Trp aporepressor engineered for fluorescence spectroscopy

The tryptophan repressor from Escherichia coli binds to the trp operator in the presence of L- tryptophan, thereby inhibiting the biosynthesis of L-tryptophan. Site-directed mutagenesis was used to change tryptophan-19 and tryptophan-99 to leucine and methionine, respectively. This mutant protein without tryptophan in its amino acid sequence has wild-type repressor activity and is a suitable model for fluorescence studies of corepressor binding. Both steady-state and time-resolved fluorescence spectroscopy have been used to compare the binding of L- tryptophan, indole-3-propionic acid, indole-3-butyric acid, and indole. In all cases, binding to the mutant aporepressor results in a large blue shift and a change in the intensity of the ligand fluorescence. The decay of the total fluorescence intensity from the complex indicates the presence of three distinct bound states of the ligand. The distribution of ligand binding modes is influenced by the substituent at the 3-position of the indole ring. The rotational correlation time of the complexes formed with L-tryptophan or indole-3-propionic acid indicate that the protein is present as a dimer, whereas with indole or indole-3-butyric acid the correlation times are much lower, suggesting that the protein is present as a monomer.