Journal article

A Biosensor of Src Family Kinase Conformation by Exposable Tetracysteine Useful for Cell-Based Screening

Sevgi Irtegun, Rebecca Wood, Kurt Lackovic, Joerg Schweiggert, Yasmin M Ramdzan, David CS Huang, Terrence D Mulhern, Danny M Hatters



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..

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Awarded by Australian National Health and Medical Research Council

Funding Acknowledgements

The work was funded in part by grants to D.M.H. and T.D.M. from the Australian National Health and Medical Research Council and the Australian Research Council. D.M.H. was a Grimwade Fellow, supported by the Miegunyah Trust and currently an ARC Future Fellow. The Huang lab is supported by the Australian National Health and Medical Research Council (Program Grant 1016701; Independent Research Institutes Infrastructure Support Scheme grant 361646), the Leukemia and Lymphoma Society (SCOR grants) and a Victorian State Government Operational Infrastructure Support (OIS) Grant.