THE INTERACTIONS OF ESCHERICHIA-COLI TRP REPRESSOR WITH TRYPTOPHAN AND WITH AN OPERATOR OLIGONUCLEOTIDE NMR-STUDIES USING SELECTIVELY N-15-LABELED PROTEIN

The effects of the binding of the corepressor L-tryptophan and an oper ator oligonucleotide to Escherichia coli trp repressor have been studi ed, using selective N-15 labelling to permit observation of the backbo ne amide resonances of 50 of the 107 residues of the protein monomer. Repressor molecules selectively labelled in turn with [N-15]alanine, [ N-15]glutamate, [N-15]isoleucine, [N-15]leucine and [N-15]methionine w ere prepared by isolating them from prototrophic E. coli cells grown i n media containing a mixture of unlabelled and the appropriate N-15-en riched amino acids. Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in r esolving the crosspeaks of, for example, the 19 leucine and 12 glutama te residues. All 50 residues studied show measurable changes in amide H-1 and/or N-15 chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has eff ects which are transmitted throughout almost the whole protein. Large chemical shift changes on ligand binding are seen in residues in the t ryptophan binding site and in the 'helix-turn-helix' DNA-binding domai n, but also in residues in helices C and F remote from the ligand bind ing sites. On operator binding there is selective broadening of the si gnals of residues in the N-terminal region of the protein and in the D NA-binding domain, perhaps reflecting a conformational equilibrium.