Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute and §Department of Medical Biology, University of Melbourne , Melbourne, Victoria 3010, Australia.
ACS Chem Biol. 2014 Jul 18;9(7):1426-31. doi: 10.1021/cb500242q. Epub 2014 May 15.
We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.
我们开发了一种新方法，可在活细胞中区分不同的蛋白质构象。该方法涉及将工程化的四半胱氨酸基序插入靶蛋白中，靶蛋白的构象状态可使双砷酸盐染料结合具有条件访问能力。XTC 用于区分 Src 家族激酶的开放和闭合调节构象。通过在三个 Src 家族激酶（c-Src、Lck、Lyn）的保守 SH2 结构域中仅用四个残基替换为半胱氨酸，就可以根据与双砷酸盐染料 FlAsH 或 ReAsH 的结合差异来监测开放和闭合构象。将激酶与荧光蛋白融合可跟踪激酶的存在，而 XTC 方法可同时评估调节状态。c-Src XTC 生物传感器用于激酶抑制剂的精品筛选，该筛选揭示了六种诱导构象闭合的化合物。XTC 方法为针对疾病和生物学中关键蛋白质构象变化的测定法提供了新的潜力。
