Sokolovski Miri, Cveticanin Jelena, Hayoun Déborah, Korobko Ilia, Sharon Michal, Horovitz Amnon
Department of Structural Biology, Weizmann Institute of Science, Rehovot, 761001, Israel.
Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 761001, Israel.
Nat Commun. 2017 Aug 9;8(1):212. doi: 10.1038/s41467-017-00285-1.
The strength and specificity of protein complex formation is crucial for most life processes and is determined by interactions between residues in the binding partners. Double-mutant cycle analysis provides a strategy for studying the energetic coupling between amino acids at the interfaces of such complexes. Here we show that these pairwise interaction energies can be determined from a single high-resolution native mass spectrum by measuring the intensities of the complexes formed by the two wild-type proteins, the complex of each wild-type protein with a mutant protein, and the complex of the two mutant proteins. This native mass spectrometry approach, which obviates the need for error-prone measurements of binding constants, can provide information regarding multiple interactions in a single spectrum much like nuclear Overhauser effects (NOEs) in nuclear magnetic resonance. Importantly, our results show that specific inter-protein contacts in solution are maintained in the gas phase.Double mutant cycle (DMC) analyses can provide the interaction energies between amino acids at the interface of protein complexes. Here, the authors determine pairwise interaction energies using high-resolution native mass spectroscopy, offering a straightforward route for the DMC methodology.
蛋白质复合物形成的强度和特异性对大多数生命过程至关重要，且由结合伴侣中残基之间的相互作用决定。双突变循环分析提供了一种研究此类复合物界面处氨基酸之间能量耦合的策略。在此，我们表明，通过测量由两种野生型蛋白质形成的复合物、每种野生型蛋白质与突变型蛋白质形成的复合物以及两种突变型蛋白质形成的复合物的强度，可从单个高分辨率天然质谱中确定这些成对相互作用能。这种天然质谱方法无需对结合常数进行容易出错的测量，与核磁共振中的核Overhauser效应（NOE）类似，能在单个光谱中提供有关多种相互作用的信息。重要的是，我们的结果表明，溶液中特定的蛋白质间接触在气相中得以维持。双突变循环（DMC）分析可提供蛋白质复合物界面处氨基酸之间的相互作用能。在此，作者使用高分辨率天然质谱确定成对相互作用能，为DMC方法提供了一条直接途径。
