Chui Ashley J, López Carlos J, Brooks Evan K, Chua Katherina C, Doupey Tonia G, Foltz Gretchen N, Kamel Joseph G, Larrosa Estefania, Sadiki Amissi, Bridges Michael D
Department of Chemistry and Biochemistry, California State University Fullerton , Fullerton, California 92831-6866, United States.
Biochemistry. 2015 Mar 10;54(9):1717-28. doi: 10.1021/bi500894q. Epub 2015 Mar 2.
The intrinsically disordered protein (IDP) 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.
内在无序蛋白（IDP）——微管相关蛋白1（stathmin）通过其与微管蛋白和微管的螺旋结合，在细胞骨架维持中发挥重要调节作用。然而，在没有结合伴侣的情况下，它缺乏稳定的折叠结构。尽管在过去二十年中stathmin一直是研究的焦点，但对这种IDP的溶液相构象动力学了解甚少。据报道，stathmin在溶液中完全是单体形式，并且具有一个持续折叠的短螺旋区域，该区域可能在C端方向充当螺旋折叠的成核位点。在此，我们报告了一项关于stathmin中该区域局部结构平衡的综合研究，该研究与这两种说法相矛盾。通过对溶液相和固定在琼脂糖固体支持物上的自旋标记stathmin突变体进行电子顺磁共振（EPR）光谱技术研究，我们发现stathmin螺旋成核区域中的所有位点都表现出多个光谱成分，这些成分对应于不同迁移率和稳定性的动态状态。重要的是，每个光谱中迁移率相对较低的状态占主导，平均丰度大于50%，我们认为这对应于蛋白质的寡聚化状态。这与丰度较低、迁移率较高的状态形成对比，后者可能代表stathmin的螺旋折叠单体状态，以及高度迁移的状态，我们认为这是蛋白质的无规卷曲构象。使用天然和SDS聚丙烯酰胺凝胶电泳、凝胶过滤色谱以及多角度和动态光散射技术对蛋白质进行进一步表征，证实了我们对EPR数据的解释，所有这些技术都表明溶液中存在寡聚的stathmin。总体而言，这些数据表明stathmin在整个所谓的螺旋成核区域中以多种状态的平衡存在，并且这种IDP表现出寡聚化的倾向。
