The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide. NMR studies using selectively 15N-labelled protein.
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.
Department of Biochemistry, University of Leicester, England.
V
V
Ramesh
R O
RO
Frederick
S E
SE
Syed
C F
CF
Gibson
J C
JC
Yang
G C
GC
Roberts
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins