title: Partly native epitopes are already present on early intermediates in the folding of tryptophan synthase.
abstract: Two monoclonal antibodies directed against the native beta 2 subunit of Escherichia coli tryptophan synthase [L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20], one recognizing the C-terminal F1 domain and the other the N-terminal F2 domain, were used to detect immunoreactive intermediates in the folding of the protein. For that purpose, the association of the monoclonal antibodies with either the beta 2 subunit or its isolated domains was studied by using fluorescence energy transfer between tryptophan residues of the antibodies and a dansyl group covalently linked to the antigen. It is shown that the association of both monoclonal antibodies with the antigen occurs within a few seconds after initiation of the renaturation, whereas complete refolding of the beta 2 subunit requires several minutes under the same experimental conditions. Thus, immunoreactive intermediates appear to be formed at an early stage of the folding process. While the isolated F1 domain alone is able to rapidly refold into a conformational intermediate already well recognized by the anti-native-beta 2 antibody, it cannot, in the absence of the F2 domain, reach its native conformation. However, its association with the anti-native-beta 2 antibody induces a structural change of F1 that brings it closer to the conformation it normally adopts when interacting with F2 inside the native beta 2 subunit.
mesh_terms: D000911:Antibodies, Monoclonal; D004735:Energy Transfer; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014367:Tryptophan Synthase / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014367:Tryptophan Synthase