(Received for publication, November 9, 1994; and in revised form, December 9, 1994)
From the
The epitope recognized by a monoclonal antibody (mAb19) directed
against the subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9
of the
chain. The affinities of mAb19 for peptides of different
lengths containing the 2-9 sequence were close to 0.6
10
M
, the affinity of mAb19 for
native
. In view of these results, a model is proposed
to account for the kinetics of appearance of the epitope during in
vitro renaturation of
(Murry-Brelier, A., and
Goldberg, M. E.(1988) Biochemistry 27, 7633-7640). A
mutant producing
chains lacking residues 1-9
(
) was prepared. The
protein was able to fold into a heat
stable homodimer resembling wild type
. Isolated
had no detectable enzymatic activity. It
could bind
chains extremely weakly and be slightly activated. In
the presence of the 1-9 peptide, the
protein could bind
chains much more strongly and generate a
50% active enzyme. Thus, although having little role in the overall
folding and stability of the protein, the 1-9 sequence of the
chain appears strongly involved in the
-
interactions
and in the enzymatic activity.
In an attempt to use monoclonal antibodies as conformational
probes to investigate the structure, conformational changes,
conformational dynamics, and folding pathway of proteins, we have
prepared a panel of monoclonal antibodies directed against different
proteins and developed a variety of experimental methods for the
quantitative analysis of antigen-antibody interactions(1) . For
most of these studies, the model protein we used was the (
)subunit of Escherichia coli tryptophan
synthase. The properties of this enzyme have been recently reviewed in
detail(2) . We shall summarize here those which are directly
relevant to the present study. The natural form of tryptophan synthase
is the heterotetramer
, which can be
readily dissociated into monomeric
chains and the dimeric
subunit. The
subunit can, in the
presence of its coenzyme pyridoxal-5`-phosphate, catalyze two distinct
reactions: indole + L-serine
L-tryptophan
+ H
O and L-serine + H
O
pyruvate + ammonia.
In the presence of chains, the
serine deaminase reaction is completely abolished, while the specific
activity of
in the tryptophan synthase reaction is
increased by a factor about 30. The three-dimensional structure of the
enzyme from Salmonella typhimurium has been
solved(3) . Because of the very strong amino acid sequence
homology between the enzymes from E. coli and S.
typhimurium, there is no doubt that the three-dimensional
structures of both enzymes also must be extremely similar. The in
vitro refolding of denatured
chains to regenerate native
subunits has been extensively
investigated(4) . In particular, one monoclonal antibody,
mAb19, turned out to detect an early folding step during the in
vitro renaturation of
(5, 6) .
It was shown that the specific epitope was not recognized by mAb19 at
early stages of the folding process and became immunoreactive as a
result of a folding step with a kinetic constant of 0.06 s
at 12 °C(5) . This intramolecular reaction occurs
early on the folding pathway, at a stage where the protein is still a
molten globule(4) . Furthermore, the affinity of mAb19 for the
early folding intermediate was shown to be of the same order of
magnitude as that of mAb19 for the native protein(5) . This
suggested that the conformation of the epitope was the same in the
early intermediate as it is in the native protein. It seemed of
interest to understand in more detail the nature of the folding step(s)
detected by mAb19, and for that purpose to characterize as precisely as
possible the epitope recognized by this antibody.
The present report
describes experiments aimed at identifying the residues involved in the
antigenic site recognized by mAb19 and at assessing their role in the
folding, stability, association with chains, and enzymatic
activity of the
subunit.
For Western blots, the buffer was TBS (0.05 M Tris base, 0.15 M NaCl, pH 7.2).
Monoclonal antibodies were obtained (10) and purified from
ascitic fluids (11) as described earlier. Their concentration,
expressed in terms of binding sites, was estimated from the absorbance
at 280 nm, using the standard value of 1.5 cm/mg (12) as specific extinction coefficient. For affinity
measurements with the radioactivity assay, the mAb19 solution was
titrated as described earlier(13) .
