NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.

与光学方法相比，核磁共振波谱法在记录蛋白质的展开转变方面具有某些独特的优势。它能够对每个残基进行监测并单独检测折叠和展开状态以及可能的平衡中间体。这样可以详细查看感兴趣蛋白质的折叠状态和协同性，并正确解释后续实验。在这里，我们详细总结了此类实验的实践和理论方面。可以避免某些陷阱，并且可以在分析过程中进行有意义的简化。尤其是对感兴趣系统的 NMR 交换机制和弛豫特性的良好理解是有益的。我们通过对 GB1 折叠和展开状态信号的全局分析展示了如何提取准确的展开值以及限制不同 NMR 检测和展开方法的因素。例如，常用的可交换酰胺可能导致系统性的蛋白质热力学稳定性测定不足。我们从几个角度阐述了如何处理更复杂的蛋白质，以及残基分辨率下蛋白质稳定性的知识如何帮助了解拥挤条件下、相分离期间和高压下的蛋白质特性。