Analysis of the thermodynamic linkage of DNA binding and ion binding for dimeric and tetrameric forms of the lac repressor.

The salt concentration dependences of the observed association constants (Kobs) for the binding of wild-type lac repressor tetramer and the dimeric lacI-18 mutant repressor to lactose operator DNA were compared. For both proteins, the data are consistent with a class of linkage models in which ion binding by the protein is driven by differences in the ionic concentrations in bulk solution and in the volume near the DNA surface. The models that best agree with the data are those in which ion-binding reactions are cooperative. In spite of close agreement between these models and the data, the determination of ion stoichiometries and apparent ion-binding affinities requires additional mechanistic or structural information. The simplest ion-binding mechanism consistent with the data is compatible with a current structural model of the repressor-operator complex. At salt concentrations in excess of 50 mM, at which cation displacement from the DNA and anion displacement from the protein are expected to dominate, similar ion stoichiometries are found for the DNA binding of dimeric and tetrameric repressors. This supports the notion that the DNA contacts of these proteins are homologous. At lower salt concentrations, in which cation binding by the proteins is expected to be significant, the net ion stoichiometry of wild-type repressor binding is slightly greater than that of the lacI-18 mutant. This difference may reflect the availability of ion-binding sites in the distal subunits of tetramer that are not present in the dimer, or may be a consequence of the involvement of ion binding in the dimer/monomer equilibrium.