©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Importance of Residues 29 in the Immunoreactivity, Subunit Interactions, and Activity of the Subunit of Escherichia coli Tryptophan Synthase (*)

(Received for publication, November 9, 1994; and in revised form, December 9, 1994)

Amiel Navon (1) Andreas J. Schulze (3) Yvonne Guillou (3) Catherine A. Zylinski (3) Françoise Baleux (2) Nicole Expert-Bezançon (3) Bertrand Friguet (3) Lisa Djavadi-Ohaniance (3) Michel E. Goldberg (3)(§)

From the  (1)Department of Life Sciences, Bar Ilan University, Ramat Gan, Israel and the (2)Unité de Chimie Organique (CNRS, URA 487) and the (3)Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris, Cedex 15, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The epitope recognized by a monoclonal antibody (mAb19) directed against the beta(2) subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the beta chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 times 10^9M, the affinity of mAb19 for native beta(2). In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of beta(2) (Murry-Brelier, A., and Goldberg, M. E.(1988) Biochemistry 27, 7633-7640). A mutant producing beta chains lacking residues 1-9 (beta) was prepared. The beta protein was able to fold into a heat stable homodimer resembling wild type beta(2). Isolated beta had no detectable enzymatic activity. It could bind alpha chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the beta protein could bind alpha 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 beta chain appears strongly involved in the alpha-beta interactions and in the enzymatic activity.


INTRODUCTION

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 beta(2)(^1)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 alpha(2) beta(2), which can be readily dissociated into monomeric alpha chains and the dimeric beta(2) subunit. The beta(2) subunit can, in the presence of its coenzyme pyridoxal-5`-phosphate, catalyze two distinct reactions: indole + L-serine L-tryptophan + H(2)O and L-serine + H(2)O pyruvate + ammonia.

In the presence of alpha chains, the serine deaminase reaction is completely abolished, while the specific activity of beta(2) 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 beta chains to regenerate native beta(2) 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 beta(2)(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 alpha chains, and enzymatic activity of the beta(2) subunit.


MATERIALS AND METHODS

Reagents and Buffer

The standard buffer used was 0.1 M potassium phosphate at pH 7.8, 2 mM EDTA, and 5 mM 2-mercaptoethanol.

For Western blots, the buffer was TBS (0.05 M Tris base, 0.15 M NaCl, pH 7.2).

Protein and Proteolytic Fragment

The native beta(2) subunit of E. coli tryptophan synthase was prepared and cleaved with trypsin, and the resulting N-terminal F1 and C-terminal F2 fragments were separated by heat denaturation as described previously(7) . The solubilized F1 fragment was purified by gel filtration on a S-200HR Sephacryl column equilibrated with standard buffer. The concentrations of beta(2) and of F1 were determined spectrophotometrically using specific extinction coefficients at 280 nm of 0.58 and 0.67 cm^2/mg, respectively. The enzymatic activity of the beta(2) subunit was measured in the indole to tryptophan reaction, either in the presence or in the absence of alpha subunit, as described previously (8) , and in the serine deamination reaction(9) .

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^2/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.

Epitope Library Construction, Screening, and Characterization

The epitope library was constructed, screened for clones expressing the epitope of mAb19, and the nucleotide sequence at the beginning of each insert as well as its length were determined as described by Friguet et al.(15) .

Cyanogen Bromide Cleavage of F1

The lyophilized F1 fragment (40 mg) was dissolved in 1 ml of 70% formic acid, 100 mg (a 20-fold molar excess over F1) of cyanogen bromide were added, and the resulting solution was incubated overnight in the dark at room temperature. The mixture was then diluted 10-fold with water and lyophilized.

Chemical Synthesis of Peptides

Peptides were synthesized by the Merrifield (16) solid-phase method using an Applied 430 synthesizer. They were purified by gel filtration and preparative C18 reverse phase HPLC, lyophilized, and stored at 4 °C. The final purity of the peptides (>98%) was checked by analytical C18 reverse phase HPLC. Amino acid composition was determined on a Beckman 6300 Amino Acid Analyzer after 6 N HCl hydrolysis. The extinction coefficients at 280 nm of the peptides were estimated from their tyrosine content, using an extinction coefficient of 1420 M cm for tyrosine at pH 6.