The polypeptide 1-102 was prepared (14) and kindly supplied by Dr. Alexei Fedorov.
For analysis of peptides produced by in vitro translation, a logarithmic 10-27% acrylamide gradient was used(22) .
The peptides produced by in vitro translation were transferred to nitro-cellulose membranes by electrotransfer at 200 milliamperes for 2 h(23) .
In order to identify the region of the chain that binds
mAb19, 13 independent clones expressing recombinant peptides recognized
by mAb19 were isolated from a gene library and their DNA was sequenced.
All the polypeptides encoded by these clones had in common residues
1-85, which localized the epitope of mAb19 within the 85 first
residues of the
chain. The N-terminal F1 fragment of the
chain was then submitted to cyanogen bromide cleavage and the resulting
peptides analyzed by SDS-PAGE. Staining of the gel revealed a major
peptide which, from its migration on the gel and its N-terminal
sequence determined by microsequencing (Pro-Ala-Leu), was identified as
the 23-101 peptide. This peptide did not give rise to any
detectable reaction with mAb19, while peptide 1-102 distinctly
reacted with mAb19 and not with a control antibody. These results
suggested that the 1-22 sequence was essential for building up
the epitope.
Figure 1:
Western blots of the
chain carrying the 2-9 deletion. Bacteria carrying the
plasmid encoding the
chain, either complete or carrying the
2-9 deletion, were grown overnight in rich medium supplemented
with ampicillin, washed, sonicated, and centrifuged. The supernatants
(15 µl) were subjected to SDS-PAGE on a 10-27% acrylamide
gel, electrotransferred, and stained as indicated under
``Materials and Methods.'' Left panel (A):
Western blot with mAb19. Slot 1,
carrying the 2-9
deletion; slot 2, wild type
; slot 3, molecular
weight markers. Right panel (B): Western blot with a
specific monoclonal antibody that recognizes the sequence 273-283
of the
chain. Slot 4,
carrying the 2-9
deletion; slot 5, wild type
; slot 6, molecular
mass (indicated in kDa) markers.
This raised the question of why screening of the
epitope library did not pick up any immunoreactive clone expressing a
protein that would end before residue 85. To test the possibility of a
cloning artifact, recombinant plasmids carrying the trypB gene
with stop codons at various places were prepared by site-directed
mutagenesis. The stop codons were introduced at positions such that
polypeptide chains starting at position 1 and ending at positions 39,
50, 60, 69, and 79 should be produced. Two positive controls were used.
One was a plasmid encoding the sequence 1-102(14) . The
second was a plasmid encoding the 1-102 fragment with a deletion
of residues 15-21. The latter plasmid thus encoded a 95 residue
fragment (1-14.22-102) carrying the 2-9
immunoreactive sequence. The corresponding mRNAs were prepared and used
as templates in a wheat germ cell-free protein synthesis system. The
proteins produced were subjected to SDS-PAGE and Western blotting.
Although considerably blurred by the presence of an endogenous antigen
likely to be the wheat germ tryptophan synthase, the results obtained
(not shown) clearly indicated that fragments extending from the
N-terminal to anywhere between residues 39 and 79 of the chain
could not be detected in the Western blots with mAb19 as performed
here. On the contrary, longer fragments containing the intact
N-terminal sequence of
could clearly be seen. Thus, rather than a
cloning artifact, the failure to detect shorter immunoreactive
fragments in the epitope library seemed to be due to a screening
artifact.
