The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio‐frequency power to lossy aqueous samples, refolding of barstar from its cold‐denatured state can be followed by real‐time NMR spectroscopy. Since temperature‐induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real‐time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low‐temperature‐unfolded state, which can potentially induce multiple folding pathways. The high time resolution real‐time 2D‐NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state‐correlated spectroscopy, an alternative folding pathway circumventing the rate‐limiting cis‐trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.

中文翻译：

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射频加热引起的冷变性Barstar的折叠：一种通过实时NMR光谱研究蛋白质折叠的新方法。

已显示barstar的C40A / C82A双突变体在水凝固点以上发生冷变性。通过将射频功率快速施加到有损耗的水性样品中，barstar从冷变性状态重新折叠后，可以进行实时NMR光谱分析。由于温度诱导的解折叠和重折叠对于该双突变体是可逆的，因此可以利用多次循环获得二维实时NMR数据。Barstar包含两个脯氨酸残基，它们在低温未折叠状态下采用顺式和反式构型的混合，可能潜在地诱导多种折叠途径。此处报道的高分辨率高分辨率实时2D-NMR测量显示了脯氨酸异构化相关的多种折叠途径的证据，并且存在稳定的中间体。通过应用高级加热循环和状态相关光谱，可以观察到另一种绕开速率限制的顺反异构化的折叠途径。动力学数据揭示了慢速和快速折叠路径上的中间体。