Abstract 0352


Kummer, L., Hsu, C. W., Dagliyan, O., Macnevin, C., Kaufholz, M., Zimmermann, B., Dokholyan, N. V., Hahn, K. M., and Plückthun, A. (2013). Knowledge-Based Design of a Biosensor to Quantify Localized ERK Activation in Living Cells. Chem. Biol. 20, 847-856.


Investigation of protein activation in living cells is fundamental to understanding how proteins are influenced by the full complement of upstream regulators they experience. Here, we describe the generation of a biosensor based on the DARPin binding scaffold suited for intracellular applications. Combining library selection and knowledge-based design, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK with a solvatochromatic merocyanine dye, whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. The DARPin-based biosensor will serve as a useful tool for studying biological functions of ERK in vitro and in vivo.


Supplement 0352

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