Determination of the equilibrium association constant between tet repressor and tetracycline at limiting Mg 2+ concentrations: A generally applicable method for effector-dependent high-affinity complexes

Abstract

An analytical method for determining very high binding constants at equilibrium for reactions requiring an effector is proposed and applied to study the interaction of tetracycline with the repressor of the tetracycline resistance gene from Tn 10. In this method complex formation is limited by low concentrations of the effector, which is Mg 2+ for the interaction of tetracycline and Tet repressor. The binding of Mg 2+ to tetracycline and subsequent formation of the ternary repressor-Mg 2+-tetracycline complex are coupled reactions yielding a dependence of repressor-tetracycline-Mg 2+ complex formation on the concentration of free Mg 2+. The binding constants can be determined from the quantitative analysis of ternary complex formation with increasing Mg 2+ concentrations. This method allows the determination of very high association constants at equilibrium in a large range of protein concentrations. In the case of repressor and tetracycline, the same affinity constant of 3 ± 2 × 10 9 m −1 was found in the range of 0.1 to 5 μ m of repressor. This result indicates that no association or dissociation of the repressor subunits occurs upon binding of tetracycline. Furthermore, the results show that a repressor dimer binds two effector molecules without significant cooperativity.