Predicting coupling limits from an experimentally determined energy landscape.
Predicting coupling limits from an experimentally determined energy landscape.
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根据实验确定的能量景观预测耦合极限。
DOI：
10.1073/pnas.0608756104
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发表时间：
2007
影响因子：
11.1
通讯作者：
Barrick,Doug
中科院分区：
文献类型：
--
作者：
Street,TimothyO;Bradley,ChristinaM;Barrick,Doug
重复蛋白由串联的结构模块组成，在这些模块中，紧密接触不会超出相邻的重复序列。尽管这些密切接触具有局部性质，但重复蛋白通常作为一个单一的、高度耦合的单位展开。以前对Notch ankyrin结构域的研究表明，这种缺乏平衡的展开中间体既是由于稳定了每个重复之间的界面，也是由于稳定性在折叠能量格局中大致均匀分布。为了研究这一想法，我们产生了Notch ankyrin结构域的15个变体，这些变体具有单一和多个不稳定的取代，使能量格局不均匀。通过对这些变体应用自由能可加性分析，我们量化了重复6和7与重复1-5脱钩的失稳阈值。这种可加性分析提出的自由能耦合极限(≈4千卡/摩尔)也反映在M值分析中，以及所有15个变体的CD和荧光监测的平衡展开跃迁之间的差异。所有这些观测都是通过分析实验确定的能源格局对日益加剧的不均衡性的反应来定量预测的。这些结果突出了均匀分布的局部稳定性对于实现合作展开的重要性。
Repeat proteins are composed of tandem structural modules in which close contacts do not extend beyond adjacent repeats. Despite the local nature of these close contacts, repeat proteins often unfold as a single, highly coupled unit. Previous studies on the Notch ankyrin domain suggest that this lack of equilibrium unfolding intermediates results both from stabilizing interfaces between each repeat and from a roughly uniform distribution of stability across the folding energy landscape. To investigate this idea, we have generated 15 variants of the Notch ankyrin domain with single and multiple destabilizing substitutions that make the energy landscape uneven. By applying a free energy additivity analysis to these variants, we quantified the destabilization threshold over which repeats 6 and 7 decouple from repeats 1–5. The free energy coupling limit suggested by this additivity analysis (≈4 kcal/mol) is also reflected inm-value analysis and in differences among equilibrium unfolding transitions as monitored by CD versus fluorescence for all 15 variants. All of these observations are quantitatively predicted by analyzing the response of the experimentally determined energy landscape to increasing unevenness. These results highlight the importance of a uniform distribution of local stability in achieving cooperative unfolding.