Quantifying Protein Interface Footprinting by Hydroxyl Radical Oxidation and Molecular Dynamics Simulation: Application to Galectin-1
Quantifying Protein Interface Footprinting by Hydroxyl Radical Oxidation and Molecular Dynamics Simulation: Application to Galectin-1
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DOI：
10.1016/j.jasms.2008.07.013
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发表时间：
2008-11-01
影响因子：
3.2
通讯作者：
Woods, Robert J.
中科院分区：
文献类型：
--
作者：
Charvatova, Olga;Foley, B. Lachele;Woods, Robert J.
生物分子表面映射方法提供了一种重要的替代方法，用于表征蛋白质-蛋白质和蛋白质-配体相互作用的情况下，它是不可能确定高分辨率的三维（3D）结构的复合物。与传统的化学衍生法相比，羟基自由基足迹法在足迹分辨率方面具有显著的进步。在这里，我们提出的足迹进行的结果与产生的纳秒时间尺度上的过氧化氢的激光诱导光解离的羟基自由基。我们将这种新兴的方法应用于碳水化合物结合蛋白，凝集素-l。由于半乳糖凝集素-1作为同源二聚体出现，足迹法被用来表征单体亚基的界面。使用最近开发的ByOnic（Palo Alto，CA）软件实现了氧化蛋白质质谱数据的有效分析，该软件经过修改以处理大量由侧链氧化引起的修饰。氧化水平的定量已通过采用光谱强度为每个残留物的基础上观察到的所有氧化态。通过检查与半乳糖凝集素-1氧化和胰蛋白酶消化后存在的合成肽相关的合成肽混合物，确定光谱强度可达到的准确度水平。出现了侧链溶剂可及性和氧化水平之间的直接关系，这使得能够预测给定蛋白质的3D结构的氧化水平。这种关系的精度提高，通过使用平均溶剂accessories计算从10 ns的分子动力学模拟的蛋白质。（J Am Soc Mass Spectrom 2008，19，1692 - 1705）（C）2008年美国质谱学会
Biomolecular surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, g lectin-l. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein. (J Am Soc Mass Spectrom 2008, 19, 1692-1705) (C) 2008 American Society for Mass Spectrometry