Screening of candidate substrates and coupling ions of transporters by thermostability shift assays

Homa Majd, Martin S. King, Shane M. Palmer, Anthony C. Smith, Liam D. H. Elbourne, Ian T. Paulsen, David Sharples, Peter J. F. Henderson, Edmund R. S. Kunji

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Article number38821
Pages (from-to)1-17
Number of pages17
JournaleLife
Volume7
DOIs
Publication statusPublished - 15 Oct 2018

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Bibliographical note

Copyright the Author(s) 2018. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • H+ SYMPORT PROTEIN
  • ESCHERICHIA-COLI
  • MEMBRANE-TRANSPORT
  • MITOCHONDRIAL CARRIERS
  • GLUCOSE TRANSPORTERS
  • METABOLOME DATABASE
  • LIGAND RECOGNITION
  • PHOSPHATE CARRIER
  • CRYSTAL-STRUCTURE
  • MAMMALIAN-CELLS

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