Differential scanning calorimetry demonstrates that the tryptophan repressor of Escherichia coli is unusually resistant to thermal denaturation. The dimeric protein undergoes reversible dissociative unfolding at pH 7.5 centered at about 900C. The thermal stability may be due in part to the unusual structure of the protein, which is composed of two identical intertwined polypeptide chains.The tryptophan (trp) repressor from Escherichia coli is involved in regulating the expression of biosynthetic enzymes for tryptophan in response to the level of this amino acid in the cell. Tryptophan binds to the repressor and increases its affinity for the operator target site within the trp promoter, thereby preventing transcription initiation. The trp repressor protein is a dimer of molecular weight 24,700 (1) with two binding sites for tryptophan and a single operator DNA binding site. The three-dimensional structure of this protein has been determined (2). The monomer structure consists of six helical regions, which are intertwined with the corresponding helices ofthe companion monomer to form the dimeric structure. The significant intersubunit contacts seen in the crystallographic analysis suggest that a stable dimeric structure might be anticipated for this protein. We report here the results of differential scanning calorimetric (DSC) experiments that show trp repressor to be remarkably thermally stable and will report elsewhere more detailed data of the effects on the thermal denaturation of variations in pH, the addition of ligands, and other experimental conditions as well as the replacement of single amino acid residues. MATERIALS AND METHODSThe tryptophan repressor was isolated in the following manner. Bacterial cells containing a multicopy plasmid (pJPR2) that overproduces the trp repressor were obtained from C. Yanofsky (Stanford University). Procedures for cell growth were those described by Paluh and Yanofsky (3). The protein was purified by using the procedure of Joachimiak et al. (4) with the changes delineated below. The cell suspension was sonicated 30 ml at a time for a total duration of 3 min (the cycle was sonication for 30 s followed by cooling for 30 s). The ammonium sulfate pellet was suspended in 0.01 M potassium phosphate, pH 7.4/0.1 mM EDTA/0.2 M KCl, and the suspension was dialyzed against this buffer. After dialysis, the sample was applied directly to a phosphocellulose column equilibrated in sample buffer, and the column was developed with the same buffer. The trp repressor was eluted by sample buffer containing 0.5 M KCl. The fractions containing the repressor were pooled and concentrated to :8 mg/ml. Polyacrylamide gel electrophoresis of the purified protein yielded a single component of the expected molecular weight; no contaminants were detected when the gel was overloaded.Before introduction into the calorimeter, the protein was dialyzed against 10 mM potassium phosphate/100 mM KCl, pH 7.5. The dialysate served as the reference solution in the calorimeter. Protein concentrations w...

No abstract

No abstract

No abstract

No abstract