Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

大多数技术允许通过跟踪单个报告者的行为或作为平均全有或全无过程来检测蛋白质展开。我们最近将 2D NMR 光谱添加到成熟的技术中，该技术能够使用蛋白质疏水核心中残基的共振获得有关展开过程的信息。在这里，我们质疑对每个共振的个体稳定性曲线的分析是否可以提供额外的特定于站点的信息。我们使用的 Yfh1 蛋白具有独特的特性，可以在低于水冰点的温度下以低离子强度进行冷变性和热变性。我们表明，与疏水核心残基的平均 NMR 曲线不一致的稳定性曲线主要包括暴露的异常值，但这些异常值确实提供了宝贵的信息。