To find out whether this screening artifact was due to an
abnormal interaction of the short peptides with the membrane onto which
they were blotted, or to a particular conformation that short peptides
might adopt and that would render the epitope unreactive, peptides
labeled with [S]methionine were synthesized in vitro as above, using mRNA with the various stop codons as
templates, and their interaction with mAb19 (10
M) in solution was investigated by competition
RIA(12) . The qualitative results of this experiment indicated
that, in solution, all the peptides investigated (starting at position
1 and ending at positions 39, 50, 60, 69, and 79, respectively) reacted
strongly with mAb19. This was quantitatively verified by measuring the
affinities of mAb19 for the peptides 1-60 and 1-102 in
solution, using the competition RIA method(12) . The affinity
obtained with peptide 1-60 from the data shown in Fig. 2was 0.75
10
and a very similar value
(0.7
10
M
) was obtained
with fragment 1-102 (Table 1). Thus, while the antigenic
determinant of the 1-60 fragment was not detected by Western
blotting, in solution it was recognized by mAb19 in the 1-60
fragment as well as in the 1-102 fragment. Moreover, the values
found for the affinities were only slightly smaller than that reported
above for the 2-9 peptide and very close to that observed
previously for native
(14) . This indicated
that residues 2-9 did not adopt an abnormal conformation in the
short peptides obtained in solution after in vitro cell-free
synthesis. Thus, the apparently poor reactivity of the short peptides
when immobilized on membranes seemed to result primarily from an
artifact in their immunodetection with mAb19 on the blots, showing that
care should be taken in interpreting Western blots when negative
results are obtained.
Figure 2:
Determination of the affinity of mAb19 for
the fragment 1-60. The S-labeled peptide 1-60
was synthesized in a wheat germ cell-free protein synthesis extract. 2
µl of the synthesis mixture were diluted in 50 µl of standard
buffer supplemented with 10 µl of caseine (1 mg/ml solution
filtered on a 0.45-µm Millipore filter) and 1-3 µl of
various dilutions of mAb19 to obtain the desired antibody
concentration. This mixture was incubated for 1 h at 4 °C, and 40
µl of a suspension of Sepharose beads coupled to mAb19 and
saturated with casein were added, incubated for 5 min with gentle
shaking, centrifuged, and washed. The immunoadsorbed proteins were
dissolved, subjected to SDS-PAGE, and the amount of radioactive
fragment in each slot was determined by scanning in a
-Imager as
described by Friguet et al.(19) . A,
bidimensional scan for the gel with fragment 1-60. Slot
1, molecular weight markers; the mAb19 concentrations in the
mixtures were as follows. Slot 2, 0; slot 3, 2.3
10
M; slot 4, 6.8
10
M; slot 5, 2.3
10
M; slot 6, 6.8
10
M; slot 7, 2.3
10
M; slot 8, 6.8
10
M. B, the total radioactivity
contained in each band at the position of fragment 1-60 in the
diagram in panel A was obtained by use of the
-Imager
software, and the data obtained were plotted as described by Friguet et al.(19) . The experimental points were fitted to a
straight line by linear regression. The slope of this line provides the
equilibrium dissociation constant (see Table 1).
Visual
inspection of the Coomassie Blue-stained gels and of the Western blots
indicated that the amount of chains produced by the cells
carrying the wild type trpB gene was much larger than that
produced by the deletion mutant. This was verified by quantitative
competition ELISA using the antibody mAb93 which recognizes an epitope
carried by the C-terminal domain of
(10) . We
found that about 1 mg of
protein was
produced per gram (wet weight) of mutant bacteria. This was about
10-fold less than the amount found for wild type
chains. The
amount of
chains produced with the plasmid carrying the deletion
was also smaller (as judged from the Coomassie Blue-stained gels) than
with the wild type plasmid. This strongly suggested that the low
amounts of
protein obtained with the
mutant resulted from a low level of synthesis rather than from a
post-translational event.
Moreover, the ratios of immunoreactive
protein detected by Western blotting in the supernatant and in the
pellet were of the same order of magnitude for the
protein and for the wild type. Thus, the
protein appeared able to fold in
vivo without giving rise to inclusion bodies.
Figure 3:
Gel filtration of a crude extract
containing the protein. 100 µl of
the extract (22 mg/ml of protein) were injected in a FPLC Superose-6
column previously equilibrated with standard buffer, and the elution
was achieved at room temperature with the same buffer at a flow rate of
0.5 ml/min. 0.25-ml fractions were collected. A competition ELISA (see
``Materials and Methods'') with mAb93 (open circles)
or mAb164 (open squares) was performed to measure the relative
amounts of the
protein in each fraction.
