Mapping α-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy
Mapping α-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy
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DOI：
10.1002/prot.22125
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发表时间：
2008-12-01
影响因子：
2.9
通讯作者：
Longhi, Sonia
中科院分区：
文献类型：
--
作者：
Belle, Valerie;Rouger, Sabrina;Longhi, Sonia
利用位点定向自旋标记EPR光谱，我们绘制了麻疹病毒核蛋白（N-TAIL）内部无序c端结构域（N-TAIL）发生诱导折叠的区域。除了四个自旋标记的N-TAIL变体（S407C, S488C， L496C和V517C） （Morin et al. (2006), J Phys Chem 110: 2059620608）外，还设计了10个新的单位点半胱氨酸变体，从大肠杆菌中纯化，并进行了自旋标记。这14个自旋标记的变体使我们能够详细绘制N-TAIL在二级结构稳定剂2,2,2-三氟乙醇或病毒磷蛋白的c端结构域X （XD）存在下的刚性增益。N-TAIL的不同区域对XD的结合有不同程度的贡献，而接枝在407和460位置的自旋标签的迁移率不受XD的影响；接枝在488-502和505-522区域内的自旋标签分别有严重和中度降低。此外，在30%蔗糖的存在下，EPR实验使我们能够精确地映射到残基488-502，N-TAIL区域正在进行螺旋折叠。488-502区域的流动性被发现即使在没有伙伴的情况下也受到限制，这种行为可以用一个短暂填充折叠态的存在来解释。最后，我们发现505-522区域在与XD结合时的受限运动是由于在488-502区域内发生的螺旋跃迁，而不是与XD的直接相互作用。
Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N-TAIL) that undergoes induced folding. In addition to four spin-labeled N-TAIL variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 2059620608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N-TAIL in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N-TAIL were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N-TAIL region undergoing at-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the at-helical transition occurring within the 488-502 region and not to a direct interaction with XD.