Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells

Bin Wu, Yan Chen, Joachim D. Müller

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Measuring the binding curve and stoichiometry of protein complexes in living cells is a prerequisite for quantitative modeling of cellular processes. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions, but lacks suitable methods for quantifying protein heterointeractions in the cell. We address this challenge by introducing heterospecies partition (HSP) analysis for protein heterointeractions of the type D + nA ↔ DAn. HSP directly identifies the hetero-interacting species from the sample mixture and determines the binding curve and stoichiometry of the protein complex. The HSP method is applied to provide the first direct characterization of the ligand-dependent binding of the retinoic X receptor to the coactivator transcription intermediate factor 2. A previous study based on protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. This study provides proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein heterointeractions by dual-color fluorescence fluctuation spectroscopy in living cells.

Original languageEnglish (US)
Pages (from-to)4117-4122
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number9
DOIs
StatePublished - Mar 2 2010
Externally publishedYes

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Proteins
Fluorescence Spectrometry
Color
Transcription Factors
Ligands

Keywords

  • Coactivator
  • Fluorescence correlation spectroscopy
  • Fluorescence fluctuation spectroscopy
  • Nuclear receptor
  • Time-integrated fluorescence cumulant analysis

ASJC Scopus subject areas

  • General

Cite this

Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells. / Wu, Bin; Chen, Yan; Müller, Joachim D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 9, 02.03.2010, p. 4117-4122.

Research output: Contribution to journalArticle

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