Biozentrum , University of Basel , Klingelbergstrasse 50/70 , CH-4056 Basel , Switzerland.
J Phys Chem B. 2019 Dec 5;123(48):10181-10191. doi: 10.1021/acs.jpcb.9b08816. Epub 2019 Nov 22.
Thermal and chemical unfolding of lysozyme in the presence of the guanidine HCl denaturant is a model system to compare the conventional two-state model of protein unfolding with the multistate Zimm-Bragg theory. The two-state model is shown to be the noncooperative limit of the Zimm-Bragg theory. In particular, the Zimm-Bragg theory provides a molecular interpretation of the empirical linear extrapolation method (LEM) of the two-state model. Differential scanning calorimetry (DSC) experiments reported in the literature are analyzed with both methods. Lysozyme unfolding is associated with a large endothermic enthalpy that decreases significantly upon addition of guanidine HCl. In contrast, the Gibbs free energy of unfolding is small, negative, and independent of the guanidine HCl concentration, contradicting, in part, the conclusions of the LEM. The unfolding enthalpy is compensated by an even larger entropy term. The multistate Zimm-Bragg theory predicts a larger conformational enthalpy and a smaller Gibbs free energy than the two-state model. The Zimm-Bragg theory provides the protein cooperativity parameter, the average length of independently folding protein domains, and the Gibbs free energy of unfolding of individual amino acid residues. Guanidine HCl binding to lysozyme is exothermic and counteracts the endothermic unfolding enthalpy. The number of bound denaturant molecules is determined from the decrease in enthalpy and is extrapolated to the guanidine HCl-to-amino acid stoichiometry at complete lysozyme unfolding. Chemical unfolding isotherms measured with circular dichroism (CD) spectroscopy are analyzed with both models. The chemical Zimm-Bragg theory is a cooperative molecular model, yielding the guanidine HCl binding constant and the protein cooperativity parameter. It allows a quantitative comparison between thermal and chemical protein unfolding. The two reactions have almost identical changes in Gibbs free energy. However, thermal unfolding is significantly more cooperative than chemical unfolding. Finally, distinct differences are observed in thermal unfolding between DSC and CD spectroscopy.
在盐酸胍变性剂存在的情况下，溶菌酶的热变性和化学变性是一个模型系统，用于比较蛋白质变性的传统两态模型和多态 Zimm-Bragg 理论。两态模型被证明是 Zimm-Bragg 理论的非协同极限。特别是，Zimm-Bragg 理论为两态模型的经验线性外推法（LEM）提供了分子解释。用两种方法分析了文献中报道的差示扫描量热法（DSC）实验。溶菌酶的变性伴随着一个很大的吸热焓，当加入盐酸胍时，这个焓会显著降低。相比之下，蛋白质变性的吉布斯自由能很小，为负值，且与盐酸胍浓度无关，这在一定程度上与 LEM 的结论相矛盾。展开焓由一个更大的熵项补偿。多态 Zimm-Bragg 理论预测的构象焓比两态模型大，吉布斯自由能小。Zimm-Bragg 理论提供了蛋白质协同参数、独立折叠蛋白质结构域的平均长度以及单个氨基酸残基的变性吉布斯自由能。盐酸胍与溶菌酶的结合是放热的，与吸热的变性焓相反。从焓的降低可以确定结合的变性剂分子数，并将其外推到溶菌酶完全变性时盐酸胍与氨基酸的化学计量比。用圆二色性（CD）光谱法测量的化学 Zimm-Bragg 理论是一个协同的分子模型，给出了盐酸胍的结合常数和蛋白质协同参数。它允许在热变性和化学变性之间进行定量比较。这两个反应的吉布斯自由能变化几乎相同。然而，热变性比化学变性协同得多。最后，在 DSC 和 CD 光谱学之间观察到热变性的明显差异。
