Lane A N, Jardetzky O
Eur J Biochem. 1987 Apr 15;164(2):389-96. doi: 10.1111/j.1432-1033.1987.tb11070.x.
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).
利用荧光、圆二色性和质子磁共振波谱法研究了来自大肠杆菌的色氨酸阻遏物的变性。两个色氨酸残基的荧光发射对尿素浓度的依赖性并不相同。碘化物对色氨酸荧光的猝灭对尿素浓度的依赖性也排除了两态转变。随着尿素的加入，222nm处的圆二色性在两个阶段降低。通过1H NMR观察到的不同残基的归一化曲线也不一致，并且需要至少一种稳定中间体的存在。对变性曲线对蛋白质浓度依赖性的分析表明，第一次转变是二聚体阻遏物的部分展开，导致螺旋含量损失约25%。第二次转变是部分展开的二聚体的解离和展开。在高浓度蛋白质（500μM）下，约73%的阻遏物在4M尿素中以中间体形式存在。表观解离常数约为10^(-4)M；亚基可能通过亚基相互作用得到强烈稳定。天然阻遏物在至少70℃时是稳定的，而在4M尿素中形成的中间体可以通过加热可逆地变性（解链温度约为60℃，ΔH约为230kJ/mol）。
