Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance.
The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).
D001426:Bacterial Proteins; D002942:Circular Dichroism; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D012097:Repressor Proteins; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014157:Transcription Factors; D014364:Tryptophan; D014508:Urea; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014364:Tryptophan; D014508:Urea; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins