Francisco Olga A, Clark Courtney J, Glor Hayden M, Khajehpour Mazdak
Department of Chemistry, University of Manitoba Canada.
University of Manitoba 468 Parker Bldg. Winnipeg Manitoba R3T2N2 Canada
RSC Adv. 2019 Jan 28;9(6):3416-3428. doi: 10.1039/c8ra10303h. eCollection 2019 Jan 22.
It has long been known that large soft anions like bromide, iodide and thiocyanate are protein denaturing agents, but their mechanism of action is still unclear. In this work we have investigated the protein denaturing properties of these anions using Ribonuclease A (RNase A) as a model protein system. Salt-induced perturbations to the protein folding free energy were determined using differential scanning calorimetry and the results demonstrate that the addition of sodium iodide and sodium thiocyanate significantly decreases the melting temperature of the protein. In order to account for this reduction in protein stability, we show that the introduction of salts that contain soft anions to the aqueous solvent perturbs the protein unfolding free energy through three mechanisms: (a) screening Coulomb interactions that exist between charged protein residues, (b) Hofmeister effects, and (c) specific anion binding to CH and CH moieties in the protein polypeptide backbone. Using the micellization of 1,2-hexanediol as a ruler for hydrophobicity, we have devised a practical methodology that separates the Coulomb and Hofmeister contributions of salts to the protein unfolding free energy. This allowing us to isolate the contribution of soft anion binding interactions to the unfolding process. The analysis shows that binding contributions have the largest magnitude, confirming that it is the binding of soft anions to the polypeptide backbone that is the main promoter of protein unfolding.
长期以来，人们都知道像溴离子、碘离子和硫氰酸根离子这样的大的软阴离子是蛋白质变性剂，但其作用机制仍不清楚。在这项工作中，我们以核糖核酸酶A（RNase A）作为模型蛋白质系统，研究了这些阴离子的蛋白质变性特性。使用差示扫描量热法测定了盐对蛋白质折叠自由能的扰动，结果表明，添加碘化钠和硫氰酸钠会显著降低蛋白质的解链温度。为了解释蛋白质稳定性的这种降低，我们表明，向水性溶剂中引入含有软阴离子的盐会通过三种机制扰乱蛋白质的解折叠自由能：（a）屏蔽带电蛋白质残基之间存在的库仑相互作用，（b）霍夫迈斯特效应，以及（c）特定阴离子与蛋白质多肽主链中的CH和CH基团结合。以1,2 -己二醇的胶束化作为疏水性的标尺，我们设计了一种实用的方法，将盐对蛋白质解折叠自由能的库仑贡献和霍夫迈斯特贡献区分开来。这使我们能够分离出软阴离子结合相互作用对解折叠过程的贡献。分析表明，结合贡献的幅度最大，证实了软阴离子与多肽主链的结合是蛋白质解折叠的主要促进因素。
