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The three-dimensional structures of a number of DNA-binding proteins alone and complexed with their cognate DNA sequences have recently been reported. One of these is the trp repressor from E. coli, for which the structure in the absence and the presence of the co- repressor, tryptophan, has been deduced from x-ray crystallographic data, as well as that of the ternary complex between protein, co-repressor, and operator DNA. Rather than provide a definitive answer for the mechanism of the activation of the repressor by tryptophan binding, these structures have raised even more questions concerning the basis for the affinity changes observed. DNA binding studies of this protein using a number of biochemical techniques have brought to light the existence of repressor multimer-DNA complexes at physiological concentrations, although these have not been well characterized. Recently, we have demonstrated, using fluorescence polarization techniques, that the repressor oligomerizes in absence of DNA and that co-repressor binding destabilizes the oligomers. Clearly, in this case, as in many others, there are subtle thermodynamic relationships between protein-protein, protein-ligand, and protein-DNA interactions. The role of these energetic couplings in the allosteric regulation of the repressor by tryptophan is, however, not understood. Because of the non-equilibrium nature of filter-binding, gel mobility shift, and nuclease protection assays it is not possible to fully explore these different binding phenomena over broad concentration ranges of each component and under different solution conditions. Fluorescence methodologies provide observables of the multiple binding equilibria in solution between nanomolar and micromolar concentrations. Variables such as the protein and ligand concentration dependence of repressor-DNA interactions, as well as different solution conditions (i.e., salt concentration), have been probed using these techniques. The intrinsic tryptophan fluorescence of the protein has been used to characterize dimer stability. The fluorescence polarization of a covalently bound DNS label has been used to characterize the higher order protein-protein interactions and the effect of tryptophan and salt concentration upon them. DNS polarization has also been used to probe the protein and tryptophan concentration dependence of operator DNA binding. Finally, fluorescence quenching of coumarin covalently coupled to the trp repressor protein upon binding the operator fragment has been used to study these protein- DNA complexes. The combination of appropriate fluorescence methodologies allows for a detailed characterization of binding affinities and stoichiometries in such systems.
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