The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid.
The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.
D001426:Bacterial Proteins; D002942:Circular Dichroism; D004926:Escherichia coli / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D012097:Repressor Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D007211:Indoles; D012097:Repressor Proteins; D014157:Transcription Factors; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C015292:indolepropionic acid