Screening of candidate substrates and coupling ions of transporters by thermostability shift assays.
Palmer, Shane M
Elbourne, Liam Dh
Paulsen, Ian T
eLife Sciences Publications, Ltd
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Majd, H., King, M., Palmer, S. M., Smith, A., Elbourne, L. D., Paulsen, I. T., Sharples, D., et al. (2018). Screening of candidate substrates and coupling ions of transporters by thermostability shift assays.. Elife, 7 https://doi.org/10.7554/eLife.38821
Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.
Mitochondria, Animals, Humans, Tetrahymena, Ions, Membrane Transport Proteins, Adenosine Diphosphate, Adenosine Triphosphate, Ligands, Biological Assay, Reproducibility of Results, Temperature, Substrate Specificity, Protein Stability
Medical Research Council (MC_U105663139)
Medical Research Council (MC_UU_00015/1)
Embargo Lift Date
External DOI: https://doi.org/10.7554/eLife.38821
This record's URL: https://www.repository.cam.ac.uk/handle/1810/285785
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/