Site-directed Mutagenesis

Stop codons or deletions were introduced in the trpB gene by oligonucleotide site-directed mutagenesis(17, 18) . A derivative of the plasmid pTZ19R, carrying the sequence coding for the first 102 amino acids of the tryptophan synthase beta subunit(19) , was used for the introduction of stop codons and of the 15-21 deletion in the 1-102 peptide. For the introduction of the 2-9 deletion in the complete beta chain, the plasmid used was derived from palphabetaTS21 (a plasmid containing the trpA and trpB genes, constructed by Dr. Carlos Zetina) by excising the fragment containing the B gene with ClaI and inserting it at the ClaI site of the Bluescript KS. The single-stranded DNA of the plasmids was prepared by infection of the E. coli strain RZ1032, used as a cellular host, by helper phage M13-K07(17, 18) . The single strand DNA of the pTZ19R derivative was used to introduce a stop codon at five different places (after codons 39, 50, 60, 69, and 79) or to construct the 15-21 deletion and the Bluescript derivative to construct the 2-9 deletion, using appropriate primers. The construction of the different stop codons resulted in the introduction of a new unique AflII restriction site, that of the 2-9 deletion introduced a new BamHI site, and the 15-21 deletion resulted in the loss of a RsaI site. After analysis of a few clones from each mutation, the clones with the correct restriction pattern were subjected to automated sequence analysis in an Applied Biosystem sequencer, using T7 and T3 primers and the dye termination method.

RNA Preparation

The different plasmids were prepared as a ``mini-prep'' or in ``large scale''(20) . 2 µg of the plasmid of interest were linearized with EcoRI, phenol extracted, and ethanol-precipitated. The precipitate was washed with 70% ethanol, vacuum dried, and then used for in vitro transcription from the T7 promoter(20) . An aliquot of the newly transcribed RNA was loaded on a 2% agarose gel in order to verify the quality of the RNA.

In Vitro Translation

All translation reactions were done using a wheat germ in vitro translation lysate (Promega). As a preliminary step, the optimum potassium acetate concentration was determined for each mRNA and was found to be 73 mM for all mRNAs, except for that coding the 39 residue fragment for which it was 93 mM. All reactions were performed in a final volume of 25 µl. A typical reaction mixture contained 1 µg of RNA, 1 µl of Rnasin (54,000 units/ml), 0.5 µl of 1 M potassium acetate, 2 µl of amino acid solution (1 mM of each amino acid except methionine), 1 µl of [S]methionine (Amersham 1000 Ci/mmol), 12.5 µl of wheat germ lysate, and 8 µl of water. The translation was allowed to proceed for 1 h at 25 °C. In order to check the efficiency of the translation and estimate the amount of peptides produced in each translation mixture, 1-µl aliquots were subjected to SDS-PAGE, after which the gel was stained, destained, dried, and subjected to quantitative radioactivity scanning in a beta-Imager (Biospace, Paris,) until about half a million counts were recorded. Integrating the counts for each fragment by means of the beta-Imager software, and comparing with a control sticker of known radioactivity, provided an estimate of the amount of the desired peptide in each preparation.

Gel Electrophoresis

Electrophoresis of the cyanogen bromide peptides was performed either with the Phast System (Pharmacia) using homogeneous SDS 20 gels, or according to Schägger and von Jagow (21) . The molecular weight markers used were from the 2350-46000 Da kit (Amersham).

For analysis of peptides produced by in vitro translation, a logarithmic 10-27% acrylamide gradient was used(22) .

Electrotransfer

The cyanogen bromide peptides were transferred onto Problott membranes (Applied Biosystems) by applying 50 V for 30 min at room temperature. The buffer used was 10 mM CHAPS at pH 11 in 10% methanol.

The peptides produced by in vitro translation were transferred to nitro-cellulose membranes by electrotransfer at 200 milliamperes for 2 h(23) .

Microsequencing

After staining with Amido Schwarz, the region of the membrane containing the desired band was cut with a razor blade, and three cycles of N-terminal sequence determination were carried out in an Applied Biosystem 470 gas-phase automatic sequencer. Phenylthiohydantoin amino acids were identified with an on-line Applied Biosystems 120A analyzer.

