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首页> 外文期刊>Biochemistry >Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles.
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Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles.

机译：大肠杆菌二氢叶酸还原酶的多态平衡解折叠：天然，中间和未折叠集合的热力学和光谱学描述。

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摘要

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.
机译：利用尿素和温度的组合作用作为变性剂，研究了无半胱氨酸的大肠杆菌二氢叶酸还原酶（AS-DHFR）的无半胱氨酸变异体的热力学和光谱性质。在不同尿素浓度下温度诱导的展开过程中以及在不同温度下尿素诱导的展开过程中记录了圆二色性（CD），吸收和荧光光谱。通过对每组展开光谱的奇异值分解获得的前三个向量被合并到唯一热力学模型的全局分析中。尽管可以将单个展开轮廓描述为两个状态的过程，但同时拟合99个向量需要一个三状态模型作为描述沿着两个扰动轴的展开反应的最小方案。该模型涉及原始（N），中间（I）和未折叠（U）状态，预测在15摄氏度，6千卡·摩尔（-）（1）的最大表观稳定性DeltaG度（NU）。表观m（NU）值为2 kcal mol（-）（1）M（-）（1），表观热容量变化DeltaC（p）（）（-NU）为2.5 kcal mol（-）（ 1）K（-）（1）。中间物质具有约2 kcal mol（-）（1）的最大稳定性，并且紧密度比天然的紧密，而不是未折叠的紧密。中间体的总数在50摄氏度左右最大（约70％），并低于> 2M尿素的检出限或在低于35或65摄氏度的温度下。平衡中间体的荧光性质类似于从尿素变性状态重折叠过程中检测到的过渡中间体的那些，表明腺苷结合域中的含色氨酸的疏水簇在平衡和动力学反应中都起着关键作用。原始状态的CD光谱性质表明存在两个主要的同工型，它们在配体结合亲和力和腺苷结合域的堆积方面有所不同。这些物种的相对种群随温度略有变化，并且不依赖于尿素浓度，这意味着这两种天然同工型结构良好且紧凑。对来自多个光谱探针和几种展开方法的数据进行全局分析，是揭示DHFR部分和完全折叠形式的结构和热力学性质的有力工具。

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