Screening of candidate substrates and coupling ions of transporters by thermostability shift assays
- University of Cambridge, United Kingdom
- Macquarie University, Australia
- University of Leeds, United Kingdom
- Cited 9
- Views 2,168
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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.
Article and author information
Author details
Funding
Medical Research Council (MC_UU_00015/1)
- Homa Majd
- Martin S King
- Shane M Palmer
- Anthony C Smith
- Edmund RS Kunji
Cambridge Commonwealth, European and International Trust
- Homa Majd
Leverhulme Trust (EM-2014 -045)
- Peter JF Henderson
Biotechnology and Biological Sciences Research Council (MPSI BBS/B/14418)
- David Sharples
Wellcome (JIF 062164/Z/00/Z)
- David Sharples
University of Leeds
- David Sharples
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Volker Dötsch, J.W. Goethe-University, Germany
Publication history
- Received: July 11, 2018
- Accepted: October 11, 2018
- Accepted Manuscript published: October 15, 2018 (version 1)
- Version of Record published: November 1, 2018 (version 2)
Copyright
© 2018, Majd et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Structural Biology and Molecular Biophysics
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