Western Blots

The membrane-bound peptides obtained by electrotransfer were immunodetected with mAb19 as follows. For the cyanogen bromide peptides, rabbit anti-mouse IgG antibodies coupled to alkaline phosphatase (Biosys) were used and revealed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (Sigma). For the peptides produced by in vitro translation, rabbit anti-mouse IgG antibodies conjugated to horseraddish peroxidase were used and developed with the ECL reagents of Amersham according to the manufacturer's recommended procedure.

Competition ELISA

Quantification of wild type or truncated beta(2) subunits in crude extracts was made by a competition ELISA based on the test previously described(24) . Briefly, the concentration range in which the absorbance obtained in the last step of the indirect ELISA is proportional to the amount of free antibody in solution was first determined. Then, the mAb, at a concentration near the upper limit of this range (2.5 times 10M) was incubated for 30 min with various dilutions of either the pure enzyme or the crude extract (wild type or mutant). The free antibody remaining in solution was then determined by indirect ELISA. Comparing the competition curve obtained with the crude extract with the standard curve obtained with pure beta(2) provided a quantitative estimate of the amount of enzyme in the extract. A similar competition assay was used to detect the relative amount of enzyme in the fractions obtained after gel filtration, using a 20-fold dilution of each fraction. The procedure for the indirect ELISA was as described previously(24) .

Affinity Measurements

They were made either by a competition ELISA method (13) when pure antigen was available, or by a competition RIA method (19) for antigens produced by in vitro cell-free synthesis.

Circular Dichroism

Circular dichroism spectra were recorded in a CD6 spectrodichrograph (Jobin-Yvon, Longjumeau, France) using 0.1- or 0.2-mm optical path cells. The temperature was 20 °C. The wavelength region scanned was between 193 and 255 nm. The spectral bandwidth was automatically kept at 2 nm. The wavelength increment was 1 nm/step and the accumulation time was 1 s/ step. Each spectrum resulted from averaging five successive scans. The spectrum of the solvent alone was recorded under identical conditions and substracted from the sample spectrum to generate the CD spectrum of the peptide.


RESULTS

In order to identify the region of the beta 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 beta chain. The N-terminal F1 fragment of the beta 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.

Chemical Synthesis and Immunoreactivity of Synthetic Peptides

To more precisely delineate the epitope, four peptides included in the 1-22 region were prepared by chemical synthesis, and their affinities for mAb19 were measured according to Friguet et al.(13) . The results shown in Table 1indicated that the shortest peptide recognized by mAb19 with an affinity close to that of the native protein was peptide 1-9. That peptide 2-22 also could bind mAb19 strongly showed that the N-terminal methionine of peptide 1-22 did not belong to the epitope. This is in line with the fact that, although its N-terminal methionine is cleaved off in vivo(25) the native protein reacts well with mAb19. The minimal immunoreactive sequence thus defined therefore corresponded to residues 2-9 of the beta chain. The affinity values obtained for all the synthetic peptides containing the 2-9 sequence were in the range of 0.1-0.3 times 10^9M (see Table 1). These affinities are only slightly smaller than that (0.6 times 10^9M) found for the native beta(2) subunit, strongly suggesting that the antigenic determinant of beta(2) recognized by mAb19 resides in the 2-9 sequence, and that the rest of the polypeptide chain might not contribute to the antigen-antibody recognition.



Preparation and Characterization of Mutants with Deletions or Stop Codons in the tryp B Gene

To ascertain the latter conclusion a plasmid encoding a beta chain lacking residues 2-9 was prepared, and the immunoreactivity of the corresponding truncated protein was investigated by Western blotting with mAb19. Fig. 1shows that the protein carrying the 2-9 deletion had no detectable reactivity toward mAb19, while it reacted strongly with mAb164, an antibody directed against the sequence 273-283 of the beta chain(26) . This confirmed that the 2-9 sequence is essential for the immunoreactivity of the beta(2) protein toward mAb19.


