Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA3
Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA3
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DOI：
10.3934/biophy.2018.3.166
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发表时间：
2018-01-01
期刊：
影响因子：
1.5
通讯作者：
Fanucci, Gail E.
中科院分区：
文献类型：
--
作者：
Dunleavy, Katie M.;Milshteyn, Eugene;Fanucci, Gail E.
IA(3)是一种本质上无序的蛋白质(IDP)，当结合到酵母蛋白酶A(YPRA)或在辅助稳定剂2，2，2-三氟乙醇(TFE)存在时，它会变成螺旋。本文用定点自旋标记(SDSL)、连续波电子顺磁共振(CW-EPR)和圆二色谱(CD)研究了TFE诱导的IA(3)的螺旋构象，包括一系列自旋标记的半胱氨酸扫描结构和不同的氨基酸取代。结果表明，IA(3)的N端凹螺旋面是与YPRA结合时的埋藏界面，它可以被自旋标记或大体积氨基酸取代而不稳定。相反，当自旋标记被引入到IA(3)的凸起表面，即暴露于溶剂的表面时，IA(3)的螺旋倾向被增强。在C-末端区域内没有观察到自旋标记的影响。这项工作进一步证明了SDSL CW-EPR在国内流离失所者研究中的实用性和敏感性。一般来说，必须注意确保自旋标记不会干扰天然的螺旋倾向，这些研究为我们提供了在哪里包含自旋标记的知识，以便将来对IA(3)的SDSL研究。
IA(3) is an intrinsically disordered protein (IDP) that becomes helical when bound to yeast proteinase A (YPRA) or in the presence of the secondary stabilizer 2,2,2-trifluoroethanol (TFE). Here, site-directed spin-labeling (SDSL) continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy and circular dichroism (CD) are used to characterize the TFE-induced helical conformation of IA(3) for a series of spin-labeled cysteine scanning constructs and varied amino acid substitutions. Results demonstrate that the N-terminal concave helical surface of IA(3), which is the buried interface when bound to YPRA, can be destabilized by the spin-label or bulky amino acid substitutions. In contrast, the helical tendency of IA(3) is enhanced when spin-labels are incorporated into the convex, i.e., solvent exposed, surface of IA(3). No impact of the spin-label within the C-terminal region was observed. This work further demonstrates the utility and sensitivity of SDSL CW-EPR for studies of IDPs. In general, care must be taken to ensure the spin-label does not interfere with native helical tendencies and these studies provide us with knowledge of where to incorporate spin-labels for future SDSL investigations of IA(3).