Ionescu R M, Smith V F, O'Neill J C, Matthews C R
Department of Chemistry, Life Sciences Consortium and Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park 16802, USA.
Biochemistry. 2000 Aug 8;39(31):9540-50. doi: 10.1021/bi000511y.
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.
利用尿素和温度作为变性剂的联合效应，研究了大肠杆菌二氢叶酸还原酶无半胱氨酸变体（AS-DHFR）的热力学和光谱性质。在不同尿素浓度下温度诱导的去折叠过程以及不同温度下尿素诱导的去折叠过程中，记录了圆二色性（CD）、吸收光谱和荧光光谱。通过对每组去折叠光谱进行奇异值分解得到的前三个向量被纳入到一个独特热力学模型的全局分析中。尽管单个去折叠曲线可以描述为两态过程，但对99个向量的同时拟合需要一个三态模型作为沿两个扰动轴描述去折叠反应的最小方案。该模型涉及天然态（N）、中间态（I）和去折叠态（U），预测在15℃时最大表观稳定性ΔG°（NU）为6 kcal mol⁻¹，表观m（NU）值为2 kcal mol⁻¹ M⁻¹，表观热容变化ΔC(p)()(-NU)为2.5 kcal mol⁻¹ K⁻¹。中间物种的最大稳定性约为2 kcal mol⁻¹，其紧密程度更接近天然态而非去折叠态。中间态的丰度在50℃左右最大（约70%），在>或 =2 M尿素或温度<35℃或>65℃时降至检测限以下。平衡中间态的荧光性质类似于从尿素变性态重折叠过程中检测到的瞬态中间态，这表明腺苷结合结构域中含色氨酸的疏水簇在平衡和动力学反应中都起关键作用。天然态的CD光谱性质揭示了存在两种主要异构体，它们在配体结合亲和力和腺苷结合结构域的堆积方面存在差异。这些物种的相对丰度随温度略有变化，且不依赖于尿素浓度，这意味着两种天然异构体结构良好且紧密。对来自多个光谱探针的数据和几种去折叠方法进行全局分析是揭示DHFR部分折叠和完全折叠形式的结构和热力学性质的有力工具。
