Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland, 21201.
Protein Sci. 2018 Jun;27(6):1047-1056. doi: 10.1002/pro.3408. Epub 2018 Apr 14.
Hydroxyl radical footprinting (HRF) is a nonspecific protein footprinting method that has been increasingly used in recent years to analyze protein structure. The method oxidatively modifies solvent accessible sites in proteins, which changes upon alterations in the protein, such as ligand binding or a change in conformation. For HRF to provide accurate structural information, the method must probe the native structure of proteins. This requires careful experimental controls since an abundance of oxidative modifications can induce protein unfolding. Fast photochemical oxidation of proteins (FPOP) is a HRF method that generates hydroxyl radicals via photo-dissociation of hydrogen peroxide using an excimer laser. The addition of a radical scavenger to the FPOP reaction reduces the lifetime of the radical, limiting the levels of protein oxidation. A direct assay is needed to ensure FPOP is probing the native conformation of the protein. Here, we report using enzymatic activity as a direct assay to validate that FPOP is probing the native structure of proteins. By measuring the catalytic activity of lysozyme and invertase after FPOP modification, we demonstrate that FPOP does not induce protein unfolding.
羟基自由基足迹法（HRF）是一种非特异性蛋白质足迹法，近年来越来越多地用于分析蛋白质结构。该方法氧化修饰蛋白质中溶剂可及的位点，这些修饰会因配体结合或构象改变而发生变化。为了使 HRF 提供准确的结构信息，该方法必须探测蛋白质的天然结构。这需要仔细的实验控制，因为大量的氧化修饰会诱导蛋白质展开。快速光化学氧化蛋白质（FPOP）是一种 HRF 方法，它使用准分子激光通过过氧化氢的光解产生羟基自由基。向 FPOP 反应中添加自由基清除剂会缩短自由基的寿命，从而限制蛋白质氧化的水平。需要直接测定来确保 FPOP 正在探测蛋白质的天然构象。在这里，我们报告使用酶活性作为直接测定来验证 FPOP 正在探测蛋白质的天然结构。通过测量 FPOP 修饰后溶菌酶和转化酶的催化活性，我们证明 FPOP 不会诱导蛋白质展开。
