Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
Protein Sci. 2011 Jan;20(1):150-9. doi: 10.1002/pro.547.
Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA₃ is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA₃ has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h((+1))/h(₀), the local tumbling volume (V(L)), and the percent change of the h(₋₁) intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h((+1)/)h(₀) as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA₃. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA₃. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.
电子顺磁共振（EPR）光谱学与定点自旋标记（SDSL）相结合，是一种用于表征蛋白质的流动性和构象变化的有价值的工具，但很少应用于固有无序蛋白质（IDP）。在这里，IA₃被用作模型系统，证明了 SDSL-EPR 对小 IDP 系统构象变化的表征。IA₃ 有 68 个氨基酸，在溶液中无结构，在加入二级结构稳定剂 2,2,2-三氟乙醇（TFE）时变为α-螺旋。生成了两个单点半胱氨酸取代物，一个在 N 端（S14C），一个在 C 端（N58C），并用三个不同的氮氧自由基自旋标记物进行标记。比较了每个标记的 EPR 线形状，并报告了加入 TFE 后流动性的变化。具体来说，利用谱线形状参数 h((+1))/h(₀)、局部翻滚体积 (V(L)) 和 h(₋₁)强度的变化百分比来定量监测 TFE 诱导的构象变化。h((+1)/)h(₀)随 TFE 滴定的变化呈 S 型，用双态 Boltzmann 模型拟合，得到过渡中点的值，从而报告 IA₃ 的全局构象变化。其他参数提供了特定于位点的信息，并表明 S14C-SL 发生构象变化，导致运动受限程度高于 N58C-SL，这与先前使用 NMR 和圆二色性光谱学研究获得的结果一致，表明 IA₃ 的 N 端片段具有更高的α-螺旋倾向。总的来说，这些结果为 SDSL 研究 IDP 中诱导的无规卷曲到螺旋构象的数据分析提供了一个框架。
