Screening of candidate substrates and coupling ions of transporters by thermostability shift assays.
Screening of candidate substrates and coupling ions of transporters by thermostability shift assays.
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DOI：
10.7554/elife.38821
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发表时间：
2018-10-15
期刊：
影响因子：
7.7
通讯作者：
Kunji ER
中科院分区：
文献类型：
--
作者：
Majd H;King MS;Palmer SM;Smith AC;Elbourne LD;Paulsen IT;Sharples D;Henderson PJ;Kunji ER
大多数转运蛋白的底物尚未确定，限制了我们对其在生理和疾病中的作用的理解。传统的鉴定方法使用放射性化合物的输运分析，但它们在技术上具有挑战性，而且许多化合物无法以放射性形式获得或过于昂贵，因此无法进行大规模试验。在这里，我们提出了一种高通量筛选方法，可以从未标记的化合物文库中识别候选底物。该分析是基于转运蛋白通过特定相互作用识别底物的原理，这导致热稳定性转移分析中转运蛋白群体的稳定性增强。测试了三种不同转运蛋白（超）家族的代表，它们在结构、转运和离子耦合机制上都有所不同。在每种情况下，底物都是从大量化学相关化合物（包括立体异构体）中正确识别出来的。在某些情况下，离子进一步增强了底物结合的稳定性，为能量耦合机制提供了可测试的假设。
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.