Wilson Christian A M, Corrêa Camila G
Department of Biochemistry and Molecular Biology, Faculty of Chemistry and Pharmaceutical Sciences, Universidad de Chile, Santiago, Chile.
Faculty of Science, Universidad de Valparaíso, Valparaíso, Chile.
Biophys Rev. 2025 Apr 22;17(2):231-245. doi: 10.1007/s12551-025-01310-0. eCollection 2025 Apr.
Free energy is a critical parameter in understanding the equilibrium in chemical reactions. It enables us to determine the equilibrium proportion between the different species in the reaction and to predict in which direction the reaction will proceed if a change is performed in the system. Historically, to calculate this value, bulk experiments were performed where a parameter was altered at a gradual rate to change the population until a new equilibrium was established. In protein folding studies, it is common to vary the temperature or chaotropic agents in order to change the population and then to extrapolate to physiological conditions. Such experiments were time-consuming due to the necessity of ensuring equilibrium and reversibility. Techniques of single-molecule manipulation, such as optical/magnetic tweezers and atomic force microscopy, permit the direct measurement of the work performed by a protein undergoing unfolding/refolding at particular forces. Also, with the development of non-equilibrium free energy theorems (Jarzynski equality, Crooks fluctuation theorem, Bennett acceptance ratio, and overlapping method), it is possible to obtain free energy values in experiments far from equilibrium. This review compares different methodologies and their application in optical tweezers. Interestingly, in many proteins, discrepancies in free energy values obtained through different methods suggest additional complexities in the folding pathway, possibly involving intermediate states such as the molten globule. Further studies are needed to confirm their presence and significance.
The online version contains supplementary material available at 10.1007/s12551-025-01310-0.
自由能是理解化学反应平衡的一个关键参数。它使我们能够确定反应中不同物种之间的平衡比例，并预测如果系统发生变化，反应将向哪个方向进行。从历史上看，为了计算这个值，进行了大量实验，其中一个参数以逐渐变化的速率改变，以改变粒子数，直到建立新的平衡。在蛋白质折叠研究中，通常通过改变温度或变性剂来改变粒子数，然后外推到生理条件。由于需要确保平衡和可逆性，此类实验非常耗时。单分子操纵技术，如光学/磁性镊子和原子力显微镜，允许直接测量蛋白质在特定力下展开/重新折叠所做的功。此外，随着非平衡自由能定理（雅津斯基等式、克鲁克斯涨落定理、贝内特接受比和重叠方法）的发展，有可能在远离平衡的实验中获得自由能值。这篇综述比较了不同的方法及其在光学镊子中的应用。有趣的是，在许多蛋白质中，通过不同方法获得的自由能值存在差异，这表明折叠途径存在额外的复杂性，可能涉及中间状态，如熔球态。需要进一步的研究来证实它们的存在和意义。
在线版本包含可在10.1007/s12551-025-01310-0获取的补充材料。
