Kinetics of Protein Complex Dissociation Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry.
Kinetics of Protein Complex Dissociation Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry.
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DOI：
10.1021/acs.analchem.5b03123
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发表时间：
2015-12-01
影响因子：
7.4
通讯作者：
Vachet RW
中科院分区：
文献类型：
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作者：
Zhang Z;Vachet RW
蛋白质聚集性疾病的重要性日益增加，需要开发新的方法来阐明负责早期蛋白质-蛋白质相互作用的分子特征。蛋白质-蛋白质结合/解离反应的动力学信息对于揭示机制的洞察力特别有价值，但是能够提供这些信息的强大工具在某种程度上缺乏。在这项工作中，我们描述了一种基于氢/氘交换（HDX）的方法，该方法提供了蛋白质低聚物解离的速率常数信息，使用已被充分研究的β-乳球蛋白（βLG）二聚体作为模型系统来验证我们的方法。通过使用自顶向下串联质谱法测量蛋白质不同区域的交换速率，并将结果数据拟合到适当的数学模型中，我们能够提取二聚体的解离速率常数。我们利用了这样一个事实，即作为蛋白质-蛋白质界面一部分的蛋白质区域具有与非界面区域不同的交换模式。这一观察结果表明，HDX/MS方法不仅可以提供动力学信息，同时还可以提供有关界面的结构信息，这对以前未表征的蛋白质-蛋白质复合物非常有价值。
The growing importance of protein aggregation diseases requires the development of new methods to elucidate the molecular features that are responsible for the incipient protein-protein interactions. Kinetic information from protein-protein association/dissociation reactions is particularly valuable for revealing mechanistic insight, but robust tools that can provide this information are somewhat lacking. In this work, we describe a hydrogen/deuterium exchange (HDX)-based method that provides rate constant information for protein oligomer dissociation, using the well-studied β-lactoglobulin (βLG) dimer as a model system to validate our approach. By measuring the rate of exchange at different regions of the protein using top-down tandem mass spectrometry and fitting the resulting data to an appropriate mathematical model, we are able to extract the dimer’s dissociation rate constant. We exploit the fact that regions of the protein that are part of the protein-protein interface have exchange patterns that are distinct from non-interfacial regions. This observation indicates that the HDX/MS method not only provides kinetic information but could also provide structural insight about the interface at the same time, which would be very valuable for previously uncharacterized protein-protein complexes.