Thermal proteome profiling allows quantitative assessment of interactions between tetrachloroethene reductive dehalogenase and trichloroethene.
Thermal proteome profiling allows quantitative assessment of interactions between tetrachloroethene reductive dehalogenase and trichloroethene.
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DOI：
10.1016/j.jprot.2018.05.018
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发表时间：
2019-02
影响因子：
3.3
通讯作者：
Dominique Türkowsky;Patrick Lohmann;Marie Mühlenbrink;T. Schubert;L. Adrian;Tobias Goris;N. Jehmlich;M. von Bergen
中科院分区：
文献类型：
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作者：
Dominique Türkowsky;Patrick Lohmann;Marie Mühlenbrink;T. Schubert;L. Adrian;Tobias Goris;N. Jehmlich;M. von Bergen
热蛋白质分析（TPP）越来越多地应用于真核生物中，通过配体存在引起的蛋白质熔化曲线位移来研究蛋白质与配体的结合。在厌氧菌中，蛋白质-底物相互作用的鉴定是一个主要的挑战。我们将TPP应用于能够以三氯乙烯为电子受体生长的硫螺旋藻（sulfurospirillum multivorans），研究其四氯乙烯还原脱卤酶PceA与三氯乙烯的相互作用。方案中的几处修改（例如，在厌氧条件下孵育；将温度范围提高到97 °C）扩大了蛋白质检测范围，并允许对氧敏感蛋白进行研究。通过在孵育过程中省略电子供体来防止酶还原脱卤。这可以检测到PceA与三氯乙烯的相互作用，并证实三氯乙烯是该酶的底物。有趣的是，一个假定的反应调节因子显示出类似的趋势，这是其在三氯乙烯呼吸中提出的作用的第一个生化暗示。我们证明，我们的TPP方法有助于鉴定严格厌氧还原脱卤酶及其调控因子的蛋白质-底物相互作用。该策略可用于鉴定未知的底物特异性和可能的信号传感蛋白，因此有可能阐明有机盐呼吸细菌研究中尚未解决的领域之一。意义评估有机盐呼吸细菌中酶-底物或蛋白质-配体的相互作用是一个根本性的挑战。热蛋白质组分析（TPP）允许阐明蛋白质组范围的热稳定性变化依赖于现代质谱的灵敏度。这使得可以识别其他方法无法检测到的相互作用。在这项TPP研究中，我们展示了氯化底物与还原性脱卤酶的相互作用，并可能与响应调节剂相互作用，从而支持响应调节剂在有机卤化物呼吸中的作用。该策略也可用于鉴定细菌中其他酶的未知底物，这些底物难以研究或只有少量生物量可用。酶-底物相互作用的评估，可能会得出关于酶特异性的结论，这代表了TPP的一个新的应用。
Thermalproteomeprofiling(TPP) is increasingly applied in eukaryotes to investigate protein-ligand binding through protein melting curve shifts induced by the presence of a ligand. In anaerobic bacteria, identification of protein-substrate interactions is a major challenge. We applied TPP toSulfurospirillum multivorans, which is able to use trichloroethene as electron acceptor for growth, to investigate the interaction of its tetrachloroethene reductive dehalogenase PceA with trichloroethene. Several modifications in the protocol (e.g., incubation under anaerobic conditions; increasing the temperature range up to 97 °C) extended the protein detection range and allowed the investigation of oxygen-sensitive proteins. Enzymatic reductive dehalogenation was prevented by omitting the electron donor during incubations. This enabled detecting the interaction of PceA with trichloroethene and confirmed that trichloroethene is a substrate of this enzyme. Interestingly, a putative response regulator showed a similar trend, which is the first biochemical hint for its proposed role in trichloroethene respiration. We proved that our TPP approach facilitates the identification of protein-substrate interactions of strictly anaerobic reductive dehalogenases and probably their regulators. This strategy can be used to identify yet unknown substrate specificities and possible signal-sensing proteins, and therefore has the potential to elucidate one of the unresolved fields in research on organohalide-respiring bacteria.SignificanceThe assessment of enzyme-substrate or protein-ligand interactions in organohalide-respiring bacteria is a fundamental challenge.Thermal proteome profiling(TPP) allows elucidating proteome-wide thermal stability changes relying on the sensitivity of modern mass spectrometry. This gives access to the identification of interactions not detectable with other methods. In this TPP study, we demonstrate the interactions of a chlorinated substrate with a reductive dehalogenase and potentially with a response regulator, thereby supporting the response regulator's function in organohalide respiration. The strategy might also be applied to identify yet unknown substrates of other enzymes in bacteria which are difficult to investigate or for which only low amounts of biomass are available. The assessment of enzyme-substrate interactions, which might enable conclusions about enzyme specificities, represents a new application for TPP.