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  • Title: Thermal proteome profiling allows quantitative assessment of interactions between tetrachloroethene reductive dehalogenase and trichloroethene.
    Author: Türkowsky D, Lohmann P, Mühlenbrink M, Schubert T, Adrian L, Goris T, Jehmlich N, von Bergen M.
    Journal: J Proteomics; 2019 Feb 10; 192():10-17. PubMed ID: 29879467.
    Abstract:
    Thermal proteome profiling (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 to Sulfurospirillum 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. SIGNIFICANCE: The 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.
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