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 x 10(9) M-1, 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 delta 1-9) was prepared. The beta delta 1-9 protein was able to fold into a heat stable homodimer resembling wild type beta 2. Isolated beta delta 1-9 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 delta 1-9 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.
Highlights
The epitope recognized by a monoclonal antibody directed against the /32 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the /3 chain
In an attempt to use monoclonal antibodies as conformational probes to investigate the structure, conformational changes, conformational dynamics, and folding pathway ofproteins, we have prepared a panel of monoclonal antibodies directed against different proteins and developed a variety of experimental methods for the quantitative analysis of antigenantibody interactions [1]. For most of these studies, the model protein we used was the /321 subunit of Escherichia coli tryptophan synthase
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, recognizes a linear epitope
Summary
The epitope recognized by a monoclonal antibody (mAbI9) directed against the /32 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the /3 chain. In an attempt to use monoclonal antibodies as conformational probes to investigate the structure, conformational changes, conformational dynamics, and folding pathway ofproteins, we have prepared a panel of monoclonal antibodies directed against different proteins and developed a variety of experimental methods for the quantitative analysis of antigenantibody interactions [1]. For most of these studies, the model protein we used was the /321 subunit of Escherichia coli tryptophan synthase. 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
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