Using hydroxyl radical footprinting to explore the free energy landscape of protein folding.
Using hydroxyl radical footprinting to explore the free energy landscape of protein folding.
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DOI：
10.1016/j.ymeth.2015.02.018
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发表时间：
2015-11-01
期刊：
影响因子：
--
通讯作者：
Ashcroft AE
中科院分区：
文献类型：
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作者：
Calabrese AN;Ault JR;Radford SE;Ashcroft AE
蛋白质的快速光化学氧化区分蛋白质构象。氧化程度的整体增加与蛋白质展开相关。将氧化定位到肽/氨基酸水平提供了进一步的见解。蛋白质折叠过程中所采用的构象状态的表征，包括全局未折叠/无序结构和部分折叠的中间物种，对于了解蛋白质如何折叠至关重要。在这项工作中，我们采用快速光化学氧化的蛋白质（FPOP）映射的结构变化，发生在折叠的四螺旋细菌免疫蛋白，Im 7。氧化足迹法结合质谱法（MS）用于探测与折叠过程同时发生的氨基酸侧链溶剂可及性的变化，通过定量野生型蛋白质相对于动力学捕获的三螺旋折叠中间体和缺乏二级结构的未折叠变体所经历的氧化程度。通过FPOP-MS对未折叠变体的分析显示氧化修饰与采用具有高度溶剂可及性的溶液构象的物种一致。相比之下，折叠中间体仅在被引入的氨基酸取代不稳定的区域中经历相对于野生型天然蛋白质增加的氧化水平。结果表明，FPOP-MS的实用性，以确定蛋白质变体在不同的构象状态，并提供洞察蛋白质折叠机制，是互补的测量，如氢/氘交换标记和Φ值分析。
Fast photochemical oxidation of proteins discriminates between protein conformers. Global increases in the extent of oxidation correlate with protein unfolding. Localisation of oxidation to the peptide/amino acid level provides further insights. Characterisation of the conformational states adopted during protein folding, including globally unfolded/disordered structures and partially folded intermediate species, is vital to gain fundamental insights into how a protein folds. In this work we employ fast photochemical oxidation of proteins (FPOP) to map the structural changes that occur in the folding of the four-helical bacterial immunity protein, Im7. Oxidative footprinting coupled with mass spectrometry (MS) is used to probe changes in the solvent accessibility of amino acid side-chains concurrent with the folding process, by quantifying the degree of oxidation experienced by the wild-type protein relative to a kinetically trapped, three-helical folding intermediate and an unfolded variant that lacks secondary structure. Analysis of the unfolded variant by FPOP–MS shows oxidative modifications consistent with the species adopting a solution conformation with a high degree of solvent accessibility. The folding intermediate, by contrast, experiences increased levels of oxidation relative to the wild-type, native protein only in regions destabilised by the amino acid substitutions introduced. The results demonstrate the utility of FPOP–MS to characterise protein variants in different conformational states and to provide insights into protein folding mechanisms that are complementary to measurements such as hydrogen/deuterium exchange labelling and Φ-value analysis.