Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy
Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy
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DOI：
10.1021/jp063708u
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发表时间：
2006-10-19
影响因子：
3.3
通讯作者：
Longhi, Sonia
中科院分区：
文献类型：
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作者：
Morin, Benjamin;Bourhis, Jean-Marie;Longhi, Sonia
我们利用定点自旋标记电子顺磁共振(EPR)谱研究了麻疹病毒核蛋白(N-Tail)固有无序的C-末端结构域的诱导折叠。制备了位于3个保守区的4个单位点N-尾突变体(S407C、S488C、L496C和V517C)，并用氮氧化物顺磁探针进行标记。我们可以监测N-Tail在二级结构稳定剂2，2，2-三氟乙醇(TFE)或其生理伙伴之一，即病毒磷蛋白的C-末端结构域(XD)存在时所经历的刚性增加。当添加XD时，接枝在位置488、496和517处的自旋标记的迁移率显著降低，与结合到位置407的自旋标记的迁移率相反，位置407不受影响。此外，自旋标记的S488C和L496C在30%蔗糖存在下与XD结合的EPR谱表明在自旋标记附近形成了α-螺旋。之前的生化和结构研究已经确定了这种阿尔法螺旋。使用TFE，我们揭示了在N-Tail的N-末端区域内以前未被检测到的结构倾向，并表明其C-末端区域即使在高TFE浓度下也“抵制”获得结构。最后，我们首次证明了在XD存在下N-Tail经历的诱导折叠过程的可逆性。这些结果强调了位点定向自旋标记EPR光谱学在识别与结合和折叠事件有关的蛋白质区域方面的适用性，同时提供了对残基水平的洞察。
We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N-TAIL). Four single-site N-TAIL mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N-TAIL undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N-TAIL and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N-TAIL undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.