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

Abstract

We isolated five monoclonal antibodies (mAbs) made against tetracycline repressor (TetR), one against the TetR tetracycline complex (Tc) and two against theTetR‐tet operator (tetO) complex. The epitopes of the anti‐TetR mAbs are localized in the α‐helix‐turn‐α‐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 binding pocket. The other anti‐TetR‐tetO mAb binds to an epitope within the HTH. Quantitative immunoprecipitation and competitive ELISA employing TetR, TetR‐Tc, or TetR‐tetO revealed different affinities of the mAbs for TetR in these functional states. Binding of the two mAbs to epitopes in the HTH was identical for TetR and TetR‐Tc indicating the same conformation in both forms. The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR‐Tc, supporting the idea of different conformations 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 have the highest affinities for free TetR, somewhat reduced affinity for TetR‐tetO and the lowest affinities for TetR‐Tc. The anti‐TetR‐Tc mAb has a discontinuous epitope, formed in TetR‐Tc, which is less well bound in TetR and not bound in the TetR‐tetO complex. One anti‐TetR‐tetO mAb does not recognize TetR‐Tc. Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational differences of the epitopes in these forms of TetR.

The Tetracycline Repressor—A Paradigm for a Biological Switch

“…[37,38] Crystallographic analyses show that they are (Figure 4). [28] The water molecules are linked by cooperative hydrogen bonds and tether a 4 , a 4' , and the DNA binding domains in a position that abolishes or prevents the specific interactions with operator DNA described in [35] in a pendulumlike movement (see Figure 3).…”

In vivo Activation of Tetracycline Rep ressor by Cre/<i>lox</i>-Mediated Gene Assembly

“…To assay the amounts of TetR (or the shorter tlklt -encoded product) of the respective Bacillus strains, Western Blot analyses were conducted. Figure 3 (lower part) depicts the results obtained with the monoclonal antibody TOP19, whose epitope maps to the helix-turn- [Pook et al, 1998], which is identical to that of TetR. The approximately equally strong signals obtained with soluble protein extracts of WH557, WH558 and RAB100 contrast to the weaker band in case of RAB102.…”

“…Immunodetection of TetR in soluble B. subtilis extracts was performed using 75 g of total protein, and either serum of TOP19 monoclonal antibody raised against TetR(B) [Pook et al, 1998] or a 1: 20,000 dilution of rabbit polyclonal antibodies recognizing TetR in conjunction with ECL Plus Western Blotting Detection Reagents (GE Healthcare, Munich, Germany) as described in detail previously [Kamionka et al, 2005].…”

Der Tetracyclin-Repressor – das Musterbeispiel für einen biologischen Schalter

“…[37,38] Kristallographische Analysen zeigen, dass diese ¾nderungen durch zickzackförmige Ketten von je acht Wassermolekülen pro Monomer des (TetR´[MgTc] ) 2 -Komplexes stabilisiert werden (Abbildung 4). [37,38] Kristallographische Analysen zeigen, dass diese ¾nderungen durch zickzackförmige Ketten von je acht Wassermolekülen pro Monomer des (TetR´[MgTc] ) 2 -Komplexes stabilisiert werden (Abbildung 4).…”