Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
J Mol Biol. 2013 Jan 23;425(2):257-72. doi: 10.1016/j.jmb.2012.11.004. Epub 2012 Nov 12.
The kinetic folding of β(2)-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH7.5 to pH2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH2.0 to pH7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5-6 s(-1), while the molecule with the Pro32 trans isomer refolds more slowly (pH7.5 and 25°C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001-0.002 s(-1)) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7-9% under a more physiological condition (pH7.5 and 37°C).
使用停流双跃技术，通过中断展开和中断折叠实验研究了β(2)-微球蛋白从酸变性状态的动力学折叠。在中断展开中，我们首先通过 pH 从 7.5 跃变到 2.0 将蛋白质展开，并通过监测色氨酸荧光进行反向 pH 跃变来进行动力学折叠测定。同样，在中断折叠中，我们首先通过 pH 从 2.0 跃变到 7.5 将蛋白质折叠，并使用盐酸胍（GdnHCl）浓度跃变以及反向 pH 跃变作为展开测定。基于这些实验，折叠可以用平行途径模型来表示，其中具有正确 Pro32 顺式异构体的分子快速折叠，速率常数为 5-6 s(-1)，而具有 Pro32 反式异构体的分子折叠较慢（pH7.5 和 25°C）。在折叠的最后一步，在后一种途径上产生的类似天然的反式构象非常缓慢地异构化为类似天然的顺式构象（0.001-0.002 s(-1))。在中断折叠的 GdnHCl 诱导展开测定中，类似天然的反式构象的展开速度明显快于类似天然的顺式构象，并且直接的 GdnHCl 诱导展开也是两相的，表明在天然条件下存在类似天然的反式构象的显著水平。折叠的一维 NMR 和实时 NMR 实验进一步表明，在更生理条件下（pH7.5 和 37°C），反式构象的比例增加到 7-9%。
