A mutational approach shows similar mechanisms of recognition for the isolated and integrated versions of a protein epitope.
Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase. It recognizes a synthetic peptide, P11, constituted of residues 273-283 of TrpB, with high affinity. We introduced 16 single and 3 double mutations in each of the two contexts, TrpB2 and P11, and used them as local probes to study the cross-reactivity of mAb164 toward these two antigens. The equilibrium constant, KD, of dissociation from mAb164 was measured for each of the mutant derivatives of TrpB2 and P11 by a competition enzyme-linked immunosorbent assay and compared with the wild type one. The variation of the free energy of interaction, DeltaDeltaG, covered nearly 8 kcal/mol for the different mutations. The values of DeltaDeltaG for the mutant derivatives of TrpB2 and for those of P11 were close and the two sets of values were strongly correlated (r = 0.96). This correlation showed that mAb164 recognized the integrated and isolated versions of residues 273-283 with very similar mechanisms. A few significant differences between the recognitions of TrpB2 and P11 by mAb164 suggested some adaptability of the interaction. The results were compatible with a recognition of residues 273-283 of TrpB in a loop conformation, close to their structure in the crystals of the complete tryptophan synthase, TrpA2TrpB2.
Rondard
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Groupe d'Ingénierie des Protéines (CNRS URA 1129), Unité de Biochimie Cellulaire, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Bedouelle
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D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D004252:DNA Mutational Analysis; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000201:enzymology; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000939:Epitopes; D009842:Oligopeptides; D014367:Tryptophan Synthase