Associated with the N-terminal Part of the
Sequence
Previous studies (19) on subdomain fragments of
PGK have shown that a fragment (1-96), corresponding to the first
half of the N-terminal domain, forms a series of oligomeric species
even at relatively low protein concentration. These species were
clearly observed using cross-linking experiments followed by SDS-PAGE
analysis. The electrophoretic mobility of the species was shown to
correspond exactly to the mobility expected for dimer, trimer, and
tetramer.
A Similar Distribution of Oligomeric Species Is Observed
Transiently during the Refolding of the Whole Protein
Previous
studies on PGK have shown that the refolding process involves at least
two kinetic phases(9, 14) . At least one fast phase
occurs during the dead time of manual mixing experiments. This phase is
followed by a slow phase, which leads to the fully refolded and active
protein. This later phase has been clearly observed using different
signals such as far UV CD, tryptophan fluorescence, titration of
engineered thiol groups(9) , fluorescence of
8-anilino-1-naphthalenesulfonic acid expulsion(14) ,
susceptibility to proteolysis(30) , binding of monoclonal
antibodies, ()and enzyme activity (9) . This phase
is not related to proline isomerization (9, 31) but
rather corresponds to the transformation of a compact but fluctuating
state in the unique and relatively rigid native structure. An important
point, established by thiol titration experiments, is that the fast
folding phase leads to a distribution of at least two different
molecular populations more flexible than the native state(9) .
Furthermore, the results described above indicate that the associated
fragments species are formed rapidly and at low protein concentrations.
These two observations suggest that the associated states observed with
the fragments might also be accessible to the whole protein as long as
the structure is not locked in the native conformation. In other words,
the associated forms might be apparent during the slow refolding phase.
The experiments reported below show that such associated forms are
indeed observed during the slow refolding phase.
4.6 10 s
) as determined by other
signals (see Fig. 5and (28) ). In control experiments (Fig. 3), the positions of the ``monomeric unfolded''
and ``monomeric native'' bands were also observed for the
unfolded protein cross-linked in the denaturant and for the native
protein cross-linked without denaturant, respectively.
, folded monomeric
protein;
, unfolded monomeric protein; , multimeric
species.
10 ± 0.3
10 s-1).
) are reported in insets.
Properties of the Transient Multimeric Species
The
refolding process of PGK from yeast or from horse muscle displays
similarities but also a difference relative to the stability of the two
domains, the C domain being more stable than the N domain in the horse
muscle protein(14) . The results of the cross-linking
experiments conducted during the refolding process of the horse muscle
enzyme, reported in Fig. 3, indicate that the formation of
multimeric species also occurs during the refolding process of this
protein. This suggests that the production of oligomeric species is not
a peculiarity of yeast PGK, but rather a conserved property between PGK
from two different species, in spite of the differences observed in the
domain stability.
/k (Fig. 7).
-tryptophan
synthase(34) . This absorbance was attributed to transient
aggregates. Transient oligomeric forms of bovine growth hormone have
been also indirectly observed by Brems(35) . It seems therefore
that such species could occur at least for a few other proteins,
although for protein concentration higher than the very low
concentration range used in the present work. However, there are no
reported direct observation of such transient multimeric species.
))
We thank Dr. N. Kellershohn for helpful advice about
the kinetic mechanism, G. Battelier for help with the
ultracentrifugation experiments, and Dr. M. Ladjimi for analysis. We
also thank Dr. Liepkalns for carefully reading the manuscript and
revising the English.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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  M. Silow and M. Oliveberg Transient aggregates in protein folding are easily mistaken for folding intermediates PNAS, June 10, 1997; 94(12): 6084 - 6086. [Abstract] [Full Text] [PDF]
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