Multiple Structural States Exist Throughout the Helical Nucleation Sequence of the Intrinsically Disordered Protein Stathmin, As Reported by Electron Paramagnetic Resonance Spectroscopy
Multiple Structural States Exist Throughout the Helical Nucleation Sequence of the Intrinsically Disordered Protein Stathmin, As Reported by Electron Paramagnetic Resonance Spectroscopy
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DOI：
10.1021/bi500894q
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发表时间：
2015-03-10
期刊：
影响因子：
2.9
通讯作者：
Bridges, Michael D.
中科院分区：
文献类型：
--
作者：
Chui, Ashley J.;Lopez, Carlos J.;Bridges, Michael D.
内在无序蛋白（LDP）安定素通过与微管蛋白和微管的螺旋结合在细胞骨架维持中起重要的调节作用。然而，在没有结合伙伴的情况下，它缺乏稳定的折叠。尽管在过去的二十年里，安定素一直是研究的焦点，但人们对这种IDP的溶液相构象动力学知之甚少。据报道，安定素在溶液中是纯单体的，它具有持久折叠的短螺旋区域，这可能在c端方向上起成核螺旋折叠的作用。在这里，我们报告了一项全面的研究，该地区的结构均衡局部的stathmin，矛盾的这两个主张。利用电子顺磁共振（EPR）技术对自旋标记的安定素突变体在溶液阶段和固定在Sepharose固体载体上时进行了研究，我们发现安定素螺旋成核区域的所有位点都表现出对应于不同迁移率和稳定性的动态状态的多个光谱成分。重要的是，迁移率相对较低的状态在每个光谱中占主导地位，平均人口大于50%，我们认为这对应于蛋白质的寡聚状态。这是相对较少的，更灵活的状态，这可能代表一个螺旋折叠的单体状态，以及一个高度移动的状态，我们提出的是蛋白质的随机线圈构象。我们对EPR数据的解释是通过使用天然和SDS PAGE、凝胶过滤色谱、多角度和动态光散射技术对蛋白质进行进一步表征来证实的，所有这些都表明溶液中存在低聚的安定素。总的来说，这些数据表明，安定素在整个螺旋成核区域以多种平衡状态存在，并且这种IDP表现出寡聚化的倾向。
The intrinsically disordered protein (LDP) stathmin plays an important regulatory role in cytoskeletal maintenance through its helical binding to tubulin and microtubules. However, it lacks a stable fold in the absence of its binding partner. Although stathmin has been a focus of research over the past two decades, the solution-phase conformational dynamics of this IDP are poorly understood. It has been reported that stathmin is purely monomeric in solution and that it bears a short helical region of persistent foldedness, which may act to nucleate helical folding in the C-terminal direction. Here we report a comprehensive study of the structural equilibria local to this region in stathmin that contradicts these two claims. Using the technique of electron paramagnetic resonance (EPR) spectroscopy on spin-labeled stathmin mutants in the solution-phase and when immobilized on Sepharose solid support, we show that all sites in the helical nucleation region of stathmin exhibit multiple spectral Components that correspond to dynamic states of differing mobilities and stabilities. Importantly, a state with relatively low mobility dominates each spectrum with an average population greater than 50%, which we suggest corresponds to an oligomerized state of the protein. This is in contrast to a less populated, more mobile state, which likely represents a helically folded monomeric state of stathmin, and a highly mobile state, which we propose is the random coil conformer of the protein. Our interpretation of the EPR data is confirmed by further characterization of the protein using the techniques of native and SDS PAGE, gel filtration chromatography, and multiangle and dynamic light scattering, all of which show the presence of oligomeric stathmin in solution. Collectively, these data suggest that stathmin exists in a diverse equilibrium of states throughout the purported helical nucleation region and that this IDP exhibits a propensity toward oligomerization.