The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide

Abstract

The effects of the binding of the corepressor l‐tryptophan and an operator oligonucleotide to Escherichia coli trp repressor have been studied, using selective 15N labelling to permit observation of the backbone amide resonances of 50 of the 107 residues of the protein monomer. Repressor molecules selectively labelled in turn with [15N]alanine, [15N]glutamate, [15N]isoleucine, [15N]leucine and [15N]methionine were prepared by isolating them from prototrophic E. coli cells grown in media containing a mixture of unlabelled and the appropriate 15N‐enriched amino acids. Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in resolving the crosspeaks of, for example, the 19 leucine and 12 glutamate residues. All 50 residues studied show measurable changes in amide 1H and/or 15N chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has effects which are transmitted throughout almost the whole protein. Large chemical shift changes on ligand binding are seen in residues in the tryptophan binding site and in the ‘helix‐turn‐helix’ DNA‐binding domain, but also in residues in helices C and F remote from the ligand binding sites. On operator binding there is selective broadening of the signals of residues in the N‐terminal region of the protein and in the DNA‐binding domain, perhaps reflecting a conformational equilibrium.

DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase

“…The switch from high to low salt buffer resulted in substitution of a fast chemical exchange between free and complexed protein, for one of intermediate exchange, typically occurring on the millisecond timescale (34,35). Intermediate exchange line-broadening for resonance signals emanating from the recognition helices of helix-turn-helix motifs is a recurrent theme in protein-DNA complexes (36)(37)(38), and has also been noted to occur with Ada-N (39). Such localised linebroadening has been attributed to flexibility within the protein-DNA interface (40).…”

Specific Isotopic Labeling and Photooxidation-linked Structural Changes in the Manganese-stabilizing Subunit of Photosystem II

“…[ 13 C]Glutamate labeling is expected to be more difficult, when compared with aspartic acid labeling (39,53). However, we found reasonable (41%) amounts of labeling from […”

“…This took about 3 h. At this time, 0.2 ml of 0.2 M isopropyl-1-thio-␤-D-galactopyranoside was added to induce MSP production. In addition, a 100-ml solution of 10% lactose was added; lactose was found to improve the labeling efficiency (39). After induction, the cells were grown for an additional 4 h and harvested.…”

NMR and Molecular Modelling Studies of the Binding of Amicetin Antibiotic to Conserved Secondary Structural Motifs of 23S Ribosomal RNAs

“…An alternative possibility is that there are exchange processes which specifically involve the non-hydrogen bonded imino protons of unpaired G and U bases located in the flexible regions of the RNA motif (such as the bulge in the middle) and further detailed studies are required to identify the specific motions responsible for the observed line broadening. This phenomenon is paralleled by the selective line-broadening observed for the backbone peptide NH protons in the spectra of selectively labelled 15 N-trp repressor on binding to the operator oligonucleotide [29]. Here the broadening selectively affects residues, most notably, in the flexible DNA-binding helix-turn-helix domain.…”