Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain.
Methods Mol Biol. 2022;2376:3-30. doi: 10.1007/978-1-0716-1716-8_1.
The analysis of protein folding reactions by monitoring the kinetic effects of specifically designed single-point mutations, the so-termed phi-value analysis, has been a favorite technique to experimentally probe the mechanisms of protein folding. The idea behind phi-value analysis is that the effects that mutations have on the folding and unfolding rate constants report on the energetic/structural features of the folding transition state ensemble (TSE), which is the highest point in the free energy surface connecting the native and unfolded states, and thus the rate limiting step that ultimately defines the folding mechanism. For single-domain, two-state folding proteins, the general procedure to perform the phi-value analysis of protein folding is relatively simple to implement in the lab. Once the mutations have been produced and purified, the researcher needs to follow a few specific guidelines to perform the experiments and to analyze the data so produced. In this chapter, a step-by-step description of how to measure and interpret the effects induced by site-directed mutations on the folding and unfolding rate constants of a protein of interest is provided. Some possible solutions to the most typical problems that arise when performing phi-value analysis in the lab are also provided.
通过监测专门设计的单点突变的动力学效应（即所谓的φ 值分析）来分析蛋白质折叠反应，这已经成为实验探测蛋白质折叠机制的一种常用技术。φ 值分析的思路是，突变对折叠和展开速率常数的影响报告了折叠过渡态集合（TSE）的能量/结构特征，折叠过渡态集合是连接天然态和展开态的自由能表面上的最高点，也是最终定义折叠机制的限速步骤。对于单结构域、两态折叠蛋白，在实验室中执行φ 值分析的一般程序相对简单。一旦产生并纯化了突变体，研究人员就需要遵循一些具体的指导方针来进行实验并分析由此产生的数据。本章提供了如何测量和解释定点突变对感兴趣蛋白质的折叠和展开速率常数的影响的逐步描述。还提供了在实验室中进行φ 值分析时出现的最常见问题的一些可能解决方案。
