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Importance of Residues 2–9 in the Immunoreactivity, Subunit Interactions, and Activity of the β2 Subunit of Escherichia coli Tryptophan Synthase

1995; Elsevier BV; Volume: 270; Issue: 9 Linguagem: Inglês

10.1074/jbc.270.9.4255

1083-351X

Amiel Navon, Andreas Schulze, Yvonne Guillou, Catherine A. Zylinski, Françoise Baleux, Nicole Expert-Bezançon, Bertrand Friguet, Lisa Djavadi‐Ohaniance, Michel Goldberg,

Glycosylation and Glycoproteins Research

The epitope recognized by a monoclonal antibody (mAb19) directed against the β2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the β chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 × 109M−, the affinity of mAb19 for native β2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of β2 (Murry-Brelier, A., and Goldberg, M. E.(1988) Biochemistry 27, 7633-7640). A mutant producing β chains lacking residues 1-9 (βΔ1–9) was prepared. The βΔ1–9 protein was able to fold into a heat stable homodimer resembling wild type β2. Isolated βΔ1–9 had no detectable enzymatic activity. It could bind α chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the βΔ1–9 protein could bind α 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 β chain appears strongly involved in the α-β interactions and in the enzymatic activity. The epitope recognized by a monoclonal antibody (mAb19) directed against the β2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the β chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 × 109M−, the affinity of mAb19 for native β2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of β2 (Murry-Brelier, A., and Goldberg, M. E.(1988) Biochemistry 27, 7633-7640). A mutant producing β chains lacking residues 1-9 (βΔ1–9) was prepared. The βΔ1–9 protein was able to fold into a heat stable homodimer resembling wild type β2. Isolated βΔ1–9 had no detectable enzymatic activity. It could bind α chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the βΔ1–9 protein could bind α 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 β chain appears strongly involved in the α-β interactions and in the enzymatic activity. INTRODUCTIONIn 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(1Goldberg M.E. Trends Biochem. Sci. 1991; 16: 358-362Abstract Full Text PDF PubMed Scopus (75) Google Scholar). For most of these studies, the model protein we used was the β2 1The abbreviations used are: β2, F1, F2, and β, β2 subunitthe N- and C-terminal proteolytic domains, and the polypeptide chain, respectively, of Escherichia coli tryptophan synthaseELISAenzyme-linked immunosorbent assaymAbmonoclonal antibodyPAGEpolyacrylamide gel electrophoresisRIAradioimmunoassayCHAPS3-cyclohexylamino-1-propanesulfonic acidHPLChigh performance liquid chromatography. subunit of Escherichia coli tryptophan synthase. The properties of this enzyme have been recently reviewed in detail(2Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172PubMed Google Scholar). We shall summarize here those which are directly relevant to the present study. The natural form of tryptophan synthase is the heterotetramer α2β2, which can be readily dissociated into monomeric α chains and the dimeric β2 subunit. The β2 subunit can, in the presence of its coenzyme pyridoxal-5′-phosphate, catalyze two distinct reactions: indole + L-serine → L-tryptophan + H2O and L-serine + H2O → pyruvate + ammonia.In the presence of α chains, the serine deaminase reaction is completely abolished, while the specific activity of β2 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(3Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar). 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 β chains to regenerate native β2 subunits has been extensively investigated(4Goldberg M.E. Semisotnov G.V. Friguet B. Kuwajima K. Ptitsyn O.B. Sugai S. FEBS Lett. 1990; 263: 51-56Crossref PubMed Scopus (100) Google Scholar). In particular, one monoclonal antibody, mAb19, turned out to detect an early folding step during the in vitro renaturation of β2(5Murry-Brelier A. Goldberg M.E. Biochemistry. 1988; 27: 7633-7640Crossref PubMed Scopus (22) Google Scholar, 6Blond-Elguindi S. Goldberg M.E. Biochemistry. 1990; 29: 2409-2417Crossref PubMed Scopus (42) Google Scholar). 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(5Murry-Brelier A. Goldberg M.E. Biochemistry. 1988; 27: 7633-7640Crossref PubMed Scopus (22) Google Scholar). This intramolecular reaction occurs early on the folding pathway, at a stage where the protein is still a molten globule(4Goldberg M.E. Semisotnov G.V. Friguet B. Kuwajima K. Ptitsyn O.B. Sugai S. FEBS Lett. 1990; 263: 51-56Crossref PubMed Scopus (100) Google Scholar). 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(5Murry-Brelier A. Goldberg M.E. Biochemistry. 1988; 27: 7633-7640Crossref PubMed Scopus (22) Google Scholar). 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 α chains, and enzymatic activity of the β2 subunit.MATERIALS AND METHODSReagents and BufferThe 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 FragmentThe native β2 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(7Högberg-Raibaud A. Goldberg M.E. Biochemistry. 