Son Ikbae, Chalikian Tigran V
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , 144 College Street, Toronto, Ontario M5S 3M2, Canada.
Biochemistry. 2016 Nov 29;55(47):6475-6483. doi: 10.1021/acs.biochem.6b00805. Epub 2016 Nov 15.
We report the first experimental characterization of the full thermodynamic profile for binding of urea to a native protein. We measured the volumetric parameters of lysozyme at pH 7.0 as a function of urea within a temperature range of 18-45 °C. At neutral pH, lysozyme retains its native conformation between 0 and 8 M urea over the entire range of temperatures studied. Consequently, our measured volumetric properties reflect solely the interactions of urea with the native protein and do not involve contributions from urea-induced conformational transitions. We analyzed our data within the framework of a statistical thermodynamic analytical model in which urea-protein interactions are viewed as solvent exchange in the vicinity of the protein. The analysis produced the equilibrium constant, k, for an elementary reaction of urea-protein binding with a change in standard state free energy (ΔG° = -RT ln k) at each experimental temperature. We used the van't Hoff equation to compute from the temperature dependence of the equilibrium constant, k, changes in enthalpy, ΔH°, and entropy, ΔS°, accompanying binding. The thermodynamic profile of urea-protein interactions, in conjunction with published molecular dynamics simulation results, is consistent with the picture in which urea molecules, being underhydrated in the bulk, form strong, enthalpically favorable interactions with the surface protein groups while paying a high entropic price. We discuss ramifications of our results for providing insights into the combined effects of urea, temperature, and pressure on the conformational preferences of proteins.
我们报告了尿素与天然蛋白质结合的完整热力学曲线的首次实验表征。我们在18 - 45 °C的温度范围内，测量了pH 7.0条件下溶菌酶的体积参数随尿素浓度的变化。在中性pH条件下，在所研究的整个温度范围内，溶菌酶在0至8 M尿素之间保持其天然构象。因此，我们测量的体积性质仅反映了尿素与天然蛋白质的相互作用，不涉及尿素诱导的构象转变的贡献。我们在统计热力学分析模型的框架内分析了我们的数据，在该模型中，尿素 - 蛋白质相互作用被视为蛋白质附近的溶剂交换。分析得出了每个实验温度下尿素 - 蛋白质结合基本反应的平衡常数k，以及标准状态自由能变化（ΔG° = -RT ln k）。我们使用范特霍夫方程，根据平衡常数k的温度依赖性计算出结合过程中伴随的焓变ΔH°和熵变ΔS°。尿素 - 蛋白质相互作用的热力学曲线，结合已发表的分子动力学模拟结果，与这样的图景一致：即尿素分子在本体中处于欠水合状态，与蛋白质表面基团形成强烈的、焓有利的相互作用，同时付出高昂的熵代价。我们讨论了我们的结果对于深入了解尿素、温度和压力对蛋白质构象偏好的综合影响的意义。