Figure 1: Western blots of the beta chain carrying the 2-9 deletion. Bacteria carrying the plasmid encoding the beta 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, beta carrying the 2-9 deletion; slot 2, wild type beta; slot 3, molecular weight markers. Right panel (B): Western blot with a specific monoclonal antibody that recognizes the sequence 273-283 of the beta chain. Slot 4, beta carrying the 2-9 deletion; slot 5, wild type beta; 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 beta 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 beta 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 (10M) 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 times 10^9 and a very similar value (0.7 times 10^9M) 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 beta(2)(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 beta-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 times 10M; slot 4, 6.8 times 10M; slot 5, 2.3 times 10M; slot 6, 6.8 times 10M; slot 7, 2.3 times 10M; slot 8, 6.8 times 10M. 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 beta-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).



Far UV Circular Dichroism of the 1-9 Antigenic Peptide

In an attempt to characterize the conformation of the immunoreactive isolated antigenic 1-9 nonapeptide, the far UV CD spectrum of a 0.1 mg/ml solution of the peptide was recorded. The spectrum obtained was quite atypical, mainly in the region below 200 nm where the residue ellipticity was particularly low (about -12,000 degbulletcmbulletdmol). Such a spectrum could by no means be generated by a random conformation or by any of the classical secondary structures of a polypeptide backbone. Moreover, the amplitude of the mean residue ellipticity increased with the peptide concentration. This suggested that aggregates are responsible for the abnormal far UV CD spectrum, thus precluding any reliable conclusion to be made concerning the conformation of the isolated nonapeptide at the much lower concentrations used for affinity measurements.

Expression and Folding of beta Chains

Because the 1-9 sequence of beta chains was not visible in the three-dimensional structure of tryptophan synthase as initially determined(3) , it could be thought that this region of the molecule may be disordered and therefore may have little role in the folding, stability, and biological properties of the native enzyme. To test this hypothesis, the beta protein was expressed in the E. coli strain MD33 transfected with a plasmid in which the 2-9 deletion had been introduced in the trpB gene and placed under the control of the tryptophanase promotor (see ``Materials and Methods''). After growth in rich medium, cells were washed, sonicated, and the insoluble material was separated by centrifugation. Both the pellet and the soluble extract were subjected to SDS-PAGE. As a control, the pellet and soluble extract from cells carrying the plasmid with the wild type trpB gene were run on the same gel. After Coomassie Blue staining a distinct band, migrating at practically the same position as wild type beta chains, was visible in the soluble extract of the beta mutant and, although very faint, also in the pellet. Western blotting using mAb164 (see above, section 2) showed that the corresponding protein reacted strongly with this antibody. Minor immunoreactive bands were also detected at positions corresponding to molecular weights smaller than that of beta chains. They very probably corresponded to fragments produced by partial proteolytic cleavage and appeared relatively more abundant with the mutant than with the wild type protein. This suggested that the truncated protein was more sensitive to proteases than the wild type protein (see below). The major band could be clearly seen both in the soluble fraction and in the pellet, but the amount contained in the soluble fraction was by far larger than in the pellet.

Visual inspection of the Coomassie Blue-stained gels and of the Western blots indicated that the amount of beta 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 beta(2)(10) . We found that about 1 mg of beta protein was produced per gram (wet weight) of mutant bacteria. This was about 10-fold less than the amount found for wild type beta chains. The amount of alpha 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 beta 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 beta protein and for the wild type. Thus, the beta protein appeared able to fold in vivo without giving rise to inclusion bodies.

State of Association of the beta Protein in the Soluble Extract

Since the beta protein appeared able to fold into a soluble conformation, it was of interest to compare this conformation with that of native wild type beta(2), and in particular to know whether beta chains can associate with themselves to form dimers, and with alpha chains to form the same type of heterotetramer as the wild type protein. This was done by submitting the soluble extract of the mutant to gel filtration on a Superose 6 column and determining the elution volume by competition ELISA with two antibodies, mAb93 that can bind either free beta(2) or the alpha(2)beta(2) complex and mAb164 that can bind free beta(2) only. The results are shown in Fig. 3. The peaks obtained with both antibodies were centered at the same position as that of wild type beta(2) and well behind that of the alpha(2)beta(2) complex. Thus, in spite of the presence of alpha chains in the soluble fraction, the beta protein migrated on the column independently from the alpha chain. This was confirmed (data not shown) by gel filtration of a mixture of 10M of purified beta protein (see below) with a large excess (2.10M) of alpha chains and 10M pyridoxal-5`-phosphate. Monitoring the absorption at 280 nm showed that the elution volume of the pure beta protein coincided with that of the wild type beta(2) subunit in the absence of alpha (data not shown). Under the same conditions (i.e. when preincubated with alpha and pyridoxal-5`-phosphate), wild type beta(2) migrated as the alpha(2)beta(2) complex. This demonstrated that, even in the presence of a considerable excess of alpha chains, the beta protein migrated as the dimer and not as the alpha(2)beta(2) heterotetramer, and probably explains why the truncated protein is more sensitive to proteases than the wild type protein.


