Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli' and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy.
Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden.
Int J Mol Sci. 2021 Jan 15;22(2):828. doi: 10.3390/ijms22020828.
Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted 'energetic coupling' describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein-ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.
定量测量蛋白质结构中分子内和分子间相互作用是一项艰巨的任务，在实验上不容易解决。这种现象被称为“能量耦合”，描述了蛋白质系统中两个残基之间的短程和长程相互作用。一种用于识别和定量描述蛋白质或蛋白质-配体复合物中的长程相互作用和变构网络的强大方法称为双突变体循环分析。在这篇综述中，我们描述了双突变体循环方法学的热力学原理和基本方程、其应用领域和最新应用，以及实验者必须考虑的注意事项和陷阱。特别是，我们展示了双突变体循环如何成为研究蛋白质结合反应中的变构机制以及蛋白质折叠途径中难以捉摸状态的有力工具。
