Morin Benjamin, Bourhis Jean-Marie, Belle Valérie, Woudstra Mireille, Carrière Frédéric, Guigliarelli Bruno, Fournel André, Longhi Sonia
Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Universités Aix-Marseille I et II, Campus de Luminy, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France.
J Phys Chem B. 2006 Oct 19;110(41):20596-608. doi: 10.1021/jp063708u.
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.
我们使用定点自旋标记电子顺磁共振（EPR）光谱法研究麻疹病毒核蛋白（N(TAIL)）内在无序的C末端结构域的诱导折叠。制备了位于三个保守区域的四个单点N(TAIL)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）或其生理伴侣之一（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。添加XD后，接在位置488、496和517处的自旋标记的流动性显著降低，而接在位置407处的自旋标记则不受影响。此外，在30%蔗糖存在下与XD结合的自旋标记S488C和L496C的EPR光谱表明在自旋标记附近形成了α-螺旋。先前的生化和结构研究已经鉴定出这样的α-螺旋。使用TFE，我们揭示了N(TAIL) N末端区域内先前未检测到的结构倾向，并表明其C末端区域即使在高TFE浓度下也“抵抗”获得结构。最后，我们首次展示了N(TAIL)在XD存在下所经历的诱导折叠过程的可逆性。这些结果突出了定点自旋标记EPR光谱法在识别参与结合和折叠事件的蛋白质区域方面的适用性，同时在残基水平上提供了见解。
