Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, 06120, Halle (Saale), Germany.
J Biomol NMR. 2022 Apr;76(1-2):3-15. doi: 10.1007/s10858-021-00389-3. Epub 2022 Jan 4.
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.
NMR 光谱学在记录蛋白质的展开转变方面具有某些独特的优势，例如与光学方法相比。它能够进行残基监测，并分别检测折叠态和展开态以及可能的平衡中间体。这使得可以详细了解感兴趣的蛋白质的折叠状态和协同性，并正确解释随后的实验。在这里，我们详细总结了此类实验的实际和理论方面。在分析过程中，可以避免某些陷阱并进行有意义的简化。特别是对感兴趣的系统的 NMR 交换机制和弛豫特性有很好的了解是有益的。我们通过对 GB1 的折叠态和展开态信号的全局分析，展示了如何准确提取展开值，以及不同的 NMR 检测和展开方法的限制。例如，通常使用的可交换酰胺可能导致热力学蛋白质稳定性的系统低估。我们提供了几种处理更复杂蛋白质的方法，以及关于残基分辨率下蛋白质稳定性的知识如何帮助理解拥挤条件下、相分离过程中和高压下的蛋白质性质。