In this assay, a decrease in the absorbance at 405 nm reflects an
increase in the concentration of antigen. The elution pattern of the
wild type protein was obtained by injecting 100 µl of pure
on the same column, eluting as above, and measuring
the absorbance at 280 nm in each fraction (filled diamonds).
The left arrow indicates the elution volume determined for the
pure
complex.
Figure 4:
Irreversible heat inactivation of the
protein. Aliquots of crude extracts
containing 0.1 mg/ml of either wild type
or of the
truncated protein were heated for 10 min at the temperature indicated
on the abscissa. After cooling on ice and centrifugation for
15 min at 10,000
g and 4 °C, the amount of soluble
(open squares) or
protein (open circles) remaining in the supernatant was
determined by competition ELISA with mAb93, substracting the absorbance
measured in the supernatant (A) from the absorbance (A
) in the control obtained without addition of
supernatant.
Figure 5:
Purification of the
protein. Aliquots of the preparation at
the different steps of the purification described under
``Results'' were analyzed by SDS-PAGE followed by Coomassie
Blue staining. A: slot 1, pure
as a
marker; slot 2, crude extract; slot 3, protamine
sulfate supernatant; slot 4, supernatant after heating at 63
°C and centrifugation; slot 5, 41% ammonium sulfate
precipitate; slot 6, supernatant of the 41% ammonium sulfate
step; slots 7 and 8, after unsuccessful
crystallization with 0.4 M ammonium sulfate. B: slot 1, pure
as marker; slot 2,
precipitate; and slot 3, supernatant, after 1 M ammonium sulfate.
In order to
ascertain that this protein was the expected one, it was subjected to
five cycles of N-terminal sequencing. The sequence of amino acids
obtained (Gly-Glu-Phe-Gly-Gly) was identical to that of residues
10-14 of the chain, which indicated that the purified
protein was indeed the expected truncated
chain with its
N-terminal methionine cleaved off. That it started at residue 10,
justified the name ``
protein''
it was given.
The experiments described above were initially aimed at
mapping the epitope recognized by mAb19. They lead to the unexpected
finding that mAb19, previously reported to be a ``conformation
dependent'' monoclonal antibody, in fact recognizes a linear
epitope. This epitope is carried by the sequence of residues 2-9
(Thr-Thr-Leu-Leu-Asn-Pro-Tyr-Phe) on the N-terminal extremity of the
chain.
Although short peptides sometimes have a preferred
conformation in aqueous solution, such conformations are only
marginally stable and the corresponding peptides are very flexible.
Moreover, when applied to the 1-9 peptide, the usual secondary
structure prediction programs find only a small propensity to form a
strand limited to residues 1-3, while residues 4-9
are predicted to be ``random.'' Therefore, it is very likely
that the isolated 1-9 peptide in aqueous buffer is devoid of any
stable ordered structure. This sequence was also thought to be
disordered in the native protein since, in the original x-ray structure
of native
(3) , residues
1-8 were not visible. However, the structure of the
complex has now been refined and
recent unpublished results (
)show that residues 3-9
are in fact well ordered and that residues 7-9 form a
strand that belongs to a four stranded antiparallel
sheet. Yet,
the affinities of mAb19 for native
and for the
peptides 1-9, 1-60, and 1-102 do not differ from one
another by more than a factor 2. This indicates that the conformation
of the epitope is very similar in all these antigens. Assuming that the
1-9 region has the same conformation in the isolated
subunit as in the
complex,
this would suggest that the isolated 1-9 peptide might adopt in
solution a stable conformation coinciding with that it has in native
. As discussed above, this is quite
unlikely. An alternative explanation is that the 2-9 region would
be disordered in the isolated
subunit (for which
mAb19 has a high affinity in the absence of
) and would become
ordered in the
complex (which mAb19
fails to recognize). This seems quite plausible for the following
reasons. In
, the side chain of
residue 8, as well as the bulk of residue 12 and of several others just
beyond, form extensive contacts with the
subunit.
