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.
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Research Article|
May 15 2003
Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy
Véronique CALLEJA;
Véronique CALLEJA
∗Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, Lincoln's Inn Fields Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.
†Advanced Technology Development Group, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middx. HA6 2JR, U.K.
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Simon M. AMEER-BEG;
Simon M. AMEER-BEG
†Advanced Technology Development Group, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middx. HA6 2JR, U.K.
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Borivoj VOJNOVIC;
Borivoj VOJNOVIC
†Advanced Technology Development Group, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middx. HA6 2JR, U.K.
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Rudiger WOSCHOLSKI;
Rudiger WOSCHOLSKI
‡Department of Biological Sciences, Biochemistry Building, South Kensington Campus, Imperial College London, London SW7 2AZ, U.K.
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Julian DOWNWARD;
Julian DOWNWARD
§Signal Transduction Laboratory, London Research Institute, Cancer Research UK, Lincoln's Inn Fields Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.
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Banafshé LARIJANI
Banafshé LARIJANI
1
∗Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, Lincoln's Inn Fields Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.
1To whom correspondence should be addressed (e-mail b.larijani@cancer.org.uk).
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Publisher: Portland Press Ltd
Received:
March 05 2003
Revision Received:
March 27 2003
Accepted:
March 27 2003
Accepted Manuscript online:
March 27 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 372 (1): 33–40.
Article history
Received:
March 05 2003
Revision Received:
March 27 2003
Accepted:
March 27 2003
Accepted Manuscript online:
March 27 2003
Connected Content
A correction has been published:
Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy
Citation
Véronique CALLEJA, Simon M. AMEER-BEG, Borivoj VOJNOVIC, Rudiger WOSCHOLSKI, Julian DOWNWARD, Banafshé LARIJANI; Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. Biochem J 15 May 2003; 372 (1): 33–40. doi: https://doi.org/10.1042/bj20030358
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