1977; 16: 4014-4020Crossref PubMed Scopus (104) Google Scholar). The solubilized F1 fragment was purified by gel filtration on a S-200HR Sephacryl column equilibrated with standard buffer. The concentrations of β2 and of F1 were determined spectrophotometrically using specific extinction coefficients at 280 nm of 0.58 and 0.67 cm2/mg, respectively. The enzymatic activity of the β2 subunit was measured in the indole to tryptophan reaction, either in the presence or in the absence of α subunit, as described previously (8Faeder E.J. Hammes G.G. Biochemistry. 1970; 25: 4043-4049Crossref Scopus (80) Google Scholar), and in the serine deamination reaction(9Crawford I.P. Ito J. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 391-397Crossref Scopus (47) Google Scholar).Monoclonal antibodies were obtained (10Djavadi-Ohaniance L. Friguet B. Goldberg M.E. Biochemistry. 1984; 23: 97-104Crossref PubMed Scopus (57) Google Scholar) and purified from ascitic fluids (11Friguet B. Djavadi-Ohaniance L. Goldberg M.E. Res. Immunol. 1989; 140: 355-376Crossref PubMed Scopus (43) Google Scholar) 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 cm2/mg (12Onoue K. Yagi Y. Grossberg A.L. Pressman D. Immunochemistry. 1965; 2: 401-415Crossref PubMed Google Scholar) as specific extinction coefficient. For affinity measurements with the radioactivity assay, the mAb19 solution was titrated as described earlier(13Friguet B. Chaffotte A.F. Djavadi-Ohaniance L. Goldberg M.E. J. Immunol. Methods. 1985; 77: 305-319Crossref PubMed Scopus (1123) Google Scholar).The polypeptide 1-102 was prepared (14Fedorov A.N. Friguet B. Djavadi-Ohaniance L. Alakhov Y.B. Goldberg M.E. J. Mol. Biol. 1992; 228: 351-358Crossref PubMed Scopus (58) Google Scholar) and kindly supplied by Dr. Alexei Fedorov.Epitope Library Construction, Screening, and CharacterizationThe 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.(15Friguet B. Fedorov A.N. Djavadi-Ohaniance L. J. Immunol. Methods. 1993; 158: 243-249Crossref PubMed Scopus (5) Google Scholar).Cyanogen Bromide Cleavage of F1The 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 PeptidesPeptides were synthesized by the Merrifield (16Merrifield R.B. J. Am. Chem. Soc. 1963; 85: 2149-2154Crossref Scopus (6610) Google Scholar) 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 MutagenesisStop codons or deletions were introduced in the trpB gene by oligonucleotide site-directed mutagenesis(17Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4543) Google Scholar, 18Messing J. Vieira J. Gene (Amst.). 1982; 19: 269-276Crossref PubMed Scopus (1717) Google Scholar). A derivative of the plasmid pTZ19R, carrying the sequence coding for the first 102 amino acids of the tryptophan synthase β subunit(19Friguet B. Fedorov A.N. Serganov A.A. Navon A. Goldberg M.E. Anal. Biochem. 1993; 210: 344-350Crossref PubMed Scopus (10) Google Scholar), 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 β chain, the plasmid used was derived from pαβTS21 (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(17Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4543) Google Scholar, 18Messing J. Vieira J. Gene (Amst.). 1982; 19: 269-276Crossref PubMed Scopus (1717) Google Scholar). 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 PreparationThe different plasmids were prepared as a "mini-prep" or in "large scale"(20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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(20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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 TranslationAll 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 [35S]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 β-Imager (Biospace, Paris,) until about half a million counts were recorded. Integrating the counts for each fragment by means of the β-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 ElectrophoresisElectrophoresis 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 (21Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10439) Google Scholar). 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(22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206016) Google Scholar).ElectrotransferThe 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(23Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5867) Google Scholar).MicrosequencingAfter 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 BlotsThe 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 ELISAQuantification of wild type or truncated β2 subunits in crude extracts was made by a competition ELISA based on the test previously described(24Djavadi-Ohaniance L. Friguet B. Butler J.E. The Immunochemistry of Solid-phase Immunoassay. CRC Press Inc., Roca Baton, FL1991: 201-206Google Scholar). 