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J Biol Chem. 2019 Feb 9. pii: jbc.RA119.007685. doi: 10.1074/jbc.RA119.007685. [Epub ahead of print]

Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.

Author information

1
RIKEN Quantitative Biology Center, Japan.
2
National Institutes of Natural Sciences, National Institute for Basic Biology, Japan.
3
Graduate School of Biostudies, Kyoto University, Japan.
4
Division of Quantitative Biology, National Institutes of Natural Sciences, National Institute for Basic Biology, Japan.

Abstract

Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (Kd). However, only a limited number of parameters have been measured experimentally in living cells. Here, we describe an approach for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy (FCCS). First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy (FCS) to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, FCCS analyses revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor (EGF) stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.

KEYWORDS:

RSK; dissociation constant; extracellular-signal-regulated kinase (ERK); fluorescence correlation spectroscopy (FCS); fluorescence cross-correlation spectroscopy (FCCS); live cell analysis; nuclear translocation; protein-protein interaction; single cell analysis

PMID:
30739083
DOI:
10.1074/jbc.RA119.007685
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