The binding of the corepressors tryptophan and 5-methyltryptophan and of the inducers 3-indolepropionate, 3-indoleacrylate and 5-methylindole to the Escherichia coli trp repressor have been studied by 1H-NMR spectroscopy. Identification of the resonances of the protons of bound ligands and their NOEs to protons of the protein (measured as transferred NOE) was greatly facilitated by the use of samples of the protein in which the hydrogens of all residues except alanine, isoleucine and threonine was replaced by deuterium. Chemical-shift changes of protein-backbone resonances and side-chain-amide resonances on ligand binding were measured with generally or selectively 15N-labelled protein. The patterns of changes in the chemical shifts of protein resonances and, particularly, ligand resonances distinguish the corepressors from the inducers, indicating, in agreement with earlier work, that corepressors and inducers bind to the protein in different ways. The NOEs observed for the bond ligands have been used to determine the position of the ligands in the crystallographically determined binding site, by means of a simulated-annealing molecular-dynamics protocol. The structures obtained show that the orientation in the binding site of the indole rings of tryptophan and 5-methyltryptophan and of 3-indolepropionate and 3-indoleacrylate differ by approximately 180 degrees in solution (in agreement with the crystallographic data for complexes of the trp repressor with tryptophan or with 3-indolepropionate). The value and limitations of calculating ligand positions based on transferred NOE are discussed.
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