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 × 10-10M) 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 β2 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(24Djavadi-Ohaniance L. Friguet B. Butler J.E. The Immunochemistry of Solid-phase Immunoassay. CRC Press Inc., Roca Baton, FL1991: 201-206Google Scholar).Affinity MeasurementsThey were made either by a competition ELISA method (13Friguet B. Chaffotte A.F. Djavadi-Ohaniance L. Goldberg M.E. J. Immunol. Methods. 1985; 77: 305-319Crossref PubMed Scopus (1123) Google Scholar) when pure antigen was available, or by a competition RIA method (19Friguet B. Fedorov A.N. Serganov A.A. Navon A. Goldberg M.E. Anal. Biochem. 1993; 210: 344-350Crossref PubMed Scopus (10) Google Scholar) for antigens produced by in vitro cell-free synthesis.Circular DichroismCircular 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.RESULTSIn order to identify the region of the β 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 β chain. The N-terminal F1 fragment of the β 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 PeptidesTo 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.(13Friguet B. Chaffotte A.F. Djavadi-Ohaniance L. Goldberg M.E. J. Immunol. Methods. 1985; 77: 305-319Crossref PubMed Scopus (1123) Google Scholar). The results shown in Table 1 indicated 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(25Crawford I.P. Decastel M. Goldberg M.E. Biochem. Biophys. Res. Commun. 1978; 85: 309-316Crossref PubMed Scopus (16) Google Scholar) the native protein reacts well with mAb19. The minimal immunoreactive sequence thus defined therefore corresponded to residues 2-9 of the β chain. The affinity values obtained for all the synthetic peptides containing the 2-9 sequence were in the range of 0.1-0.3 × 109M− (see Table 1). These affinities are only slightly smaller than that (0.6 × 109M−) found for the native β2 subunit, strongly suggesting that the antigenic determinant of β2 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.Tabled 1Preparation and Characterization of Mutants with Deletions or Stop Codons in the tryp B GeneTo ascertain the latter conclusion a plasmid encoding a β chain lacking residues 2-9 was prepared, and the immunoreactivity of the corresponding truncated protein was investigated by Western blotting with mAb19. Fig. 1 shows 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 β chain(26Larvor M.-P. Djavadi-Ohaniance L. Friguet B. Baleux F. Goldberg M.E. Mol. Immunol. 1991; 28: 523-531Crossref PubMed Scopus (13) Google Scholar). This confirmed that the 2-9 sequence is essential for the immunoreactivity of the β2 protein toward mAb19.Figure 1:Western blots of the β chain carrying the 2-9 deletion. Bacteria carrying the plasmid encoding the β 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, β carrying the 2-9 deletion; slot 2, wild type β; slot 3, molecular weight markers. Right panel (B): Western blot with a specific monoclonal antibody that recognizes the sequence 273-283 of the β chain. Slot 4, β carrying the 2-9 deletion; slot 5, wild type β; slot 6, molecular mass (indicated in kDa) markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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(14Fedorov A.N. Friguet B. Djavadi-Ohaniance L. Alakhov Y.B. Goldberg M.E. J. Mol. Biol. 1992; 228: 351-358Crossref PubMed Scopus (58) Google Scholar). 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 β 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 β 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 [35S]methionine were synthesized in vitro as above, using mRNA with the various stop codons as templates, and their interaction with mAb19 (10-8M) in solution was investigated by competition RIA(12Onoue K. Yagi Y. Grossberg A.L. Pressman D. Immunochemistry. 1965; 2: 401-415Crossref PubMed Google Scholar). 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(12Onoue K. Yagi Y. Grossberg A.L. Pressman D. Immunochemistry. 1965; 2: 401-415Crossref PubMed Google Scholar). The affinity obtained with peptide 1-60 from the data shown in Fig. 2 was 0.75 × 109 and a very similar value (0.7 × 109M−) 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 β2(14Fedorov A.N. Friguet B. Djavadi-Ohaniance L. Alakhov Y.B. Goldberg M.E. J. Mol. Biol. 1992; 228: 351-358Crossref PubMed Scopus (58) Google Scholar). 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 35S-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 β-Imager as described by Friguet et al.(19Friguet B. Fedorov A.N. Serganov A.A. Navon A. Goldberg M.E. Anal. Biochem. 1993; 210: 344-350Crossref PubMed Scopus (10) Google Scholar). 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 × 10-10M; slot 4, 6.8 × 10-10M; slot 5, 2.3 × 10-9M; slot 6, 6.8 × 10-9M; slot 7, 2.3 × 10-8M; slot 8, 6.8 × 10-8M. 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 β-Imager software, and the data obtained were plotted as described by Friguet et al.(19Friguet B. Fedorov A.N. Serganov A.A. Navon A. Goldberg M.E. Anal. Biochem. 1993; 210: 344-350Crossref PubMed Scopus (10) Google Scholar). 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Far UV Circular Dichroism of the 1-9 Antigenic PeptideIn 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 deg•cm-2•dmol−). 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.Expressi