Furthermore, another monoclonal antibody, mAb164-2, also
recognizes the isolated synthetic peptide corresponding to its epitope
(residues 273-283) with an affinity nearly as as high as that it
has for native
(26) . This sequence contains
the C-terminal
-hairpin of the four-stranded
-sheet in which
the epitope of mAb19 is also involved. This suggests that the
four-stranded
-sheet may be disorganized in the isolated
subunit. Thus, interactions with the
subunit
seem to significantly stabilize the four-stranded
-sheet
suggesting that the 3-9 sequence may loose its ordered structure
in the absence of these interactions. This possibility is also
indirectly supported by our observations that
, although it lacks residues 1-9,
can fold into a conformation close to that of the native wild type
protein, and that it has a heat stability very similar to that of
normal holo-
. Indeed, these observations demonstrate
that, in the
subunit alone, the 1-9 region
affects neither the overall conformation nor the stability of the rest
of the polypeptide chain. Thus, the 1-9 region appears to form
only weak intrasubunit interactions in the absence of
chains, and
hence its conformational stability is likely low. Moreover, although
the 1-9 region forms only one visible interaction with
in
the wild type
complex (a hydrogen
bond involving the side chain of Tyr-8), the truncated protein binds
extremely weakly and addition of the synthetic peptide 1-9
considerably strengthens this binding. This indicates that the
1-9 region is indirectly, yet strongly, involved in interactions
responsible for the stability of the
complex. These interactions must in turn stabilize the
conformation of the 1-9 region which, in their absence, is likely
to become disordered. Although this would be definitely supported only
if the three-dimensional structure of the isolated
subunit could be solved, the converging pieces of evidence
discussed above lead us to believe that, in isolated
,
the 1-9 region is highly flexible and that mAb19 probably
recognizes a region of
which, prior to its
association with the antibody, has no or little defined structure.
This raises the intriguing question of the nature of the folding
step that controls the immunoreactivity regain of chains during
their in vitro renaturation(5, 6) . Indeed,
since mAb19 reacts well with the short, very probably disordered,
1-9 peptide it should also react well with unfolded
chains.
Yet, that a relatively slow folding step (k = 0.06
s
at 12 °C) is required for the appearance of
the epitope during the refolding of
chains in vitro has
been unambiguously demonstrated by earlier
studies(5, 6) . What is this isomerization reaction? A
plausible model is that the hydrophobic N-terminal sequence of
might get very rapidly, but only transiently, buried inside the protein
core. The regain of immunoreactivity would then occur at a later stage
of the folding, when the 2-9 antigenic sequence would get
expelled from the interior of the protein into the solvent, as a result
of a tighter packing of the hydrophobic core.
This tentative model
might suggest that the transient burying of residues 2-9 is an
essential folding step. That the protein
is able to fold into a native-like conformation clearly rules out this
possibility. Rather, it seems that the 1-9 region is involved in
interactions that occur at a late stage of the folding, as a final
adjustment of the structure that leads to the active conformation. This
view is supported by our observation that the 1-9 peptide can
complement the already folded
protein in
the presence of the
subunit.
That a short peptide can
complement a polypeptide chain truncated on its N-terminal end has been
reported for several proteins. The case which resembles most that
reported here is the complementation in E. coli
-D-galactosidase (30) for which it has been
recently shown that the
(N-terminal) peptide is essentially
involved in the formation of intersubunit association areas (31) . The results we report here, together with the refined
three-dimensional structure of the
complex, suggest that the same is true for the 1-9 peptide
in tryptophan synthase. Equilibrium and kinetic studies are currently
conducted in our laboratory to understand how this peptide bridges the
and the
subunits into a stable complex and
leads to the formation of the functional protein.