Screening of candidate substrates and coupling ions of transporters by thermostability shift assays
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Authors
Majd, Homa
Palmer, Shane
Elbourne, Liam
Paulsen, Ian
Sharples, David
Henderson, Peter
Kunji, Edmund RS
Publication Date
2018-10-12Journal Title
eLife
ISSN
2050-084X
Publisher
eLife Sciences Publications Ltd
Type
Article
This Version
VoR
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Majd, H., King, M., Palmer, S., Smith, A., Elbourne, L., Paulsen, I., Sharples, D., et al. (2018). Screening of candidate substrates and coupling ions of transporters by thermostability shift assays. eLife https://doi.org/10.7554/eLife.38821
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.
Sponsorship
MRC (MC_U105663139)
Identifiers
External DOI: https://doi.org/10.7554/eLife.38821
This record's URL: https://www.repository.cam.ac.uk/handle/1810/287627