Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy

Véronique Calleja, Simon M. Ameer-Beg, Borivoj Vojnovic, Rudiger Woscholski, Julian Downward, Banafshé Larijani

Research output: Contribution to journalArticlepeer-review

Abstract

To be able to detect in situ changes in protein conformation without perturbing the physiological environment would be a major step forward in understanding the precise mechanism occurring in protein interaction. We have developed a novel approach to monitoring conformational changes of proteins in intact cells. A double-labelled fluorescent green fluorescent protein-yellow fluorescent protein (GFP-YFP) fusion protein has been constructed, allowing the exploitation of enhanced-acceptor-fluorescence (EAF)-induced fluorescence resonance energy transfer (FRET). Additionally, a novel fusion partner, YFPdark, has been designed to act as a sterically hindered control for EAF-FRET. Any conformational changes will cause a variation in FRET, which, in turn, is detected by fluorescence lifetime imaging microscopy ('FLIM'). Protein kinase B (PKB)/Akt, a key component of phosphoinositide 3-kinase-mediated signalling, was selected for this purpose. Although conformational changes in PKB/Akt consequent to lipid binding and phosphorylation have been proposed in models, its behaviour in intact cells has not been tractable. We report here that platelet-derived-growth-factor ('PDGF') stimulation of NIH3T3 cells expressing the GFP-Akt-YFP construct resulted in a loss of FRET at the plasma membrane and hence a change in PKB/Akt conformation. We also show that the GFP-Akt-YFP construct conserves fully its functional integrity. This novel approach of monitoring the in situ conformational changes has broad application for other members of the AGC kinase superfamily and other proteins.

Original languageEnglish
Pages (from-to)33-40
Number of pages8
JournalBiochemical Journal
Volume372
Issue number1
DOIs
Publication statusPublished - 15 May 2003

Keywords

  • AGC kinases
  • Conformational change
  • Dark yellow fluorescent protein (YFP)
  • Enhanced acceptor fluorescence
  • Fluorescence resonance energy transfer (FRET)
  • Protein kinase B (PKB)/Akt

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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