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

Lutz Kummer, Chia Wen Hsu, Onur Dagliyan, Christopher MacNevin, Melanie Kaufholz, Bastian Zimmermann, Nikolay Dokholyan, Klaus M. Hahn, Andreas Plückthun

Research output: Contribution to journalArticle

32 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)847-856
Number of pages10
JournalChemistry and Biology
Volume20
Issue number6
DOIs
StatePublished - Jun 20 2013

Fingerprint

Biosensing Techniques
Biosensors
Chemical activation
Cells
Fibroblasts
Mitogens
Scaffolds
Libraries
Conformations
Proteins
Phosphotransferases
Embryonic Structures
Fluorescence

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

Cite this

Kummer, L., Hsu, C. W., Dagliyan, O., MacNevin, C., Kaufholz, M., Zimmermann, B., ... Plückthun, A. (2013). Knowledge-based design of a biosensor to quantify localized ERK activation in living cells. Chemistry and Biology, 20(6), 847-856. https://doi.org/10.1016/j.chembiol.2013.04.016
Kummer, Lutz ; Hsu, Chia Wen ; Dagliyan, Onur ; MacNevin, Christopher ; Kaufholz, Melanie ; Zimmermann, Bastian ; Dokholyan, Nikolay ; Hahn, Klaus M. ; Plückthun, Andreas. / Knowledge-based design of a biosensor to quantify localized ERK activation in living cells. In: Chemistry and Biology. 2013 ; Vol. 20, No. 6. pp. 847-856.
@article{7b267e0258424dfbac3c687683f71647,
title = "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.",
author = "Lutz Kummer and Hsu, {Chia Wen} and Onur Dagliyan and Christopher MacNevin and Melanie Kaufholz and Bastian Zimmermann and Nikolay Dokholyan and Hahn, {Klaus M.} and Andreas Pl{\"u}ckthun",
year = "2013",
month = "6",
day = "20",
doi = "10.1016/j.chembiol.2013.04.016",
language = "English (US)",
volume = "20",
pages = "847--856",
journal = "Cell Chemical Biology",
issn = "2451-9448",
publisher = "Elsevier Inc.",
number = "6",

}

Kummer, L, Hsu, CW, Dagliyan, O, MacNevin, C, Kaufholz, M, Zimmermann, B, Dokholyan, N, Hahn, KM & Plückthun, A 2013, 'Knowledge-based design of a biosensor to quantify localized ERK activation in living cells', Chemistry and Biology, vol. 20, no. 6, pp. 847-856. https://doi.org/10.1016/j.chembiol.2013.04.016

Knowledge-based design of a biosensor to quantify localized ERK activation in living cells. / Kummer, Lutz; Hsu, Chia Wen; Dagliyan, Onur; MacNevin, Christopher; Kaufholz, Melanie; Zimmermann, Bastian; Dokholyan, Nikolay; Hahn, Klaus M.; Plückthun, Andreas.

In: Chemistry and Biology, Vol. 20, No. 6, 20.06.2013, p. 847-856.

Research output: Contribution to journalArticle

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

AU - Hahn, Klaus M.

AU - Plückthun, Andreas

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.

UR - http://www.scopus.com/inward/record.url?scp=84879376222&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84879376222&partnerID=8YFLogxK

U2 - 10.1016/j.chembiol.2013.04.016

DO - 10.1016/j.chembiol.2013.04.016

M3 - Article

C2 - 23790495

AN - SCOPUS:84879376222

VL - 20

SP - 847

EP - 856

JO - Cell Chemical Biology

JF - Cell Chemical Biology

SN - 2451-9448

IS - 6

ER -

Kummer L, Hsu CW, Dagliyan O, MacNevin C, Kaufholz M, Zimmermann B et al. Knowledge-based design of a biosensor to quantify localized ERK activation in living cells. Chemistry and Biology. 2013 Jun 20;20(6):847-856. https://doi.org/10.1016/j.chembiol.2013.04.016