PT  - JOURNAL ARTICLE
AU  - Homa Majd
AU  - Martin S. King
AU  - Shane M. Palmer
AU  - Anthony C. Smith
AU  - Liam D.H. Elbourne
AU  - Ian T. Paulsen
AU  - David Sharples
AU  - Peter J. F. Henderson
AU  - Edmund R. S. Kunji
TI  - Screening of candidate substrates and coupling ions of transporters by thermostability shift assays
AID  - 10.1101/367805
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 367805
4099  - http://biorxiv.org/content/early/2018/07/13/367805.short
4100  - http://biorxiv.org/content/early/2018/07/13/367805.full
AB  - 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.