RT Journal Article
SR Electronic
T1 Screening of candidate substrates and coupling ions of transporters by thermostability shift assays
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 367805
DO 10.1101/367805
A1 Homa Majd
A1 Martin S. King
A1 Shane M. Palmer
A1 Anthony C. Smith
A1 Liam D.H. Elbourne
A1 Ian T. Paulsen
A1 David Sharples
A1 Peter J. F. Henderson
A1 Edmund R. S. Kunji
YR 2018
UL http://biorxiv.org/content/early/2018/07/13/367805.abstract
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.