Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain.
Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain.
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DOI：
10.1016/j.jmb.2013.04.015
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发表时间：
2013-07-24
影响因子：
5.6
通讯作者：
Komives, Elizabeth A.
中科院分区：
文献类型：
--
作者：
Lamboy, Jorge A.;Kim, Hajin;Dembinski, Holly;Ha, Taekjip;Komives, Elizabeth A.
先前的单分子荧光共振能量转移（smFRET）研究中，IκBα的第二个和第六个锚蛋白重复序列（AR）被FRET对标记，显示出缓慢的波动，好像IκBα AR结构域在其天然状态下展开。为了系统地探测这些缓慢的动态波动发生在哪里，我们现在呈现来自smFRET研究的数据，其中FRET标记被放置在AR 1和4（突变体命名为AR 1-4）、AR 2和5（AR 2-5）以及AR 3和6（AR 3-6）。这里呈现的结果表明，AR 6最容易从AR结构域分离/展开，在室温下经历大幅波动。AR 6具有比其他IκBα AR更少的稳定共有残基，这可能有助于AR 6“失去抓地力”。AR 5在室温下几乎没有波动，但在37 °C下有相当一部分分子显示波动。引入已知折叠AR 6的稳定突变抑制了AR 5的波动，表明AR 5的波动可能是由于AR 6的重复序列间稳定性减弱。AR 1在室温下也有一定的波动，这表明波动是AR结构域末端AR的一般行为。值得注意的是，AR 1在结合状态下仍然波动，但主要在0.6和0.9之间的FRET效率，而在游离IκBα中，波动延伸到<0.5 FRET效率。总的来说，我们的研究结果提供了一个更完整的图片的AR域的本地状态动态的能量景观。
Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1–4), at ARs 2 and 5 (AR 2–5), and at ARs 3 and 6 (AR 3–6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 “loses grip”. AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.
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发表时间：
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影响因子：
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作者：
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通讯作者：
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