Kinetic and Thermodynamic Studies of Tet Repressor−Tetracycline Interaction

Stopped-flow measurements have been employed to study the kinetics of the conformational changes in TetR (B) induced by tetracycline binding with and without Mg2+ ions. Result of stopped-flow fluorometry measurements at pH 8.0 indicate conformational changes in the helix−turn−helix motif in the N-terminal domain and in the C-terminal inducer binding domain. Binding of tetracycline (Tc) to TetR in the absence of Mg2+ can be described by a simple kinetics process, which is limited to the first step association without any unimolecular conformational change step upon Tc binding. The rate constants for this process are equal to 2.0 × 105 M-1 s-1 and 2.1 s-1 for the forward and backward reaction, respectively, and gave the binding constant K a = 0.96 × 105 M-1. The kinetics of [Tc−Mg]+ binding to TetR can be described by reactions in which the first step describes the association characterized by the rate constants k a = 1.4 × 105 M-1 s-1 and k d = 2.2 × 10-2 s-1 and binding constant K a = 6.3 × 106 M-1. The first step of [Tc−Mg]+ association is followed by at least three conformational change steps, which occur in the inducer binding site and then propagate to the surroundings of Trp75 and Trp43 residues. The rate constants for the forward, k c, and backward, k - c, reaction for each of these conformational steps have been determined. The thermodynamics of the binding of tetracycline with and without Mg2+ to TetR was investigated by isothermal titration calorimetry (ITC) at pH 8.0 and 25 °C. The measurement shows that TetR dimer possesses two equivalent binding sites for tetracycline, characterized by binding constant K a = 9.0 × 106 M-1 and K a = 7.0 × 104 M-1 for Tc with and without Mg2+, respectively. The binding of the inducer to TetR, in the presence and absence of Mg2+ ion, is an enthalpy-driven reaction characterized by ΔH = −51 kJ mol-1 and ΔH = −33 kJ mol-1, respectively. The entropy change, ΔS, for the interaction in the presence of Mg2+ is equal to −38.9 J K-1 mol-1, and for the tetracycline alone, it was estimated at −17.6 J K-1 mol-1.