A biosensor of Src family kinase conformation by exposable tetracysteine useful for cell-based screening
AuthorIRTEGUN, SEVGI; WOOD, REBECCA; LACKOVIC, KURT; Schweiggert, Jörg; Ramdzan, Yasmin M.; Huang, David C. S.; Mulhern, Terrence D.; Hatters, Danny M.
Source TitleACS Chemical Biology
PublisherAmerican Chemical Society (ACS)
University of Melbourne Author/sIRTEGUN, SEVGI; WOOD, REBECCA; Lackovic, Kurt; Schweiggert, JOERG; MOHAMED RAMDZAN, YASMIN; Mulhern, Terrence; Hatters, Daniel; Huang, David
AffiliationDepartment of Biochemistry and Molecular Biology, Faculty of Medicine, Dentistry & Health Sciences
Department of Medical Biology, Faculty of Medicine, Dentistry & Health Sciences
Biochemistry and Molecular Biology
Document TypeJournal Article
CitationsIrtegun, S., Wood, R., Lackovic, K., Schweiggert, J., Ramdzan, Y. M., Huang, D. C. S., et al. (2014). A biosensor of Src family kinase conformation by exposable tetracysteine useful for cell-based screening. ACS Chemical Biology.
Access StatusThis item is currently not available from this repository
NHMRC Grant codeNHMRC/1062104
The research outputs in this collection have been funded in whole or in part by the National Health and Medical Research Council (NHMRC).
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 based on 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.
- Click on "Export Reference in RIS Format" and choose "open with... Endnote".
- Click on "Export Reference in RIS Format". Login to Refworks, go to References => Import References