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Knowledge-based design of a biosensor to quantify localized ERK activation in living cells.


ABSTRACT: 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.

SUBMITTER: Kummer L 

PROVIDER: S-EPMC4154710 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Knowledge-based design of a biosensor to quantify localized ERK activation in living cells.

Kummer Lutz L   Hsu Chia-Wen CW   Dagliyan Onur O   MacNevin Christopher C   Kaufholz Melanie M   Zimmermann Bastian B   Dokholyan Nikolay V NV   Hahn Klaus M KM   Plückthun Andreas A  

Chemistry & biology 20130601 6


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 fluo  ...[more]

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