查看文章

Live-cell recordings of protein conformations and interactions yield novel mechanistic insights into physiologic and pathologic protein functions. We have developed an adaptable and genetically encoded bioluminescence-based biosensor platform to precisely measure signaling dynamics of full-length RAS GTPases and RAF kinases upon physiologic pathway activation, post-translational modifications, mutagenesis, and drug binding. We have recorded the impact of both patient mutations and bioactive small-molecule binding on kinase conformations and binary interactions of GTP-loaded H, N, KRAS with kinases such as RAF. Systematic profiling of αC-helix-OUT BRAF inhibitors (BRAFi) vemurafenib, dabrafenib, encorafenib, and PLX8394 using the BRAF reporter reporter platform (displaying 10 different patient mutations) revealed differences in drug specificities and efficacies. Unexpectedly, the αC-helix-OUT …