Figure 3: Gel filtration of a crude extract containing the beta 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 beta 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 beta(2) 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 alpha(2) beta(2) complex.



Stability of the beta Protein

The heat stability of the truncated protein was investigated by irreversible heat inactivation experiments. The beta and the beta(2) protein were submitted to 10 min of heating at various temperatures, and the amount of soluble protein remaining in the supernatant was assayed by ELISA with mAb93. It had been demonstrated previously that heat-inactivated beta chains precipitate and can be removed by low speed centrifugation(27) . Thus, the amount of soluble protein faithfully reflects the amount of heat stable molecules. The results of such heat inactivation studies are shown in Fig. 4. Comparing the curves obtained for the wild type and the beta proteins indicated that the thermal stability was not significantly affected by the deletion of residues 2-9.


Figure 4: Irreversible heat inactivation of the beta protein. Aliquots of crude extracts containing 0.1 mg/ml of either wild type beta(2) 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 times g and 4 °C, the amount of soluble beta(2) (open squares) or beta 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(max)) in the control obtained without addition of supernatant.



Purification of the beta Protein

Because its structural and stability properties much resembled those of the wild type beta(2) subunit, the beta protein was submitted to a purification protocol inspired from that used for purifying the wild type protein(7) . This protocol consisted of the following steps: protamine sulfate precipitation of nucleic acids, heating at 63 °C for 10 min, removal of pyridoxal-5`-phosphate by hydroxylamine, ammonium sulfate (41% saturation) precipitation of the apoenzyme, and attempts to crystallize the apoenzyme at low ammonium sulfate concentration. The progress of the purification was followed by SDS-PAGE (Fig. 5). It can be seen that, already after the heating step and ammonium sulfate precipitation, the preparation was considerably enriched (Fig. 5A). But the crystallization step with 0.4 M ammonium sulfate was ineffective since, unlike the wild type protein, the beta protein remained soluble when dialyzed against 0.4 M ammonium sulfate. A further dialysis against 1 M ammonium sulfate resulted in the precipitation of a protein in a very satisfactory state of purity (Fig. 5B). The protein thus obtained was used for further studies.


Figure 5: Purification of the beta 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 beta(2) 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 beta(2) 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 beta chain, which indicated that the purified protein was indeed the expected truncated beta chain with its N-terminal methionine cleaved off. That it started at residue 10, justified the name ``beta protein'' it was given.

Immunoreactivity of the beta Protein Toward Anti-native and Anti-denatured beta(2) Monoclonal Antibodies

In an attempt to further characterize the conformation and the stability of the beta protein, its reactivity toward monoclonal antibodies that recognize the native or the denatured protein was examined by a competition ELISA(28) . beta reacted very well with antibodies 68, 93, and 164 which also react with the native wild type protein, and failed to react with antibodies B3B5, D4B6, 14, and 172 which are anti-denatured beta(2) antibodies that also fail to react with native beta(2). From these results, one can therefore conclude that beta indeed has a conformation similar to that of the native protein. Its conformational rigidity also seems similar to that of native beta(2), as revealed by its lack of reactivity with the antibodies recognizing exclusively the denatured protein(29) .

