CHARACTERIZATION OF CHARGE CHANGE SUPER-REPRESSOR MUTANTS OF TRP REPRESSOR - EFFECTS ON OLIGOMERIZATION CONFORMATION, LIGATION AND STABILITY

We have carried out a physical characterization of mutant repressor pr oteins of the trp repressor system of Escherichia coil by circular dic hroism, chemical denaturation, and 8-anilino-1-naphthalenesulfonate bi nding. We have also probed the protein-protein interactions via fluore scence anisotropy and lifetime measurements and measured the thermodyn amics of ligand (L-tryptophan) binding by isothermal titration calorim etry. Here, we present investigations of four charge change super-repr essor mutants: EK13, EK18, DN46 and EK49, and compare these results wi th those previously obtained for wild-type trp repressor and the AV77 super-repressor mutant. These studies demonstrate that super-repressor phenotypes may result from changes in operator affinity (DN46, EK49), protein-protein interactions (EK18), as well as the coupling of foldi ng to ligand binding (AV77, EK13, EK18). Correlations between the olig omerization behavior and cooperativity of DNA binding for some mutants indicate that coupling of oligomerization to DNA modulates operator s ite occupation giving rise to the super-repressor phenotype. The prese nt results underscore the complex interplay between the multiple equil ibria in this system. Moreover, they provide insights into the structu ral basis for the mutational perturbation of the energetics of this cl assical allosterically controlled transcriptional regulator. (C) 1996 Academic Press Limited