Mizukami Takuya, Abe Yukiko, Maki Kosuke
Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan.
PLoS One. 2015 Aug 5;10(8):e0134238. doi: 10.1371/journal.pone.0134238. eCollection 2015.
In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0-2.2 M) than the formation of the native state (0-1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7-2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.
在本研究中，我们研究了脱辅基肌红蛋白中尿素诱导的动力学折叠中间体和非天然平衡态形成的动力学机制的等效性。尽管平衡中间体和动力学中间体具有相似的结构特性，但它们在不同条件下通过不同机制积累，其形成是否涉及共同或不同的动力学机制仍不清楚。为了研究潜在的形成机制，我们在pH 6.0和8°C下，通过连续和停流荧光在大约100 μs至10 s的时间范围内测量了马脱辅基肌红蛋白的重折叠和去折叠动力学，以及平衡去折叠转变，作为尿素浓度的函数。与天然态的形成（0 - 1.6 M）相比，在更宽的尿素浓度范围（0 - 2.2 M）内观察到了动力学中间体的形成。此外，在天然态和去折叠态不稳定的条件下（在约0.7 - 2 M尿素时），动力学中间体作为主要的平衡态持续存在。随着尿素浓度从0 M增加到约2 M，观察到从动力学中间体到平衡中间体的连续转变，这表明这些状态共享共同的动力学折叠机制。这一发现支持了这些中间体是等效的结论。我们的结果进而表明，在折叠的早期阶段形成了抵抗变性剂扰动的蛋白质区域，这进一步支持了瞬态中间体和平衡中间体的结构等效性。在重折叠约140 μs内积累了另一种折叠中间体，在去折叠<1 ms内积累了一种去折叠中间体。最后，通过使用定量建模，我们表明五态顺序模型适当地描述了马脱辅基肌红蛋白的折叠机制。
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