Activity of the beta Protein and Complementation by Peptide 1-9

The beta protein was found inactive in the serine deaminase reaction. Similarly, no activity was detected in the indole to tryptophane reaction when beta (10M) was assayed in the absence of alpha. When alpha was added at the concentration usually used in the assay of beta(2) (1.3 times 10M), a very small tryptophan synthase activity (about 0.5% only of that of wild type beta(2)) was observed. When alpha chains were added at larger concentrations, the specific activity of beta increased steadily. Thus, with alpha chains at 2 times 10M (the highest concentration used) in the assay mixture, the specific activity of beta was 60 units/mg, i.e. about 5% of that of wild type beta(2). The curve representing the activity as a function of alpha concentration (data not shown) showed that the saturation of beta by alpha was far from being reached in that concentration range, indicating that the dissociation constant for the alpha/beta complex is well above 2 times 10M. However, when alpha (2 times 10M), beta (1.5 times 10M monomers), the isolated 1-9 peptide (1.3 times 10M) and pyridoxal-5`-phosphate (10M) were mixed, incubated for 5 min at 20 °C, and then assayed in the indole to tryptophan reaction, the specific activity of the beta protein increased to about 700 units/mg. Furthermore, the concentration of alpha chains in the assay needed to reach half the maximum activity in the presence of the 1-9 peptide was only about 2.5 times 10M. These results indicated that the interaction between the alpha chains and the beta protein is very weak, but that it is considerably strengthened in the presence of the isolated peptide 1-9.


DISCUSSION

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 beta 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 beta 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 alpha(2)beta(2)(3) , residues 1-8 were not visible. However, the structure of the alpha(2)beta(2) complex has now been refined and recent unpublished results (^2)show that residues 3-9 are in fact well ordered and that residues 7-9 form a beta strand that belongs to a four stranded antiparallel beta sheet. Yet, the affinities of mAb19 for native beta(2) 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 beta(2) subunit as in the alpha(2)beta(2) complex, this would suggest that the isolated 1-9 peptide might adopt in solution a stable conformation coinciding with that it has in native alpha(2)beta(2). As discussed above, this is quite unlikely. An alternative explanation is that the 2-9 region would be disordered in the isolated beta(2) subunit (for which mAb19 has a high affinity in the absence of alpha) and would become ordered in the alpha(2)beta(2) complex (which mAb19 fails to recognize). This seems quite plausible for the following reasons. In alpha(2)beta(2), 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 alpha subunit.^2 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 beta(2)(26) . This sequence contains the C-terminal beta-hairpin of the four-stranded beta-sheet in which the epitope of mAb19 is also involved. This suggests that the four-stranded beta-sheet may be disorganized in the isolated beta(2) subunit. Thus, interactions with the alpha subunit seem to significantly stabilize the four-stranded beta-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 beta, 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-beta(2). Indeed, these observations demonstrate that, in the beta(2) 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 alpha chains, and hence its conformational stability is likely low. Moreover, although the 1-9 region forms only one visible interaction with alpha in the wild type alpha(2)beta(2) complex (a hydrogen bond involving the side chain of Tyr-8), the truncated protein binds alpha 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 alpha(2)beta(2) 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 beta(2) subunit could be solved, the converging pieces of evidence discussed above lead us to believe that, in isolated beta(2), the 1-9 region is highly flexible and that mAb19 probably recognizes a region of beta(2) 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 beta 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 beta 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 beta 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 beta 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 beta 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 beta protein in the presence of the alpha 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 alpha complementation in E. coli beta-D-galactosidase (30) for which it has been recently shown that the alpha (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 alpha(2)beta(2) 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 alpha and the beta(2) subunits into a stable complex and leads to the formation of the functional protein.


FOOTNOTES

*
This work was supported by the Institut Pasteur, the Paris 7 University, the Centre National de la Recherche Scientifique (U.R.A. 1129), and the Association Franco-Israélienne pour la Recherche Scientifique et Technique (AFIRST). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Unité de Biochimie Cellulaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex, France. Tel: 33-145688386; Fax: 33-140613043.

(^1)
The abbreviations used are: beta(2), F1, F2, and beta, beta(2) subunit, the N- and C-terminal proteolytic domains, and the polypeptide chain, respectively, of Escherichia coli tryptophan synthase; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; RIA, radioimmunoassay; CHAPS, 3-cyclohexylamino-1-propanesulfonic acid; HPLC, high performance liquid chromatography.

(^2)
C. Hyde, personal communication.


ACKNOWLEDGEMENTS

We are particularly grateful to Drs. David Davies and Craig Hyde (NIH) for making available the refined atomic coordinates of tryptophan synthase and providing detailed comments on its three-dimensional structure. We thank J. d'Alayer (Laboratoire de Microséquençage des Protéines-Institut Pasteur) for his expertise in carrying out the N-terminal sequence determination.


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