Mapping pH-Induced Protein Structural Changes Under Equilibrium Conditions by Pulsed Oxidative Labeling and Mass Spectrometry
Mapping pH-Induced Protein Structural Changes Under Equilibrium Conditions by Pulsed Oxidative Labeling and Mass Spectrometry
复制标题
DOI：
10.1021/ac302393g
复制
发表时间：
2012-11-06
影响因子：
7.4
通讯作者：
Konermann, Lars
中科院分区：
文献类型：
--
作者：
Vahidi, Siavash;Stocks, Bradley B.;Konermann, Lars
基于质谱（MS）的蛋白质构象研究是一个快速发展的领域。部分无序构象的表征是特别感兴趣的，因为这些物种是不服从经典的高分辨率技术。这种平衡中间体通常可以通过暴露于温和的酸性pH值来填充。羟基自由基（中心点OH）在溶剂可接近的侧链处引入氧化修饰，而掩埋的位点受到保护。中心点OH可以通过H2 O2的激光光解（蛋白质的快速光化学氧化-FPOP）产生。由此产生的标记图案可以通过MS进行分析。部分无序的中间体的表征通常涉及在不同溶剂条件下的比较测量。将结构诱导的标记变化与pH介导的“次级”效应区分开来可能具有挑战性。FPOP中的次级效应问题以前很少受到注意。我们证明，通过适当选择条件（例如，在不存在pH依赖性中心点OH清除剂的情况下）可以几乎完全消除这种不希望的现象。使用脱辅基肌红蛋白作为模型系统，我们映射的中间体，在pH 4形成的结构。该物种保留了高度保护的螺旋G，螺旋G被部分保护的螺旋A、B和H包围。我们的研究结果表明FPOP的平衡中间体的结构表征的实用程序。与氢/氘交换MS相比，几乎不存在固有pH依赖性代表了优势。
Mass spectrometry (MS)-based protein conformational studies are a rapidly growing field. The characterization of partially disordered conformers is of particular interest because these species are not amenable to classical high-resolution techniques. Such equilibrium intermediates can often be populated by exposure to mildly acidic pH. Hydroxyl radical (center dot OH) introduces oxidative modifications at solvent-accessible side chains, while buried sites are protected. center dot OH can be generated by laser photolysis of H2O2 (fast photochemical oxidation of proteins-FPOP). The resulting labeling pattern can be analyzed by MS. The characterization of partially disordered intermediates usually involves comparative measurements under different solvent conditions. It can be challenging to separate structurally induced labeling changes from pH mediated "secondary" effects. The issue of secondary effects in FPOP has received little prior attention. We demonstrate that with a proper choice of conditions (e.g., in the absence of pH-dependent center dot OH scavengers) such undesired phenomena can be almost completely eliminated. Using apomyoglobin as a model system, we map the structure of an intermediate that is formed at pH 4. This species retains a highly protected helix G that is surrounded by partially protected helices A, B, and H. Our results demonstrate the utility of FPOP for the structural characterization of equilibrium intermediates. The near absence of an intrinsic pH dependence represents an advantage compared to hydrogen/deuterium exchange MS.