Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Biochemistry. 2010 Jan 19;49(2):365-71. doi: 10.1021/bi9014055.
Investigations of irreversible protein unfolding often assume that alterations to the unfolded state, rather than the nature of the native state itself, are the cause of the irreversibility. However, the present study describes a less common explanation for the irreversible denaturation of pepsin, a zymogen-derived aspartic peptidase. The presence of a large folding barrier combined with the thermodynamically metastable nature of the native state, the formation of which depends on a separate prosegment (PS) domain, is the source of the irreversibility. Pepsin is unable to refold to the native state upon return from denaturing conditions due to a large folding barrier (24.6 kcal/mol) and instead forms a thermodynamically stable, yet inactive, refolded state. The native state is kinetically stabilized by an unfolding activation energy of 24.5 kcal/mol, comparable to the folding barrier, indicating that native pepsin exists as a thermodynamically metastable state. However, in the presence of the PS, the native state becomes thermodynamically stable, and the PS catalyzes pepsin folding by stabilizing the folding transition state by 14.7 kcal/mol. Once folded, the PS is removed, and the native conformation exists as a kinetically trapped state. Thus, while PS-guided folding is thermodynamically driven, without the PS the pepsin energy landscape is dominated by kinetic barriers rather than by free energy differences between native and denatured states. As pepsin is the archetype of a broad class of aspartic peptidases of similar structure and function, and many require their PS for correct folding, these results suggest that the occurrence of native states optimized for kinetic rather than thermodynamic stability may be a common feature of protein design.
不可逆蛋白质变性的研究通常假设，导致不可逆性的原因是变性状态的改变，而不是天然状态本身的性质。然而，本研究描述了胃蛋白酶不可逆变性的一个不太常见的解释，胃蛋白酶是一种酶原衍生的天冬氨酸肽酶。大折叠势垒的存在与天然状态的热力学亚稳性质相结合，而天然状态的形成取决于一个单独的前肽（PS）结构域，这是不可逆性的来源。由于大折叠势垒（24.6 千卡/摩尔），胃蛋白酶无法在从变性条件下返回时重新折叠到天然状态，而是形成热力学稳定但无活性的折叠状态。天然状态通过 24.5 千卡/摩尔的解折叠激活能在动力学上得到稳定，与折叠势垒相当，这表明天然胃蛋白酶作为热力学亚稳态存在。然而，在 PS 的存在下，天然状态变得热力学稳定，PS 通过稳定折叠过渡态使胃蛋白酶折叠，使其稳定 14.7 千卡/摩尔。一旦折叠，PS 就被去除，天然构象作为动力学陷阱状态存在。因此，虽然 PS 引导的折叠是热力学驱动的，但没有 PS，胃蛋白酶的能量景观主要由动力学势垒而不是天然状态和变性状态之间的自由能差异决定。由于胃蛋白酶是类似结构和功能的广泛天冬氨酸肽酶类的原型，并且许多需要它们的 PS 来正确折叠，这些结果表明，针对动力学而不是热力学稳定性优化的天然状态的出现可能是蛋白质设计的一个共同特征。
