Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy.
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, YFP(dark), 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.
Calleja
Véronique
V
Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, Lincoln's Inn Fields Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.
Ameer-Beg
Simon M
SM
Vojnovic
Borivoj
B
Woscholski
Rudiger
R
Downward
Julian
J
Larijani
Banafshé
B
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt