Construction and characterization of monomeric tryptophan repressor: a model for an early intermediate in the folding of a dimeric protein.
Construction and characterization of monomeric tryptophan repressor: a model for an early intermediate in the folding of a dimeric protein.
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单体色氨酸阻遏物的构建和表征：二聚蛋白折叠早期中间体的模型。
DOI：
10.1021/bi9707786
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Matthews,CR
中科院分区：
文献类型：
--
作者：
Shao,X;Hensley,P;Matthews,CR
大肠杆菌色氨酸阻遏物（TR）是一种同源二聚体，其高度螺旋的亚基以复杂的方式相互交错。通过在疏水二聚体界面引入一对极性氨基酸，构建了Trp阻遏物的单体形式。超离心分析表明，谷氨酸取代39位的亮氨酸，形成单体/二聚体平衡，在25 °C和pH 7.6下，解离常数为1.11 × 10- 4 M。色氨酸荧光，近紫外和远紫外圆二色性，和NMR光谱表明，在微摩尔浓度的单体占主导地位，二级和三级结构存在。疏水染料结合实验表明，非极性表面是可访问的单体形式。尿素诱导的单体L39 E TR的平衡展开通过圆二色谱、荧光和吸收光谱进行监测。符合转换表明，尿素变性过程遵循一个简单的两态模型，涉及单体的天然和未折叠的形式。在pH7.6和25 °C条件下，无变性剂存在时，标准态的自由能为2.37 ± 0.15 kcal mol-1，去折叠转变对变性剂的敏感性为0.86 ± 0.04 kcal mol-1 M（urea）-1。热变性转变发生在一个很宽的温度范围内，这表明焓变小或中间体可能存在。动力学研究表明，单体的重折叠和解折叠在停流CD和荧光光谱的混合停滞时间（5 ms）内完成。这些结构、热力学和动力学结果与先前报道的野生型TR二聚体折叠中早期单体中间体的结果非常相似[Mann，C. J.，&马修斯，C. R.（1993）Biochemistry 32，5282 - 5290]。一个稳定的，单体形式的TR，非常类似于一个短暂的折叠中间的建设应该提供有用的见解TR折叠的早期事件的性质。
Tryptophan repressor (TR) fromEscherichia coliis a homodimer whose highly helical subunits intertwine in a complex fashion. A monomeric version of Trp repressor has been constructed by introducing a pair of polar amino acids at the hydrophobic dimer interface. Analytical ultracentrifugation was used to show that the replacement of leucine at position 39 with glutamic acid results in a monomer/dimer equilibrium whose dissociation constant is 1.11 × 10-4M at 25 °C and pH 7.6. Tryptophan fluorescence, both near- and far-UV circular dichroism, and NMR spectroscopies demonstrated that, at the micromolar concentrations where the monomer predominates, secondary and tertiary structure are present. Hydrophobic dye-binding experiments showed that nonpolar surface is accessible in the monomeric form. The urea-induced equilibrium unfolding of monomeric L39E TR was monitored by circular dichroism, fluorescence, and absorbance spectroscopies. Coincident transitions show that the urea denaturation process follows a simple two-state model involving monomeric native and unfolded forms. The free energy at standard state in the absence of denaturant was estimated to be 2.37 ± 0.15 kcal mol-1, and the sensitivity of the unfolding transition to denaturant, themvalue, was 0.86 ± 0.04 kcal mol-1M(urea)-1at pH 7.6 and 25 °C. The thermal denaturation transition occurred over a broad temperature range, suggesting either that the enthalpy change is small or that intermediates may exist. Kinetic studies showed that both the refolding and unfolding of the monomer were complete in the mixing dead time of stopped-flow CD and fluorescence spectroscopy, 5 ms. These structural, thermodynamic, and kinetic results are very similar to those previously reported for an early, monomeric intermediate in the folding of the wild-type TR dimer [Mann, C. J., & Matthews, C. R. (1993)Biochemistry 32, 5282−5290]. The construction of a stable, monomeric form of TR that strongly resembles a transient folding intermediate should provide useful insights into the nature of the early events in the folding of TR.