Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains.
Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains.
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DOI：
10.1007/978-1-61779-927-3_22
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
de Prat-Gay, Gonzalo
中科院分区：
文献类型：
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作者：
Chemes, Lucia B;Alonso, Leonardo G;de Prat-Gay, Gonzalo
圆二色性（CD）光谱是一种简单而强大的技术，它允许评估蛋白质或蛋白质结构域的构象特性。如本系列所讨论的，内含子无序蛋白（IDP）与无规卷曲多肽的不同之处在于不同区域呈现特定的构象偏好，表现出动态二级结构内容[1]。这些动态二级结构元件可以通过不同的化学（溶剂、离子强度、pH）或物理（温度）试剂、通过翻译后修饰和通过配体来稳定或扰动。此信息对于定义ID性质非常重要。由于IDP呈现动态构象，圆二色性测量（以及其他方法）不应作为在独特条件下进行的单一光谱来进行，而应改变化学条件并观察行为，作为确定ID性质的一部分。在本章中，我们提出了对感兴趣的蛋白质进行远紫外CD测量以及识别和表征固有无序区域的基本方法，以及用于分析所研究的蛋白质中存在的残余二级结构的几种方案。这些技术很容易执行;它们需要最少的培训，并且可以作为更复杂的方法（如NMR）的基础。
Circular dichroism (CD) spectroscopy is a simple and powerful technique, which allows for the assessment of the conformational properties of a protein or protein domain. Intrinsically disordered proteins (IDPs), as discussed throughout this series, differ from random coil polypeptides in that different regions present specific conformational preferences, exhibiting dynamic secondary structure content [1]. These dynamic secondary structure elements can be stabilized or perturbed by different chemical (solvent, ionic strength, pH) or physical (temperature) agents, by posttranslational modifications, and by ligands. This information is important for defining ID nature. As IDPs present dynamic conformations, circular dichroism measurements (and other approaches as well) should be carried out not as single spectra performed in unique conditions, but instead changing the chemical conditions and observing the behavior, as part of the determination of the ID nature.In this chapter, we present the basic methodology for performing Far-UV CD measurements on a protein of interest and for identifying and characterizing intrinsically disordered regions, and several protocols for the analysis of residual secondary structure present in the protein under study. These techniques are straightforward to perform; they require minimal training and can be preliminary to more complex methodologies such as NMR.