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

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.

Usage Notes
Figure 2 dataValidation of the method for determining substrate specificity using three unrelated proteins.Figure 2.xlsxFigure 3 dataIdentification of substrates for a mitochondrial phosphate carrier from the thermophilic ciliate Tetrahymena thermophila.Figure 3.xlsxFigure 4 DataThe effect of coupling ions on the stabilization of transporters by substrate binding.Figure 4.xlsxFigure 1-figure supplement 1Figure 2-figure supplement 2Figure 2-figure supplement 4Figure 3-figure supplement 3

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