TY - JOUR
T1 - Knowledge-based design of a biosensor to quantify localized ERK activation in living cells
AU - Kummer, Lutz
AU - Hsu, Chia Wen
AU - Dagliyan, Onur
AU - MacNevin, Christopher
AU - Kaufholz, Melanie
AU - Zimmermann, Bastian
AU - Dokholyan, Nikolay V.
AU - Hahn, Klaus M.
AU - Plückthun, Andreas
N1 - Funding Information:
We thank A. Honegger for calculations of Gibbs free energies of protein variants. L.K. was supported by a doctoral fellowship of the Ernst Schering Foundation. We gratefully acknowledge funding from Schweizerische Nationalfonds grant 3100A0B-128671, the Swiss National Center of Competence in Research in Structural Biology, the PhosphoNetX Project in SystemsX (all to A.P.), the European Union FP7 Collaborative Project AffinityProteome (contract 222635) (to A.P., M.K., and B.Z.), and grant GM090317 from the National Institutes of Health (to K.M.H.). A.P. is a founder and shareholder of Molecular Partners, AG, which commercializes the DARPin technology.
PY - 2013/6/20
Y1 - 2013/6/20
N2 - 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.
AB - 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.
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U2 - 10.1016/j.chembiol.2013.04.016
DO - 10.1016/j.chembiol.2013.04.016
M3 - Article
C2 - 23790495
AN - SCOPUS:84879376222
SN - 1074-5521
VL - 20
SP - 847
EP - 856
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 6
ER -