Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction

We isolated five monoclonal antibodies (mAbs) made against tetracycline rep ressor (TetR), one against the TetR tetracycline complex (Tc) and two again st the TetR-tet operator (tetO) complex. The epitopes of the anti-TetR mAbs are localized in the a-helix-turn-a-helix motif (HTH), at different sites near the Tc binding pocket and at the dimerization interface. The anti-TetR -Tc and one of the anti-TetR-tetO mAbs recognize epitopes near the Tc bindi ng pocket. The other anti-TetR-tetO mAb binds to an epitope within the HTH. Quantitative immunoprecipitation and competitive ELISA employing TetR, Tet R-Tc, or TetR-tetO revealed different affinities of the mAbs for TetR in th ese functional states. Binding of the two mAbs to epitopes in the HTH was i dentical for TetR and TetR-Tc indicating the same conformation in both form s. The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR-Tc, supporting the idea of different conformation s of that epitope in these forms of TetR. The greatest affinity differences were found for epitopes around the Tc binding pocket. Two anti-TetR mAbs h ave the highest affinities for free TetR, somewhat reduced affinity for Tet R-tetO and the lowest affinities for TetR-Tc. The anti-TetR-Tc mAb has a di scontinuous epitope, formed in TetR-Tc, which is less well bound in TetR an d not bound in the TetR-tetO complex. One anti-TetR-tetO mAb does not recog nize TetR-Tc. Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational diff erences of the epitopes in these forms of TetR.