Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan.
We have examined the interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan, using both equilibrium dialysis and flow dialysis methods. Results obtained by the two procedures were equivalent and indicate that the trp aporepressor binds L-tryptophan with an equilibrium dissociation constant (Kd) of 40 microM at 25 degrees C under standard binding assay conditions (10 mM potassium phosphate, pH 7.4, 0.2 M potassium chloride, 0.1 mM EDTA, 5% glycerol). Molecular sizing of the purified trp aporepressor shows that in the absence of ligand the regulatory protein exists as a dimeric species with greater than 99% purity and an apparent molecular weight of 30,000. Under the storage and assay conditions used, the dimer appears quite stable, and essentially no monomer or higher multimeric species are detected. Analysis of binding data by Scatchard and direct linear plot methods shows two identical and independent ligand-binding sites/native trp aporepressor dimer. When examined as a function of temperature, L-tryptophan binding by trp aporepressor varied over 7-fold (Kd = 28 microM at 6.5 degrees C to Kd = 217 microM at 40 degrees C). At the optimal growth temperature for E. coli (37 degrees C), the dissociation constant was 160 microM for the ligand, L-tryptophan. From the relationship between temperature and L-tryptophan binding by trp aporepressor, the apparent enthalpy change delta H = -10.6 +/- 0.6 kcal mol-1 and the apparent entropy change delta S = -17 +/- 2 cal degree-1 mol-1 were determined.
Arvidson
D N
DN
Bruce
C
C
Gunsalus
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RP
D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics*; D005976:Glutaral / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012097:Repressor Proteins / Q000378:metabolism*; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors; D014364:Tryptophan; D005976:Glutaral