Title | Screening of candidate substrates and coupling ions of transporters by thermostability shift assays. |
Publication Type | Journal Article |
Year of Publication | 2018 |
Authors | Majd, H, King, MS, Palmer, SM, Smith, AC, Elbourne, LDh, Paulsen, IT, Sharples, D, Henderson, PJf, Kunji, ERS |
Journal | Elife |
Volume | 7 |
Date Published | 2018 10 15 |
ISSN | 2050-084X |
Keywords | Adenosine Diphosphate, Adenosine Triphosphate, Animals, Biological Assay, Humans, Ions, Ligands, Membrane Transport Proteins, Mitochondria, Protein Stability, Reproducibility of Results, Substrate Specificity, Temperature, Tetrahymena |
Abstract | 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. |
DOI | 10.7554/eLife.38821 |
Alternate Journal | Elife |
Citation Key | 10.7554/eLife.38821 |
PubMed ID | 30320551 |
PubMed Central ID | PMC6211832 |
Grant List | EM-2014 -045 / / Leverhulme Trust / International JIF 062164/Z/00/Z / / Wellcome / International MC_UU_00015/1 / / Medical Research Council / United Kingdom MPSI BBS/B/14418 / / Biotechnology and Biological Sciences Research Council / United Kingdom / / Wellcome Trust / United Kingdom JIF 062164/Z/00/Z / / Wellcome Trust / United Kingdom |